Eckert Animal Physiology: Mechanisms and Adaptations (Fourth Edition)

V O U R T H

Version

Eckert

Animal Physiology and Adaptation

david

Rand Air

U N I V E A S ~O T FW B R I T I S HC O L U M B I A

maze

B U R G G R E N

Catherine

French

U N I V E R S I TOYF C A L I F O R N I A S, A N D L E G O

MIT C O N T R I B U T I O NBSY

RUSSELL FERNALD S T N F O R DUN I V E R S I T Y

W. H. Freeman and New Ynrk Company

Acquisition Publisher: Deborah Allen Development Publisher: Kay Ueno Project Publisher: Kate Ahr Image Research: Larry Marcus Cover Designer: Michael Mendelsohn, Design 2000, Inc Cover Image: Arctic

Fox, Canada O Daniel J. Cox/Tony Stone Images

Illustration on the back: Roberto Osti

Typography: Michael Mendelsohn, Design 2000, Inc.; Victoria Tomaselli

Illustration Coordinator: Bill Page Illustration: Fine Line Illustration; Medical and Scientific Illustration, William C. Ober, MD and Claire Garrison, RN, BA Production Coordinator: Maura Studley Composition: Progressive Information Technologies Manufacturer: RR Donnelley & Sons Company

Library of Congress Publishing Cataloging Data Randall, Dav~dJ., 1938Eckert an~malphysis~ology: mechanisms and adaptations/Dav~d Randall, Warren Burggren, Kathleen French.-4. Relative P. CM References and Index. Includes blbl~ograph~cal ISBN 0-7167-2414-6 (hardcover) 1. Physiology. I. Bergeron, Warren. 11. French, Catherine. 111. Tttle. QP31.2.R36 1997 591.1-dc20 96-31713 CIP Copyright O 1978, 1983, 1988, 1997 W.H. Freeman and Company. all rights reserved. No part of this book may be reproduced for public or private use, stored in a retrieval system, transmitted, or otherwise reproduced, by mechanical, photographic, or electronic means, or in phonograms of any kind, without written permission publisher. Printed in the United States of America. Second printing, 1997, RRD

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about the author

david randle

For contributions to fish physiology. Frequent speaker at seminars on fish physiology and other topics, most recently in Brazil, France, Germany, Italy, People's Republic of China, Russia, and the United States. He has worked with the World Health Organization and the US Environmental Protection Agency on ammonia standards. Randall is the author and co-author of numerous publications in leading peer-reviewed journals and is the author of the acclaimed Fish Physiolog? (AcademicPress), 15 volumes of which are in print. Volume 16, subtitled Deep Sea Fish, was published in 1997. Among Randall's other responsibilities is serving as a co-instructor for a third-year vertebrate and environmental physiology class. His research interests relate to the interplay between gas and ion exchange between fish gills. ..................................................................... .. .. ................................ WARREN BURGGREN is subject to change as development progresses. Warren Burggren taught physiology for 23 years, actively participated in symposiums, seminars, and formal co-curricular research and training activities, and has been a professor of biological sciences at the University of Nevada, Las Vegas since 1992. He has taught courses in many countries. A co-author of The Evolution of Air, he has taught at UNLV and the University of Massachusetts. "Breath of Vertebrate Animals" (Cambridge University Press, 1981). Since serving as Setts Professor of Zoology from 1987 to 1980, Burggren has regularly contributed to the publication of the collection. Department of Physiology, including before 1991, including Human Anatomy and Physiology, Bioenergetics, Introduction to Zoology, and Comparative Animal Physiology from Comparative Press, Ecophysiology IV. Burggren's research interests include development (Wiley-Liss, 1991). Burggren co-edited Environmental physiology, Comparative Animal Physiology, and Mental Physiology of the Amphibia (Environmental and Environmental Physiology University. Special Chicago Press, 1992) and most recently co-edited Dehi's studies, focusing on respiratory Ontogeny and development of the cardiovascular system; molecular or cardiovascular systems and how systems influence regulation (Cambridge University Press, 1997). David Randall, a renowned fish physiologist and a leading expert in respiratory and circulatory physiology, collaborated with the late Roger Eckert on early questions in animal physiology and continues his contributions in this fourth issue. Randall has been a faculty member at the University of British Columbia, Vancouver, Canada, since 1963 and a full professor since 1973. He was named Associate Dean of the Graduate School in 1990. Randall was elected a Fellow of the Royal Society of Canada in 1981, a Guggenheim and Killam Fellow, and was awarded the prestigious Fry Medal for Contributions to Zoological Research by the Zoological Society of Canada in 1993. 1995 Award of Excellence from the American Fisheries Association

..................................................................... ...................................

Catherine French

Kathleen French has been a neurobiologist at UC San Diego since 1985 and has taught high school courses in embryology, mammalian physiology to medical students, and cellular neurobiology for the past 10 years. In addition, French participated in a training program at UC San Diego, teaching science teaching assistants teaching techniques and concepts. She is also an instructor for the Neuroscience and Behavior course at Woods Hole Marine Biological Laboratory in Massachusetts, an intensive course primarily for graduate students and postdoctoral fellows. French brings her expertise and love of teaching

As a co-author of this issue of Animal Physiology, along with a lifelong interest in the nervous systems of organisms from a wide range of phyla. As an associate project scientist at UC San Diego, French's research focuses on the control of neuronal development, a topic she has studied in various invertebrate species. Her current research concerns the cellular events that control the differentiation of identified neurons in medicinal leeches, with a focus on the cellular physiology of embryonic neurons and the effects of cell-cell contacts. She is the author and co-author of numerous research and review articles published in peer-reviewed journals, including the Journal of Neuroscience and the Journal of Neurophysiology.

Part I Principles of Physiology 1 Animal Physiological Studies

2 Experimental methods to explore physiology 3 Molecules, energy and biosynthesis

4 Membranes, channels and transport

Part II Physiological Processes 5 Physical Basis of Nerve Function 6 Communication Between Neurons

7 Environmental Recognition 8 Glands: Secretory Mechanisms and Costs 9 Hormones: Regulation and Action 10 Muscle and Animal Movement

351

11 Behavior: Initiation, Mode, and Control

405

Part III Physiological System Integration 465 12 Cycles

467

13 Gas Exchange and Acid-Base Equilibrium

517

14 Ionic and Osmotic Equilibrium

571

15 Energy Production: Nutrition, Digestion. and metabolism

627

16 Using energy: Environmental challenges mean

665

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Preface Acknowledgments

ninety-seven

Structural Analysis of Cells in Cell Culture

Biochemical Analysis: Component Measurement: Components present. Concentration Measurement: How Much

Part I Principles of Physiology Chapter 1 Studies in Animal Physiology

Subdisciplines of Animal Physiology Why Study Animal Physiology?

4 4

Fragrance, business, agricultural applications, insights into human physiology

Key topics in animal physiology Structure-function relationships Adaptation, adaptation and adaptation Homeostatic feedback control systems Conformation and regulation

Physiological Sciences Literature Spotlight 1-1 Concepts of Feedback Animal Experiments in Physiology Summary Reviews Recommended Reading

4 4 4

4 5 5 7 8 9 10

12 13 13 14 14

Isolating Organs and Organ System Experiments Observing and Measuring Animal Behavior Power of Behavioral Experiments Behavioral Research Methods

Importance of Physiological State in Research Summary Revlew Questions~ons Suggested Reading

Chapter 3 Molecular, energy, and biosynthetic origins of major biochemical molecules Atoms, bonds, and molecules Special roles of H, O, N, and C in life processes Water: a unique solvent Water molecular properties of water Water as a solvent

properties of the solution

Chapter 2 deals with experimental methods for studying physiology, formulating and testing hypotheses. August Crow Principle. Experimental Design and Physiological Levels

Molecular technology. Tracking molecules with radioisotopes. Tracking molecules with monoclonal antibodies. Genetic engineering

Cell Technology Using Microelectrodes and Microplates

Concentration, collective properties and activity ionization of water and aqueous solutions

Focus 3-1 Electrical terms and conventions Mixing of ions into macromolecules

content

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Biomolecules, Carbohydrates, Proteins, Nuclear Compounds, Energy in Living Cells, Energy: Concepts and Definitions, Transfer of Chemical Energy by Coupled Reactions, ATP: Cellular Temperature Energy Carriers and Reaction Rates, Enzymes: General Properties, Enzyme Specificity and Active Processes, Mechanisms of enzyme catalysis, effects of temperature and pH on enzyme reactions, cofactors, enzyme molecules, enzyme inactivation, regulation of metabolic reactions, control of enzyme synthesis, control of enzyme activity, ATP oxidative metabolic production, phosphorylation and energy transfer energy metabolism Oxygen Debt Glycolysis Regulates Cycle Efficiency Abstract Rev ~ewQuest~ons Recommended Reading

Ionic gradients as a source of coupled cellular energy transfer. membrane selectivity. Electrolyte selectivity. non-electrolyte selectivity. Mechanisms of endocytosis and exocytosis. Exocytosis mechanism

Chapter 4 Membranes, Channels, and Transport Effects of Membrane Structure and Organization on Membrane Flow Osmotic, Osmotic Pressure, and Tonicity, Effects of Electricity on Diffusion of Ions Through Membrane Channel Diffusion Through the LLP~dB Layer Focus 4-2 Artificial Bilayers Facilitate Membrane Transport Actively transporting Na+ lK+ pumps as a model for AV transport

connections between cells

Gap Junct~ons Tlight Junct~ons Epithelial cell transport Active salt transport through epithelial cells Water transport Abstract Comments New Issue Suggested Reading

Part II Physiological processes Chapter 5 Physical basis of neural function Overview of neural structure, function and organization Signal transmission in single neurons Signal transmission between neurons Organs of the nervous system Membrane excitation Measurement of membrane potential, passive and active electrical properties Dispersed film. Focus 5-1: The discovery of "animal electricity". The role of ion channels. Passive electrical properties of membranes. Membrane resistance and conductivity. membrane capacity. electrochemical potential. Nernst equation: Calculation of equilibrium potential for a single ion Focus 5-2 Quantitative considerations of charge separation between membranes Goldman equation: Calculation of equilibrium potential for multiple ions Resting potential Ionic gradient and the role of channels Active transmission Action potential General properties Ionic basis of action potential Summary of ion concentration changes during excitation of other electrically excited channels in focus 5-3 voltage clamp method

content

habit

................................... Viewing the question. recommended reading

Chapter 6 on communication between neurons. Transmission of signals in the nervous system: an overview. Information transfer within a single neuron 6-1 Extracellular hallmarks of impulse conduction Focus 6-2 Axon diameter and conduction velocity Information transfer between neurons: synapse/synaptlc structure and function: electrical synapse Synaptlc structure And Function: Chemical Synapse Fast Chemical Synapse Focusing 6-3 Pharmacological Effects Useful in Synapse Research Focusing 6-4 Reversal Potential Calculation Presynaptic Release of Neurotransmitter Quanta1 Release of Neurotransmitter Depolarization Release Coupling Chemical properties of non-pulsatile release neurotransmitters fast, direct neurotransmitters slow, indirect neurotransmitters mission postsynaptic mechanisms MS- receptors and channels in fast, direct neurotransmission receptors m slow, indirect neurotransmission neuromodulation integration at synapses synaptic plasticity homosynaptic modulation: simple homosynapt modulation: post-tetanic potentiation heterosynapt modulation "long-term potency ~for summary review~new questions Recommended reading~ngs

Coding, intensity, input-output relations, range, grading, control of sensory perception, chemical senses: taste and smell, gustatory receptor mechanism, olfactory receptor mechanism, mechanosensation, cells and vestibular apparatus, ear of vertebrates insect ear electrothermal Optical Mechanisms of Sensory Vision: Evolution and Function Compound Eye Focusing 7-1 Subjective Correlation of Primary Light Responses Photoreception in the Vertebrate Eye: Conversion of Photons into Neural Signals Focusing 7-2 Electroretinogram Focusing 7-3 Light-Limited Reception of the Senses, Limitations of sensory reception, distance and color. Revlew homework summary, recommended reading

224 225 226 226 231 232 235 238 238 241 242 248 248 250 251 252 253

256 257 261 263 268 269 270 271 271

Chapter 8 Glands: Secretion mechanisms and costs of cellular secretions Types and functions of secretions Surface secretions: Packing and transport of extracellular membranes and mucus-separating substances Spotlight 8-1 Substances with similar structures and functions deposited from different organisms Isolation of substances and general properties of storage glands Endocrine glands Energy costs of glandular activity in exocrine glands Summary of new issues Recommended reading

Chapter 7 Knowing Your Surroundings

Chapter 9 Hormones: Regulation 301 and Its Implications

General properties of sensory reception Properties of receptor cells Common mechanisms and molecules of sensory transduction From transduction to neuronal output

The endocrine system: an overview Chemical types and general functions of hormones Regulation of hormone secretion Neuroendocrine system Hypothalamic control of the pituitary gland

302 3 02 303 303 304

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content

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Glandular Hormones Released from Prostatic Glands 305 Neurohormones Released from Posterior Prostatic Glands 308 Focus 9-1 Peptide Hormones 310 Cellular Mechanisms of Hormone Action 311 Lipid Soluble Hormones and Cytoplasmic Receptors 311 Lipid Soluble Hormones and Intracellular Signaling 312 Lighting Up 9 -2 Passed Enzyme Cascade Enhancement 322 Physiological Actions of Hormones 328 Metabolic and Developmental Hormones 328 Hormones Regulating Water and Electrolyte Balance 336 Reproductive Hormones 338 Prostaglandins 342 Hormone Actions in Invertebrates 343 Summary 346 Revlew Quest 348 Recommended Reading 349

Chapter 10 Muscle and Animal Movement Structural Basis of Muscle Contraction Myofilament substructure Sarcomere Contraction: Slldlng-Flament-theory The Making of Kreuzbrücken and Kraft Spotlight 10-1 Parallel and Row Arrangement: The Geometry of Muscle Mechanics of Strength and Power Shortening Speed Spotlight 10- 2 Effect of skin muscle fiber crossbridges on the force-velocity relationship Regulation of contraction by lime in crossbridge attachment Exclusion-contraction-coupling-contraction-relaxation cycle transmission Force generation Serles Elast~ cComponent active state Twitch and tetanus muscle contraction energy muscle Utilization of ATP by globulin ATPases and calcific pumps Regeneration of ATP during muscle activity Classification of fiber types in vertebrate skeletal muscle Functional principles of different fiber types Adaptation of muscles to different activities Adaptive strength: functional diversity in jumping frogs Sex: Floating Fish Adaptation Speed: Noise Production High-Frequency Muscle: Neural Control of Muscle Contraction in Asynchronous Flight Muscles

Motor Control in Vertebrates, Motor Control in Arthropods, Myocardium, Smooth Muscle, Summary, Revlew Questions, Recommended Reading

Chapter 11 Behavior: Initiation, Modes, and Control

405

Focus 11-1 Behavior of animals lacking a nervous system 403 Development of the nervous system 408 Organization of the vertebrate nervous system 412 Major parts of the central nervous system 413 420 Autonomic nervous system Animal behavior 423 Basic behavioral concepts 4 23 42 6 Behavior of example neural circuits Features 432 Parts of the Neuronal Puzzle 433 Sensory Networks 434$ Focus 11-2 Voting Curves: Neuronal Responses to Stimulus Parameters 436 Focus 11-3 Specificity of Neuronal Connections and Interactions 447 Motor Networks 45 3 Summary 461 Review Questions 462 462 suggested answers

Part III Physiological Systems Integration Chapter 12 Circulatory System General Planning Open Circuit Closed Circuit Heart Electrical Activity Coronary Circuit Mechanical Properties of the Heart in Focus 12-1 Frank-Starling Mechanism The Pericardium of the Vertebrate Heart: Comparing Function Morphology Dynamics of laminar and turbulent flow, pressure and flow relationship, microcirculation of peripheral circulation, arterial system, venous system, capillaries and lymphatic system

... Contents xi11 ................................. The cycle and regulation of the immune response OF CIRCULATION center Vascular Control Cardiovascular Microcirculation Control Response to Extreme Conditions Exercise Hemostasis Summary New Topics Recommended Reading

Chapter 13 Gas Exchange and Acid-Base Balance General Considerations Focus 13-1 Early Experiments on Gas Exchange in Animals Oxygen and Carbon Dioxide in Blood Respiratory Organs Focus 13-2 Gas Laws Oxygen Transport in Blood Carbon in Blood D ~ o x ~ Transport Transfer Gases in and out of the blood regulate body pH Hydrogen ion production and excretion Hydrogen ion disruption between compartments Factors affecting intracellular pH Factors affecting body pH Air Gas transfer: Functional anatomy of the lungs and other systems Pulmonary circulation Focus 13- 3 Lung Volume Ventilation Lungs Pulmonary Surfactant Heat and Water Loss Through the Lungs Gas Transfer in Breast Eggs Tracheal System Water in Insects Gas Transfer: Gas Exchange between Gill Stream and Gall Bladder. Functional anatomy of the gallbladder. Regulation of gas transmission and respiration. Ventdat~on-to-Perfus~on Rat~os~a) Increased Carbon D~ox~ Levels (hypercapma) D~v~n g Air-breathing Animals Training Swim Bladder: Oxygen Accumulation Under Large Gradients

Summarize the review questions. recommended reading

Chapter 14 Ionian and Osmotic Balance Issues of Ion Exchange and Osmoregulation Between the Water Gradient Between the Animal and the Surrounding Area Surface Volume Permeability of Skin Nutrition, Metabolic Factors and Excretion Temperature, Practice and Respiration Osmoregulators and Osmoregulators Osmoregulation in Water Respiration Regulation Organ Mammalian Kidney Prehistoric Mammalian Anatomy Product Focus 14-1 Renal Clearance ANCE Regulation of pH by Renal-Urine Concentration Mechanism Focus 14-2 Countercurrent System Control of Non-mammalian Vertebrates Water Reabsorption Extrarenal Kidney Osmoregulation Vertebrate Organs Salt Glands Fish Invertebrates Osmoregulation Organs Filtrate Reabsorption System Secretion Reabsorption System Excretion of Nitrogenous Waste Mals Summary Rev~ewQuests~ons Recommended Reading

Chapter 15 Energy Acquisition: Nutrition, Digestion, and Metabolism Directed Methods Feeding Through the Extensor Surface Endocytosis Filters Nutrient Solution Ingestion of Prey Ingestion and Grazing of Herbivores to Collect Food Digestive System Overview Foregut: Foregut Ingestion: Ingestion, storage and digestion. Gut: chemical digestion and absorption

571 571 574 574 574 575 577 5 78 580 581 581 584 587 587 588 590 595 601 603

604 606 608 608 608 613 616 616 617 620 621 623 624 624 625 625

fourteen

content

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . …… . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Hindgut: Absorption and defecation of water and ions, dynamics of gut structure - effects of diet, alimentary canal motility, muscle and ciliary motility, peristalsis, motor control, gastrointestinal secretions, extravenous secretions, maximal secretions Control, Spotlight 15 - 1 Behavioral Regulation of Nutrients and Digestion and Absorption Nutrient Absorption in the Gut Blood Transport of Nutrients Gut Water and Electrolyte Balance Nutrient Requirements Energy Balance Nutrient Molecules Review Questions Abstract Recommended Reading

643 644 644 645 645 646 649 650 653

654 657 657 658 659 66 1 66 1 66 1 663 664 664

Chapter 16 Using Energy for Challenges 665 Concepts of Energy Metabolism Measuring Metabolic Rate Basal Metabolic Rate and Standard Metabolic Rate Metabol~cScope D~rectCalor~rnery Focus 16-1 Energy Units (or When Are Calories Not Calories?) Indirect Calorimetry Method - Measuring B. Fasting volume and waste excretion. Indirect measurement of metabolic rate, respiratory state, energy storage, specific kinetics, effects on height and metabolic rate. Spotlight 16-2. Reynolds number: energy effects of large and small animals on animal body temperature and metabolic rate determinants of animal body temperature classification

665 666 666 667 668

668 668 669 670 671 672 672

676 677 677 680 682

Temperature ratios in ectotherms ectotherms in freezing and cold environments, ectotherms in water and heat environments Costs and benefits of ectotherms: comparison with endothermic body temperature, fever, rest: special metabolic conditions. Rigid sleep, hibernation and hibernation Body rhythms and energy C~rcadlan Rhythms Unstructured endogenous rhythms Thermoregulation, metabolism and biorhythms , ENVIRONMENT AND EVOLUTION Abstract Rev~ewQuests~ons Recommended Reading

Appendix 1: SI Units Appendix 2: Logarithmic and Exponential Functions Appendix 3: Conversions, Formulas, Physical and Chemical Constants, Definitions References Glossary Index

It's been almost ten years since the third edition of Animals

Physiology was first published by Roger Eckert with the help of David Randall. Roger died in 1986 while revising the third edition completed by George Augustine and David Randall. This book forms the basis for the fourth edition, aptly titled Eckert's Animal Physiology. Although this new edition has been extensively revised and redesigned, it retains the approach that characterized the previous editions so successfully: the use of comparative examples to illustrate general principles, often supported by experimental data. In addition, we have highlighted homeostatic principles and updated molecular and cellular coverage. This issue retains comprehensive coverage of tissues, organs, and organ systems. The book integrates cellular and molecular themes early on in order to develop common themes to explain and compare the interactions between regulated physiological systems that produce coordinated responses to environmental changes in various animal populations. The fundamentals and mechanisms of animal physiology and the animal adaptations that allow them to exist in so many different environments form the central theme of this book. The diversity and adaptability of the millions of species that make up the animal kingdom bring endless fascination and delight to nature lovers. Last but not least, the fun comes from observing how the animal's body works. At first glance, with so many animal species adapted to such diverse lifestyles and environments, understanding and appreciating animal physiology may seem overwhelming. Fortunately (for scientists and students) there are relatively few concepts and principles that form the basis for understanding animal function, because evolution is both conservative and creative. Introduction to Physiology is challenging for both teachers and students due to the interdisciplinary nature of the subject, which incorporates chemistry, physics, and more

biology. Most of the students are eager to get involved in this subject and go on to learn more exciting modern scientific knowledge. For this reason, Eckert's Animal Physiology is organized in such a way that basic background material is presented in such a way that students can work through it on their own, then move quickly to consider animal function and understand their experimental instructions. Eckert's Animal Physiology develops key concepts in a simple and straightforward manner, emphasizes the principles and mechanisms of information assembly, and illustrates the functional strategies of animals that evolve within the range of chemical and physical possibilities. The focus is on common principles and patterns, not exceptions. Examples are selected from a wide range of animal life, intentionally illustrating similarities between organisms; for example, similar links are associated with the reproduction of humans and yeast. Therefore, the more esoteric and minor details are only cursory or paid no attention in order not to distract from the central idea. Research methods when we use narratives describing experiments to convey information.

Structure of the Book The chapters are divided into three parts for the first time, which we believe will enhance the understanding of animals as comprehensive systems at any organizational level. Each section is introduced by an opening statement to give students an idea of the material to be studied. Part I consists of four chapters and deals with the core principles of physiology. Part I1 (Chapters 5-11) discusses physiological processes, while Part I11 (Chapters 12-16) discusses how these fundamental processes are integrated in animals living in different environments. All 16 chapters have been extensively revised and reorganized to keep up with new scientific developments.

sixteen

foreword

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What's new in this issue A new methodology chapter (Chapter 2) has been added to Part 1, where some recent molecular techniques as well as traditional methods are discussed and illustrated. This emphasis on molecular coverage is carried throughout the book; for example, Chapters 5, 6, and 7 are updated with recent insights into the cellular and molecular basis of membrane excitation, synaptic transmission, and sensory transduction. Part I1 includes a new chapter (Chapter 8, Glands: Mechanisms and Costs of Secretion) summarizing information on an important but often overlooked effector system. In Part 11, Chapter 11 (Behavior: Priming, Patterns, and Control) maintains and expands on previous editions of the description of the vertebrate and invertebrate nervous One of the fastest growing fields in biology. Several concepts in neuroethology that bridge the gap between the study of pure behavior and the study of cellular function in the nervous system are introduced, along with examples of recent important neurobehavioral studies. The role of the nervous system in maintaining homeostasis by regulating all systems is integrated in Part 111, furthering the book's integrated approach. Throughout the book, greater emphasis is placed on environmental adaptation, and specific examples of environmental adaptation (such as the hydrology of elephant seals in Chapter 14) illustrate general principles of comparative physiology. New topics introduced in the fourth edition include the immune response section in Chapter 12 (Circulation) and the biological rhythm section in Chapter 13 (Harnessing Energy: Meeting Environmental Challenges).

Education Extensive use of illustrations and graphics to illustrate and complement points made in the text

legend. For the first time, full-color graphics have been added, creating a high-quality visual program to further motivate students. Spotlights provide details about experiments and people involved in significant advances in the problem, the derivation of some equations, or just historical background on the topic in question. The reflection questions in the main text of the chapter (note a) encourage problem-based learning and stimulate discussion of different aspects of the material presented. The textual narrative includes effective built-in examples to support the principles; in presenting information, it conveys a coherent scope of topics and flair for investigative methods. Citations to literature in the text and figure captions are unobtrusive, but frequent enough for students to be aware of the role of scientists and their literature to develop as a discipline. Additional teaching aids include key terms explained in the text, bolded when first mentioned, and formally defined in a helpful, comprehensive glossary. The material at the end of the chapter includes an executive summary that gives students a quick overview of the points covered in the chapter, knowledge review questions, and a notated list of recommended further reading. At the end of the book, students will find the following resources: an appendix with information on units, equations, and formulas; a glossary; and a bibliography with full citations for all references cited in the chapters. Our goal was to create a balanced, up-to-date representation of animal functionality, characterized by a clear presentation. I hope that Eckert Animal Physiology will be helpful to readers, and welcome your constructive criticism and suggestions.

September 1996

David Randall Bergren Warren Catherine French

Second

Bill Kristan, UC Sun Riverside Diego Paul Lennard, Emory University Jon E. Levine, Northwestern University Harvey B. Lillywhite, University of Florida Gainesville Duane R. McPherson, SUNY an der Geneseo Duncan S. MacKenzie, DE Texas A&M University Eric Mundall, spät, UCLA Kenneth Nagy, UCLA Richard A. Nyhoff, Calvin College Richard W. Olsen, UCLA School of Medicine C. Leo Ortiz, UC Santa Cruz Harry Peery, University of California J. Larry Renfro, University of Connecticut Marc M. Roy, Beloit College Roland Roy, Brain Institute, UCLA School of Medicine Jonathon Scholey, UC Davis C. Eugene Settle, University of Arizona Michael P. Sheetz, Gregory Snyder, Washington University School of Medicine, Joe Henry Steinbach, University of Colorado Boulder, Curt Swanson, Washington University School of Medicine, Malcolm H. Wayne State University. Taylor, University of Delaware - College of Life and Health Sciences Ulrich A. Walker, Columbia University - School of Medicine Eric P. Widmaier, Boston University Andrea H. Worthington, Siena College Ernest M. Wright, UCLA School of Medicine – Center for Science and Health

ckert Animal Physiology has benefited greatly from the contributions of several individuals, and we are very grateful for their efforts. Russell Fernald of Stanford University contributed to the planning and restructuring of the book and the initial revision of many chapters. Lawrence C. Rome of the University of Pennsylvania wrote the first draft of Chapter 10 (Muscle and Animal Movement), which contains most of the updated material except for the heart and smooth muscle and neural control sections. Harold Atwood of the University of Toronto contributed to some of the early discussions on the revision. In addition, we thank the informal comments from collaborators around the world and the formal manuscript review provided by the following colleagues from across the country:

Joseph Bastian, University of Oklahoma Robert B. Barlow, Syracuse University Sensory Institute Francisco Bezanilla, UCLA School of Medicine Health Sciences Center Phillip Brownell, Oregon State University Richard Bruch, Louisiana State University Wayne W. Carley, National Biological Association, Lehrer Ingrith Deyrup-Olsen, Dale Erskine University of Washington, A. Verdi Farmanfarmaian Lebanon Valley College, Robert Full Rutgers University, Carl Gans University of California, Berkeley, Edwin R. Griff University of Michigan, Kimberly Hammond University of Cincinnati, University of California Riverside David F. Hanes, Sonoma State University Michael Hedrick, Cal State University - Hayward James W. Hicks, University of California, Irvine Sara M. Hiebert, Swarthmore College William H. Karasov, University of Wisconsin McGrady UCLA Mark Konishi, California Institute of Technology

In the end, the common sense and kind words of our editors, Kay Ueno and Kate Ahr, greatly improved this book and ensured its publication. DAVIDRANDALL WARREN BURGGREN FRENCH KATHLEEN xvii

What you know about animal physiology is based on information (data) obtained from experiments. Because the ultimate goal of animal physiology is to understand how a process occurs in an organism, experiments must be designed to measure the animal (or its animal (allowing cells or tissues)) in a known state, such as B. at rest, during physical activity, digestion This type of experiment is particularly demanding and requires the use of a variety of techniques and methods. Many experimental techniques and measurement devices commonly used in animal physiology are "proven". These include pressure transducers for measuring pressure , catheter implants for drawing blood or injecting samples, respirometers for determining metabolic rate, etc. A description of each of these techniques is beyond the scope of this chapter, especially since these basic techniques are described in J.N. Cameron's Principles of Physiological Measurements are well described in texts such as . In this chapter we highlight some of the many molecular and cellular techniques that have recently been added to the physiologist's toolbox, describe them briefly, and illustrate their importance in physiological research. First, however, we consider the nature of the hypotheses and the general principles that apply when testing them. When you understand why and how you conduct experiments in animal physiology—whether using traditional or novel methods—you can better Understand the strengths and limitations of the information you learn in this book.

Formulating and testing hypotheses Scientists use experimental data to establish general laws of physiology—some of which are centuries old and others still emerging. In turn, these general laws serve as the basis for formulating new hypotheses, which are specific predictions that can be tested by further experiments. Examples of general "laws" supported by many existing laws

The data suggest that hydrobreathing animals regulate acid-base balance by altering the excretion of HCO- in exhaled water, whereas air-breathing animals regulate acid-base balance by altering the elimination of CO- gas in exhaled air. From this general rule, the following testable hypothesis can be drawn: the transition from HCO elimination to CO elimination occurs when water-absorbing tadpoles transform into air-breathing frogs. Although hypotheses are formulated in the form of statements rather than questions, the purpose of experiments is to test the validity of hypotheses and thus answer implicit questions. Physiological experiments should start with a well-structured, specific hypothesis focused on a particular level of analysis and applicable to testable experimental methods. While a hypothesis such as that killer whales have very high cardiac output while chasing seals may be interesting and indeed true, unless there is a viable experimental method to gather the data (evidence) needed for acceptance, making this hypothesis is only a matter of fact. an intellectual exercise) or refusal (refutation). However, finding ways to test new hypotheses has been an important impetus for the development of new experimental techniques and measurement tools. For example, currently available telemetry equipment for collecting blood flow data in small and medium-sized animals such as ducks, fish, and seals is being improved for use in larger animals.

The August Krogh Principle August Krogh is a Danish animal physiologist with broad interests in comparative physiology. Dozens of important research papers bearing his name formed the basis for further experiments in the field of respiration and gas exchange. In fact, Krogh's work eventually won him the Nobel Prize in Physiology in the late 18th and early 20th centuries. One of the reasons for Krogh's extraordinary success as a physiologist was his uncanny ability to choose just the right experimental animals to test his hypotheses on. In his view, for every definite physiological question, there is a best-fit animal that can most effectively provide the answer.

physical principles

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Experimental designs based on unusual characteristics of animals are known as the August-Krogh principle (Krebs, 1975). There are many illustrations of this principle throughout the book and throughout modern animal physiology. For example, in the 1970s, a group of animal physiologists interested in the evolution of air-breathing crustaceans studied the relatively small tide crabs, but were frustrated by the fact that the animals' small size prevented them from "giving up" biological secrets . Drawing on August Krogh's principle that there exists an ideal animal in which to conduct their research, these physiologists organized an expedition to the Palau Islands in the South Pacific. On these islands lives the 'coconut' or 'robber crab', a terrestrial hermit crab weighing up to 3kg. The animals' large size (for land crabs) made possible a number of experiments that provided important new data during the month-long expedition. As another example, animal physiologists interested in cardiac output in fish often have difficulty measuring pressure and flow, as well as blood pumping from the heart, due to the typical location of teleosts (i.e., bony fish). But seals, a mostly obscure (bottom-dwelling) marine fish that live in the deep ocean (though it's pretty ugly!), have an unusually large heart that's more accessible than other fish. By following the August-Krogh principle and using robins as the basis for their experiments, comparative cardiovascular physiologists now know more about heart function in fish than they have to continue to know about the relatively indomitable anatomy of trout, salmon, etc. Go fight catfish. Experimental Design and Physiological Levels

When designing an experiment, the physiologist must first decide at what level to analyze the physiological problem. The choice of this level determines the appropriate method (and selection of animals) for measuring the experimental variable of interest. Historically, techniques for studying physiological questions were first developed at the whole animal level; subsequently, with increasing speed over the past few decades, new techniques for experimentation at the cellular and now molecular level were developed. Add it in. Conceptually, however, we usually proceed in the reverse order: starting at the molecular level, then working our way up to the cell, tissue, organ, and finally the whole animal level, similar to what is shown in Figure 1-1. Therefore, in the following sections, we describe some representative experimental approaches to study physiological processes, starting at the molecular level. Much of the information presented in the other chapters of this book is based on experimental results obtained using these various techniques. Only by understanding how and why these methods work and their limitations will you be able to properly evaluate the information presented.

Note that no one analysis is inherently more valuable or important than the other. Indeed, the best understanding of animal physiology comes from integrating knowledge of the components involved from the molecular to the organ system level. Nonetheless, we recognize that a strong trend in animal physiology (like all biology) over the past decade has been "reductionism", that is, the study of cellular and molecular mechanisms in an attempt to explain more complex processes at a higher organizational level. Ultimately, some of the most valuable experiments are analytics-level experiments that provide insight into adjacent organizational-level processes. Although researchers and students are often interested in new and often expensive methods, meaningful results can be obtained with well-designed experiments and relatively simple instrumentation and techniques. In other words, well-thought-out experimental design can often make up for state-of-the-art equipment and techniques.

Molecular Technologies The past few decades have seen an explosion in the number and complexity of technologies available to study molecular events, with new methods and improvements emerging. The variety of molecular techniques available has had a major impact on biological research in general, and animal physiology certainly benefits from molecular approaches. In this section, we describe only some of the powerful molecular techniques used to answer questions in animal physiology. A more detailed discussion of these and related techniques can be found in textbooks such as Molecular Cell Biology by H.D. Lodish et al. Detecting molecules with radioisotopes

Physiological processes can often be better understood by understanding the movement of molecules within and between cells. For example, if we could track the movement of a particular neurohormone from its site of synthesis to its release to its site of action, we could better understand its role in regulating physiological processes. Many types of experiments that track the movement of molecules important to physiology use radioisotopes, relatively unstable, decaying radioactive isotopes of chemical elements. The natural decay of radioisotopes is accompanied by the release of high-energy particles, which can be detected with suitable instruments. With the exception of 12jI, which emits y particles, isotopes commonly used in biological research all emit p particles. Although radioisotopes occur naturally, those commonly used in experimental research are produced in nuclear reactors. The most commonly used isotopes in biological research are 32P, 12jI, 35S, 14C, 4SCa, and 3H. Radioactive isotopes of elements normally present in target molecules can be directly incorporated into molecules in vitro or in vivo

Molecules or precursor molecules that eventually transform into the desired molecule. The resulting radiolabeled molecule exhibits the same chemical and biochemical properties as the unlabeled molecule. A wide variety of so-called radiolabeled bioactive molecules (eg, amino acids, sugars, hormones, proteins) are now readily available (at high cost) from companies that specialize in their manufacture. Once a molecule is radioactively labeled, the particles emitted by the radioisotope can be used to detect the presence of the molecule, even at very low concentrations. In one type of follow-up experiment, a radiolabeled target molecule or its precursor is administered to an animal, an isolated organ, or cells grown in cell culture, and samples are taken periodically for particle emission measurements. Two types of instruments are used to detect emitted particles. A Geiger counter detects the ionization produced in a gas by emitted energy. A scintillation counter detects and counts the tiny flashes of light these particles produce as they pass through a special "scintillation fluid." The amount of radiation detected by both instruments is directly related to the amount of radiolabeled molecules present in the sample. In another type of experiment, the location of radiolabeled molecules in a tissue section is localized by autoradiography. In this technique, which literally means "photographing the radioactive isotope in the tissue," a thin section of tissue containing the radioactive isotope is placed on a photographic emulsion. Over the course of days or weeks, the particles emitted by the radioisotope expose the emulsion and produce dark grains that correspond to the location of the labeled molecules in the tissue (Figure 2-1). This qualitative record can be quantified by measuring the exposure of the emulsion in a densitometer and comparing it to the exposure elicited by known concentration standards; in this way, the actual concentration of radiolabeled molecules in the tissue or parts thereof can be determined. Autoradiography is particularly useful in neurobiology, endocrinology, immunology, and other fields of physiology that involve communication between cells. Tracking Molecules with Monoclonal Antibodies Examining biological structures in tissue sections mounted on microscope slides can be a daunting task. For example, even when the tissue is stained with dark purple nuclei and slightly lighter cell membranes, it is still difficult to see most details in the tissue. Through antibody staining, the structural details of cells can be better observed. This remarkable technique is able to locate molecules present in extremely low concentrations that are difficult to study with other techniques. Antibody staining typically involves the covalent attachment of fluorescent dyes to antibodies that recognize specific determinants on the antigen molecule. (While we often think of antigens as pathogenic microorganisms or invading foreign substances such as pollen, normal, biologically active molecules

caudal putamen

Figure 2-1 Autoradiography can reveal biochemical and structural details invisible to traditional tissue fixation and staining techniques. This autoradiograph shows the frontal lobe section of a rat brain after cannabinoid receptors have been bound by radiolabeled synthetic cannabinoids (very similar to the active ingredient in marijuana). The most radioactive areas (i.e., the areas with the most cannabinoid receptors) were most exposed on the film on which the brain slices were placed, mainly showing up as a dark area in the striatum (caudoputum) that regulates motor function. [Courtesy of Miles Herkenham, NIMH. ]

Identical antibodies produced in response to an antigen are called monoclonal antibodies; however, most natural antigens have multiple rather than a single determinant, so many different antibodies may be produced. A mixture of antibodies that recognize different determinants on the same antigen is called a polyclonal antibody. Once the antibodies are made and attached to fluorescent dyes that recognize unique sites on the target molecule, they can be injected into the target cells or tissues. Over the past decade, researchers have increasingly used a combination of monoclonal and polyclonal antibodies for antibody staining, especially in immunofluorescence microscopy (Figure 2-2). Alternatively, radiolabeled monoclonal antibodies can be used and the location of any antigen-antibody complexes formed in the sample detected by autoradiography. This method is used to localize the hormones epinephrine and norepinephrine to specific cells of the adrenal medulla, as described in Chapter 8 on the gland. Monoclonal antibodies can be used not only to detect specific molecules in cells, but also to purify them, as described below. This purified molecule is suitable for detailed studies of its structure and function. The key advance that made antibody staining possible was the development of a method to produce large quantities of monoclonal antibodies. It is impractical to isolate and purify a single type of monoclonal antibody from the antiserum of animals exposed to this antigen because each type of antibody is present in very small amounts. In addition, B

Figure 2-2 Commonly used monoclonal and polyclonal antibodies for antibody staining. In this immunofluorescent image of a rat spinal cord cultured for 10 days, a mouse monoclonal antibody (green) and a rabbit polyclonal antibody (red) specific for a single protein are combined with a blue fluorescent dye that binds directly to DNA. use together. Here we see neurons (red), astrocytes (green), and DNA (blue). [Contributed by Nancy L. Kedersha/lrnrnuno Gen]

Antibody-producing lymphocytes (or B cells) usually die within a few days and therefore cannot be cultured for long periods of time. In the mid-1970s, G. Kohler and C. Milstein discovered that normal B cells could fuse with cancerous lymphocytes called myeloma cells, which grew indefinitely in culture (i.e. formed an "immortal" cell line). The resulting hybrid cells, called hybridomas, are spread on solid growth medium in a petri dish. Each cell grows into a clone of the same cell, and each clone secretes a single monoclonal antibody. Clones are then screened to identify those secreting the desired antibody. These self-sustaining cell lines can be cultured and used to obtain large quantities of homogeneous monoclonal antibodies (Figures 2-3). While individual researchers can create and maintain their own hybridoma cell lines, many now choose to have specific monoclonal antibodies produced by companies that specialize in their production. (Next time you're in a university or college library, find Science and check the classifieds in the back.) The monoclonal antibody technology developed by Kohler and Milstein revolutionized molecular research so much that they won a Nobel Prize. Genetic Engineering

Genetic engineering includes various techniques used to manipulate the genetic material of an organism. The approach is increasingly used in agriculture and medicine, and holds promise for researchers in animal physiology. These techniques allow for the mass production of important biological molecules (such as hormones) that are usually present in very small amounts.

concentration, animals with mutations that affect certain physiological processes, and animals that synthesize above or below average levels of specific gene products. Genetic engineering begins with the identification of the structural genes that encode specific proteins in the DNA of the target organism. For example, the gene encoding human insulin can be identified in DNA isolated from human cells. A DNA fragment containing the insulin gene of interest can be "cut out" from the original, very long strand of human DNA and inserted into a cloning vector, which is a piece of DNA contained in an appropriate host cell and can be cloned independently of the host cell copy. host cell. DNA. Insertion of a foreign DNA segment (for example, the human insulin gene) into a cloning vector produces recombinant DNA, which is any DNA molecule that contains DNA from two or more different sources. Bacterial plasmids are a common cloning vector. These are extrachromosomal circular DNA molecules that replicate within bacterial cells. Under certain conditions, the recombinant plasmid containing the gene of interest is taken up by the common bacterium E. coli, a process called transformation (Figures 2-4). Usually only one plasmid molecule is taken up per bacterial cell. In transformed cells, the built-in plasmid can replicate, and when the cell divides, a set of identical cells, a clone, is formed. Each cell in the clone contains at least one plasmid containing the gene of interest. This general genetic engineering technique, called DNA or gene cloning, can be used to obtain a DNA "library" consisting of multiple bacterial clones, each specific clone containing a gene from a human or other species. Depending on the size and number of genes in the organism under test, different DNA clone variants are used. Clonal Populations for Medicine and Research Under appropriate environmental conditions, recombinant DNA in "engineered" E. coli clones is transcribed into messenger RNA, which directs the synthesis of the encoded protein. For example, commercial companies grow E. coli cells in vats that carry recombinant DNA containing genes for human insulin or other hormones; after harvesting the bacterial cells, large quantities of the human hormone can be isolated with relative ease. In the past, hormones needed to treat patients with endocrine disorders came from tissues from other mammals, such as cows and pigs. This is a time-consuming and expensive procedure due to the relatively low concentrations of the hormone. It turns out that using genetically engineered bacteria to make these hormones is much cheaper and the product is purer. In addition, hormones isolated from other mammalian species often elicit an immune response in humans, a complication that human hormones derived from genetically engineered bacteria do not. Recombinant DNA technology is also a powerful tool for basic research on human genetic diseases. By isolating and studying genes associated with inherited diseases, scientists can determine the molecular basis of these diseases. this will

Figure 2-3 Secreted hybridoma inhibitors

in Zellkultur

Polyethylene glycol fuse n\n

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splenocytes

ask

"Pure" (homogeneous) monoclonal antibodies To produce monoclonal antibodies, antibody-producing spleen cells are first fused with myeloma cells originally obtained from B lymphocytes. Hybrid cells or hybridomas secreting antibodies specific for the protein of interest are isolated. They can be administered in cell culture, where they secrete large quantities of specific antibodies, or injected into host mice, where they induce antibody production.

@@Myelomzellen

I 1

Transfer cells to selective media

select hybrid cells

Select cells producing the desired antibody

growing up in popular culture

Antibody

Surely it would lead to better control and even a cure. In recent years, many labs around the world have been working on a major project to "map" the location of all human genes on long strands of human chromosomal DNA and determine their nucleotide sequences. The Human Genome Project has provided invaluable data to researchers studying genetic diseases. DNA cloning and recombinant DNA technology also form the basis of gene therapy. This approach to treating patients with genetic disorders introduces patients to the normal form of the missing or defective gene. For example, people with cystic fibrosis have a defective CFTR gene, so they cannot produce the normal protein encoded by the gene. The consequence of this defect is a buildup of very sticky mucus in the airways of the lungs, which can lead to potentially fatal breathing problems. Molecular biologists have manipulated cold viruses with the normal CFTR gene. When some cystic fibrosis patients were infected with an engineered cold virus, the virus particles carried normal human genes into the patients' lung cells, where they took root. Subsequent synthesis

Antibody

A normal gene product helps relieve most cystic fibrosis symptoms in treated patients.

Physiological principles

................................................... ................................... Plasmid Vectors

DNA fragment

bacterial chromosome

Figure 2-4 DNA cloning is a method of isolating and preserving individual genes. In the cloning procedure presented, the specific DNA fragment to be cloned is inserted into a plasmid vector that also contains a gene that confers resistance to the antibiotic ampicillin. When the resulting recombinant plasma was mixed with E. coli cells, some cells formed a plasmid that could replicate intracellularly. When the cells were placed in culture medium containing ampicillin, only those cells that had taken up the vector grew. As each selected cell proliferates, it eventually forms a colony of cells (clones), all containing the same recombinant plasmid.

"Special" Mutants As described in Chapter 1, mutations are permanent changes to the nucleotide sequence of DNA. Mutations that occur spontaneously or can be induced experimentally are replicated when cells divide and are passed on to daughter cells. Mutated genes can tell us a lot about how biological processes work. The specific disruption of physiological processes caused by a single mutant gene can determine the function controlled by a specific gene, information that may not be revealed using traditional physiological techniques. For example, cardiovascular physiologists create and analyze the effects of mutations in zebrafish to understand heart development. In J-N. Chen and M. Fishman (1997) generated dozens of specific cardiovascular mutations in zebrafish. This process begins when adult zebrafish are exposed to powerful mutagens -- compounds that cause permanent mutations in the germ cell lineage. Subsequent mating of the F and F2 generations resulted in numerous mutations in the embryos. An embryo will rarely result in only one specific mutation in a structure or process of interest. For example, Fishman's group has identified

A mutation that causes the walls of the ventricles to be abnormally thin in one heart and narrow the outflow tract of the arteries in the other. Both diseases are similar to human disease states. Mutations usually cause abnormal effects only when homozygous (that is, when an individual acquires the mutated form of the gene from both parents). Even if a mutation causes a lethal condition inconsistent with long-term survival, it can be "preserved" in parents who are heterozygous for the mutation and carry both normal and mutated forms of the gene. Every time these parents mate, some of the offspring will be homozygous and exhibit abnormal effects. Thus, the heterozygous parents represent a "living gene pool" for these mutations. Transgenic animals Transgenic animals are another type of GMO that has the potential to make significant contributions to physiology. A transgenic animal is an animal whose genetic makeup has been experimentally modified by adding or replacing genes from other animals of the same or different species. Transgenic animals, especially mice, are at the forefront of various animal models that can help researchers understand fundamental physiological processes and disease states resulting from their dysfunction. Many techniques have been used to produce transgenic animals. In one approach, "foreign" DNA containing a gene of interest (called a transgene) is injected into the pronucleus of a fertilized egg (usually from a mouse), which is then implanted in a pseudopregnant female. The transgene integrates into the chromosomal DNA of the developing embryo at a relatively low frequency, resulting in all germ cells and somatic cells of the offspring carrying the transgene (Figures 2-5). Mice expressing the transgene are then mated to generate transgenic lines. This method is used to add functional genes, that is, extra copies of genes already present in the animal or genes that are not normally present, resulting in overexpression of the gene product. Subsequent analysis of the morphology and physiology of transgenic animals can provide important insight into physiological processes that cannot be easily studied in any other way. Transgenic animals characterized by underexpression or no expression of specific genes are equally instructive. MR Capecchi (1994) studied a method for replacing functional genes with defective ones, resulting in so-called knockout mice. These mice were unable to express the protein originally encoded by the replaced gene and thus lacked the functions mediated by the missing protein. By examining the effects of such ablation of gene function, the molecular and genetic basis of physiological processes can be determined. Knockout mice are commonly used to elucidate human physiological processes, as humans and mice are more than 98% genetically identical. To name a few, researchers are studying normal genes that regulate

Exploring Experimental Methods in Physiology

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fertilized egg from a female

Injected eggs implanted in a new woman's fallopian tubes

10-30% of offspring contain the transgene

B. Single cells, for measuring various properties of cells or injecting substances into them. Although cell physiologists use these devices in a variety of ways, the techniques used to make them are decades old. Essentially, the region in the center of the glass capillary is heated to its melting point. The ends of the tube are then pulled apart, reducing the soft spot in the glass to an invisible small diameter before it breaks and separates. The result is two micropipettes, each with a pull-down tip just a micron in diameter. When filled with a suitable solution, a micropipette can act as a microelectrode. Typically, the micropipette (or microelectrode) is mounted in a micromanipulator, a mechanical device that holds the pipette steady and allows its tip to move stepwise in three different planes. Measuring electrical properties Because neurons communicate by electrical signals, microelectrodes can be used to "eavesdrop" on their communications by measuring electrical signals across cell membranes and how those signals change under different conditions. Microelectrodes used to measure electrical potential (voltage) across cell membranes have little current flowing from the cell to the electrode. Thus, even if a neuronal component is present, there is little or no neuronal disturbance.

axQ Breed transgenic to preserve DNA in the germline

Figure 2-5. Transgenic animals are created by adding or replacing genes from another animal of the same or different species. A fraction of surviving offspring retains the transgene, which can be maintained in the germline by selective breeding.

Early cardiac development in embryos and cancer development in percussion studies in certain types of mice.

Cell Technology Understanding cells and cell behavior is the goal of many physiological experiments. Armed with knowledge of cellular behavior and communication, we can begin to understand how populations of cells function like tissues, and how tissues function like organs. Physiological analysis at the cellular level has been most intensively studied using several now standard techniques. In this section, we discuss three very common and efficient cellular techniques: microelectrode recording, microscopy, and cell culture. Use of microelectrodes and micropipettes

Many cell physiology experiments use micropipettes or various types of microelectrodes. These tiny glass "needles" can be inserted into tissue and even

Communication with the microelectrodes of neighboring cells is inductive. Signals from neurons or muscle cells are generated by filling a micropipette with an ion-conducting solution (usually potassium chloride) and connecting it to a suitable amplifier. A second electrode connected to the amplifier is placed in the liquid or organism next to the first electrode. When the tip of the first electrode is pushed across the cell membrane and into the cytoplasm, it completes an electrical circuit whose properties (voltage, current) can be measured. Our understanding of electrical activity within cells has improved dramatically since the introduction of microelectrode recording in the 1950s. One of the most revolutionary advances in microelectrode recording methods is patch clamping. Using this technique, the behavior of a single protein molecule that forms an ion channel (Latin for "in its normal position") can be recorded in situ, as shown in Figures 2-6. This approach is at the heart of the recent explosion of knowledge about membranes, including their channels and how they regulate the movement of substances across them (see Chapters 4–6). Measurement of Ion and Gas Concentration Specially designed microelectrodes can be used to measure the concentration of common inorganic ions in cells, including H+, Na+, K+, C1-, Ca2+ and Mg2+. This is because cells use the movement of ions across their membranes to communicate and work. The magnitude, direction, and timing of ion motion provide important information about certain processes. ~ Kroelectrodes to measure that

Physiological principles

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flow behavior

Both sides of the ion exchange barrier in the tip. Proton-selective microelectrodes are particularly suitable for measuring the pH of blood and other body fluids.

Measuring intracellular pressure and blood pressure Microelectrodes are now being used to measure hydrostatic pressure in single cells and in microscopic blood vessels -- any fluid-filled space into which a microelectrode tip can be inserted. To understand how this micro-pressure system works, let's look at small blood vessels. Insert the microelectrode filled with at least 0.5 M NaCl solution and mounted in the micromanipulator into the vessel to be examined. The higher pressure inside the vessel causes the interface between the plasma and the solution filling the electrodes to move into the electrodes. This leads to an increase in the resistance of the electrode tip, since the resistance of the plasma is higher than that of the NaCl solution. Changes in resistance are measured and proportional to changes in blood pressure. A motor-driven pump connected to the micropressure system generates pressure in the microelectrode, which exactly balances the pressure in the vessel. This back pressure keeps the interface in a fixed position; hence it is called a servo-null system. The desired equilibrium pressure developed in the micropressure system is then monitored using conventional pressure transducers for measuring blood pressure in the much larger blood vessel. The microprinting system has significantly increased our knowledge of the development of cardiovascular function in the developing embryo and larvae. These techniques also allow direct cardiovascular measurements in adult very small animals such as insects.

Figure 2-6 Patch clamp recordings can determine the movement of ions across small membrane regions containing transmembrane ion channels. (A) Patch clamp in-situ diagram. When a fire-polished microelectrode is placed on the cell surface, a very high resistance seal is formed between the electrode tip and the membrane. This seal allows direct measurement of membrane properties beneath the tip. Typically, only a few transmembrane ion channels lie beneath the T~pal, reducing the current through them for direct measurement (0) Micrographs showing the tip of a patch micropipette abutting the cell body of a neuron. The diameter of the tip is about 0.5 μm [Sakmann part B, 1992.1

Partial pressures of gases dissolved in liquids, such as O and CO, are now also available. The microelectrode tip used to measure the concentration of a specific ion (such as Na+) is sealed with an ion exchange resin that is only permeable to that ion. The rest of the electrode (the "barrel") is filled with a known concentration of the same ions. The potential measured by the microelectrode when no current is flowing reflects the ratio of the ion concentrations on the two

Microinjecting Materials into Cells In addition to being used as microelectrodes, micropipettes can also be used to inject substances into individual cells. These substances can be active molecules that cause measurable changes in cell or tissue function. For example, drugs that affect blood pressure and heart rate can be injected into very small blood vessels, such as those inside a bird's egg shell, or into the microscopic heart of a frog embryo. Alternatively, the injected substance could be a dye that marks the injected cells and helps reveal cellular processes or track cell division. A classic variant of this technique involves horseradish peroxidase, an enzyme derived from the horseradish plant that converts certain colorless substrates into colored products. When the enzyme is injected with a micropipette into a neuron's processes (especially axons), it is taken up and transported back to the cell body of the neuron; A colored "track" is formed between them. With this technique, peripheral nerves can be traced back to their origin in the central nervous system, a task that is challenging even for the most experienced neuroanatomist using more traditional techniques.

Cell structure analysis

Cellular function depends on cellular structure, reinforcing the central theme discussed in Chapter 1 that strong structure-function relationships govern animal physiology. Physiologists often use structural analysis at the cellular level to supplement physiological measurements to understand animal function. Such analyzes require a different type of microscope, as animal cells are typically around 10-30 μm in diameter, well below the smallest particles visible to the human eye.

Light microscopy, as the name suggests, uses photons of visible or near-visible light to illuminate specially prepared cells. Under optimal conditions, the resolution, or resolving power, of an optical microscope is a few microns; two objects that are closer than the microscope's resolution look like one object. As the resolution of microscopes has improved, so has our understanding of cellular structures and their components. Because cells derived from living animals die quickly, tissues must be prepared quickly to prevent degradation of cellular components. Fixation involves adding a special chemical, such as formalin, to kill and fix the cells

Their components, usually amino groups of proteins, are linked by covalent bonds. The fixed cells are then treated with dyes or other reagents that stain specific cellular characteristics, allowing the visualization of colorless and translucent cells. Fixing and staining large pieces of tissue is impractical and does not allow visualization of individual cells. Typically, small pieces of tissue are cut into ultrathin sections or slices, only 1 to 10 µm thick, using a special knife called a microtome. Since most tissue is fragile even after fixation, embed it in a medium (e.g., wax, plastic, gelatin) to support it during sectioning. This medium surrounds and penetrates tissue, then hardens to allow cutting. Tissue sections are then placed on glass slides for staining and subsequent microscopic observation (Figure 2-7A). In some cases, tissue embedding disrupts the structure of cells or their contents, making them impossible to stain or label with special compounds before viewing. Another approach is to freeze the tissue instead of embedding it, allowing the ice to support the tissue during subsequent sectioning. When ready, observe the tissue using a compound light microscope (the simplest light microscope) (Figure 2-7B).

Specimens embedded in paraffin or plastic resin and attached to microtome arms

metal or glass blade

'2W'

Sheet tape

Sectioning tape on glass slides, stained and mounted under coverslips

I-

Prism reflection

Objectives >Kondensorlinsen Lichtweg

3ase with light source

Figure 2-7 Preparation of specimens for light microscopy by thin sectioning and staining. (A) Cells and tissues taken from an organism are first fixed to preserve their structure and then sliced into thin sections with a metal or glass knife. The sections are mounted on glass slides, which can then be stained for later viewing

by compound light microscopy. (B) A compound light microscope sends light vertically upwards through the condenser, the sample on a glass slide, the objective lens, and finally the eyepiece in the eyepiece where the sample is viewed. [Adapted from Lodish et al., 1995.1

As available optics improved, so did shading techniques. Many organic dyes originally developed for use in the textile industry were discovered through trial and error and can selectively stain specific cellular components. Some of these dyes develop color based on charge, such as hematoxylin, which labels negatively charged molecules such as DNA, RNA, and acidic proteins. However; the basis for the specificity of many dyes is unclear. Replacing traditional dye staining with fluorescent labeling reagents increases the sensitivity of visualization. Fluorescent molecular labels absorb light of one wavelength and emit light of another, longer wavelength. When a sample treated with a fluorescent reagent is viewed through a fluorescence microscope, only cells or cellular components to which the label is bound are visible (Figure 2-8). Probably the most common and useful type of fluorescence microscopy is immunofluorescence microscopy, in which samples are treated with fluorescently labeled monoclonal and polyclonal antibodies. Figure 2-2 shows a good example of an image obtained using this technique. Due to the poor results of immunofluorescence microscopy of fixed slices, this technique is mainly applied to whole cells. However, images obtained by standard whole-cell fluorescence microscopy revealed that the emitted light came from labeled molecules located at many depths in the cell. For this reason, images are often blurred. Confocal scanning microscopy eliminates this problem and provides clear images from fluorescence microscopy.

Penny-marked samples that do not require thin sections. In this type of microscope, a sample is illuminated with stimulating light from a focused laser beam that rapidly scans different regions of the sample in a single plane. Light from this plane is combined by a computer into a composite image. Repeated scans of the sample in different planes provide data that a computer can then use to create successive sections of the fluorescence image. Figures 2-9 compare images obtained using conventional and confocal fluorescence microscopy. Visualization with other types of microscopes depends on the specimen altering one or more properties of light passing through the tissue on a slide, rather than fixation and staining. Because these methods do not

lens

incident light source

Man

barrier filter

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detection

Figure 2-8 Examination of a sample stained with a fluorescent label through a fluorescence microscope that produces images of only the structures to which the label is bound. The incident light source passes through an external filter that transmits blue light (450–490 nm). To ensure optimal illumination of the sample. The incident light hits the sample through a beamsplitter that reflects light below 510 nm downward but transmits light above 510 nm upward. The fluorescent signal from the labeled sample passes upward through a blocking filter that removes unwanted fluorescent signals that do not correspond to wavelengths emitted by the label used to stain the sample.

Figure 2-9 Conventional and confocal microscopes provide different images of biological material. These photomicrographs show lysed, mitotically fertilized sea urchin eggs. A fluorescein-labeled antibody was used to bind the antibody to tubulin, an important structural component of the mitotic spindle. (A) Conventional fluorescence microscopy showing blurred images due to fluorescein molecules above and below the focal plane. (B) Confocal microscopy, which detects fluorescence only in the focal plane, produces a sharper image of the same sea urchin egg. [From White et al., 1987.1

Exploring Experimental Methods in Physiology

................................................... ................................................ If staining is required , as long as it is thin enough, it can be used on living tissue to let enough light through. Brightfield microscopy (Figure 2-10A) reveals little detail compared to phase-contrast microscopy because different components of the sample refract light differently, and images have varying degrees of brightness and immediacy (Figure 2-10B). In Nomarski microscopy (also known as differential interference contrast microscopy), an illuminating beam of plane-polarized light is split into narrow, parallel beams before passing through a tissue sample, and the emerging beams are recombined into a single image. Small differences in the refractive index or thickness of adjacent parts of the sample translate to bright images if the light rays are in phase when recombined, or dark images if they are out of phase. The final image gives the sample an illusion of depth (Figure 2-1OC). In darkfield microscopy, light is shone onto the sample from the side, so the observer sees only scattered light from cellular components. Therefore, the image looks as if there are many light sources in the sample. In addition to direct viewing through a microscope, images can also be stored electronically after being captured by a digital camera or video camera. In a digital camera, a color image is captured in its entirety on a two-dimensional array of photosensitive elements. While digital cameras offer very high resolution, the light intensity required can be high. Alternatively, a camera that requires less light can be used to scan images according to preset scan modes. Due to the high light sensitivity of the camera, cells can be observed for longer periods of time without associated photodamage. This image enhancement is especially important for viewing live cells that contain fluorescent markers that are toxic to cells at high light intensities. A

bright field

Phase B

Compared

Figure 2-10 Different light microscopy techniques provide dramatically different images. (A) Brightfield image of a cell, typical of an unstained sample image, showing little contrast and detail when observed by compound light microscopy. (B) Phase contrast image height -

Electron Microscopy As with all imaging devices, the resolution limit is directly dependent on the wavelength of the illuminating light. That is, the shorter the illumination wavelength, the shorter the minimum distance between two distinguishable objects (i.e., the higher the resolution). In an electron microscope, a beam of high-speed electrons is used for illumination instead of visible light. Electron microscopes have better resolution because the electron beam has a much shorter wavelength than visible light. In fact, the resolution of modern transmission electron microscopes is typically 0.5 nm (5 angstroms, A), while the resolution of light microscopes is no lower than about 1000 nm (1 pm). Since the effective wavelength of an electron beam decreases with increasing speed, the resolution limit of an electron microscope depends on the voltage available to accelerate the illuminating electrons. A transmission electron microscope creates images by passing electrons through a sample and focusing the resulting image onto an electron-sensitive phosphor screen or photographic film (Figure 2-11). The electron beam is deflected by magnets that align and focus the electrons onto the sample, much like a condenser lens in a compound optical microscope. Imaging depends on differential scattering of electrons in different regions of the sample; stray electrons cannot be focused by the objective and therefore do not hit the screen. Since the electron beam penetrates the uncolored sample almost uniformly, it is almost impossible to distinguish its composition without coloring it. The most commonly used dyes in electron microscopy are heavy metal salts (such as osmium, lead, or uranium) that enhance electron scattering. Parts stained by this electron-dense material appear dark in electron micrographs. c nomarski

Enhances the visual contrast between different areas of the sample. (C) Nomarski microscopy (differential interference contrast) imparts a sense of depth to the image. [Contributed by Matthew J. Footer. ]

physical principles

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Condenser

display screen

Figure 2-11 Electron microscopes share common features of compound light microscopes, such as lenses, but use a beam of electrons instead of beams of light to illuminate the sample. In a transmission electron microscope, shown here, an image is created by passing electrons through an object and projecting them onto a fluorescent screen. In a scanning electron microscope, electrons reflected from the surface of a sample coated with a reflective metal film are collected by a lens and viewed on a cathode ray tube.

Since air deflects the focused electron beam aimed at the sample, the sample must be kept in a vacuum during imaging. Samples must be well fixed to preserve their biological structure when viewed under an electron microscope. Glutaraldehyde was used to covalently cross-link proteins and osmium tetroxide to stabilize lipid bilayers. After fixation, infiltrate the sample with plastic resin. Thin sections were then cut from the resin block, stained, and finally placed on metal grids for a transmission electron microscope. The sample must be cut into extremely thin slices (50–100 nm thick) to allow the electron beam to penetrate. Only a diamond or glass knife is sharp enough to cut tissue sections into such thin slices. Glass knives are made by breaking a square of glass 2.5 cm large and approximately 5 mm thick diagonally. Because glass is actually a slow-flowing liquid, the resulting edge is sharp enough to cut tissue for only a few hours before the flow of molecular glass dulls the edge. Although very expensive, diamond knives do not have this problem and are therefore the tool of choice for cutting thin sections.

The fine details of transmission electron microscopy can provide important insights into the structure of biological tissues (Figure 2-12A). Unfortunately, the size of the sample to be examined is very small because the slices have to be very thin. Therefore, it is difficult to understand three-dimensional structure without going through the truly tedious process of reconstructing an image from a series of discrete parts. The development of various techniques for preparing samples for transmission electron microscopy has expanded the range of objects that can be imaged and the information available in the images. Scanning electron microscopy, like transmission electron microscopy, uses electrons rather than photons to create images of samples. However, a scanning electron microscope collects electrons scattered from the surface of a specially prepared sample. The instrument provides excellent three-dimensional images of cell and tissue surfaces, but cannot detect subsurface features (Figure 2-12B). The samples are coated with a very thin film of a heavy metal, such as platinum, before being examined under a scanning electron microscope. Acid is then used to dissolve the tissue, leaving behind a metallic image of the tissue's surface that can be viewed under a microscope. The resolution of scanning electron microscopy is about 10 nm, which is significantly lower than that of transmission electron microscopy.

Cell Culture Growing cells in glass or plastic containers is called cell culture. This technology has revolutionized our ability to study cells and the physiological processes they support at the tissue and organ level. In the past, explants (small pieces of tissue removed from a donor animal) were kept alive and grown in flasks containing an appropriate mix of nutrients and other chemicals. Today, the most common procedure is to open (dissociate) small pieces of tissue, and then suspend the dissociated cells in a nutrient-rich chemical broth, where they grow and divide as independent entities. Successful growth of cells in vitro requires a suitable medium, the liquid in which the cells are suspended. Until the early 1970s, cells from all animals were routinely grown in liquid media consisting mainly of horse or fetal bovine serum (a clear component of blood plasma) or unrefined blood from crushed chicken embryos. Chemical extract composition. However, the chemical composition of these media is poorly defined and contains many unidentified compounds. Furthermore, it is difficult to predict whether cells from a given source will grow in any of these media, or which components might be added if the first attempt is unsuccessful. Growing cells in vitro is largely a matter of trial and error (and luck). The defined media available for research today are produced according to precise chemical formulas. However, successful culture of many cell types requires the addition of trace amounts (less than 5%) of horse serum to such defined media. this observation

However, many types of animal cells have not been successfully cultured. However, due to advances in media and culture techniques, the list of cells that can be cultured is constantly increasing. For example, cell lines can be grown in culture in the following tissues and organs: bone and connective tissue, skeletal muscle, cardiac muscle and smooth muscle, epithelial tissue from liver, lung, breast, skin, bladder and kidney, some nervous tissues, some endocrine Tissue glands (eg B. adrenal, pituitary, islets in pancreas) M

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Unlike normal animal cells, cancer cells often grow rapidly and uncontrollably in the body and can grow indefinitely in culture. Treating some normally cultured cells with certain drugs leads to transformation, a process that causes them to behave like cancer cells that have been isolated from tumors. Such transformed cells can also be cultured indefinitely. A homogeneous population of such "immortal" cells is called a cell line. Although normal cells differ in many ways from cancer cells and transformed cells, cell culture of the latter enables certain types of research that are not feasible with primary cell cultures of normal cells. Cell culture offers many possible applications in animal physiology. New devices such as silicon wafer sensors for measuring acidity and other variables have been combined with cell culture technology to provide important insights into the physiology of cells and organisms. For example, hormonal regulation of H+ secretion by a variety of in vitro cultured cells can be studied by stimulating the cultured cells with agonists and antagonists and measuring changes in the acidification rate of the medium. This method has also been used to study tissues and organs with abnormal rates or perturbations of SH+ secretion, such as swim bladder tissue. 1

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Figure 2-12 Transmission electron microscopy provides a view of the interior of a biological tissue, while scanning electron microscopy highlights surface features. (A) Transmission electron micrograph of mouse oviduct cilia. (B) Scanning electron micrograph of cilia in mouse oviduct. [Courtesy of E.R. Dirksen. ]

complete medium

It shows that certain growth factors in the blood are necessary for the growth and division of animal cells in vitro (Figure 2-13). Even when defined media are available, in vitro culture of animal cells is a challenging technique. Normal animal cells can usually only grow outside the body for a few days, then stop proliferating and eventually die. A relatively homogeneous population of such cells is called a cell lineage. Cultured cell lines can be used in many types of experiments, but their limited lifespan makes them unsuitable for other research. in adi

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Figure 2-13 Cells grown in culture often require specific factors to stimulate maximum rates of division and growth. In the indicated cultures, maximal cell numbers were only achieved in the presence of epidermal growth factor (EGF). Addition of EGF (arrow) to cultures lacking this substance resulted in immediate further growth of cell colonies. [Adapted from Lod~she et al., 1995.1

Physiological principles

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Biochemical Analysis Most biochemical processes take place in aqueous solutions and require gas exchange. For this reason, physiologists often need to measure the chemical composition of fluids and/or the concentrations of their constituents in different body compartments. For example, to assess whether a crab can regulate its internal salt concentration while swimming in diluted estuarine water, a physiologist needs to know the salt concentration in the water surrounding the crab, the crab's hemolymph (blood) and the urine it produces. Crab. With these data, the crab's ability to maintain homeostasis can be assessed. Biochemical analysis of biologically relevant fluids, gases, and structures is often based on the physical or chemical properties of the material of interest (e.g., Na+ in crab urine). The sensitivity and accuracy of such measurements have recently improved dramatically, allowing physiologists to ask questions about even subtle physiological functions that could not be measured before. Both qualitative and quantitative analysis are important for physiological research. The first goal is to determine the composition of the fluid or structure, ie. H. The elements, ions and compounds that make it up. The goal of the second analytical method is to measure the concentration of a specific substance in a fluid or structure of interest. Many analytical instruments and techniques provide both composition and concentration data. Measuring Composition: What's the Way? There are many methods, old and new, that can be used to measure chemical composition. Sometimes animal physiologists are only interested in knowing whether a specific substance, such as ammonia or hemoglobin, is present in a sample. At other times, you may want to identify all the different proteins, carbohydrates, or other molecular species in your sample. In other words, the nature of the question being investigated determines which component data are relevant. Few biological samples receive a complete compositional analysis like the novice chemistry lab course. Various colorimetric tests have been developed to determine the presence of certain substances in solution. These tests are based on the chemical reaction of the substance of interest, changing its ability to absorb visible or ultraviolet (W) light.

radiation of different wavelengths. This changes the transmission of light or UV radiation through the solution, which can be recorded with a spectrophotometer. Many biochemical tests use an enzyme to catalyze a reaction with a substance of interest. For example, a common test for lactate (a product of anaerobic glucose metabolism) uses an enzyme to convert lactate into products with different UV absorbing properties. To perform this test, a solution suspected of containing lactate is placed in a small reaction vessel along with the enzyme and other reaction components. After a while, place the vial in the spectrophotometer and measure the UV transmittance of the solution. The transfer of control reaction vials lacking enzyme was also measured. The presence of lactate in the sample is indicated by the difference in UV transmittance between the control tube and the test tube. Chromatography is a widely used technique for separating proteins, nucleic acids, sugars and other molecules present in a mixture. In its simplest form, paper chromatography, the components of a sample move through the paper at different rates according to their relative solubility in the solvent, as shown in Figure 2-14A. To visualize the separated components, chromatograms are often sprayed with a colorimetric reagent that produces visible colors with the components of interest. More complex mixtures can be separated by column chromatography, in which a sample solution is passed through a column packed with a porous bead matrix (Figure 2-14B). Different components of the sample pass through the column at different rates, and the resulting fractions are collected in a series of tubes. Depending on the type of sample, different tests are used to determine the presence of each component in the collected fractions. Many different types of matrices are used in column chromatography, depending on the composition of the solutions to be separated. For example, matrices can be used that classify components according to charge, size, water insolubility (hydrophobicity), or binding affinity for the matrix. The last matrix is used in affinity chromatography, where matrix beads are coated with molecules (for example, antibodies or receptors) that bind to components of interest. When the sample mixture is applied to the column, all components will pass through except those recognized by the affinity matrix. This is a very powerful technique that can be used to purify proteins and other biomolecules at very low concentrations. Electrophoresis is a general technique for separating molecules in a mixture based on their speed of motion in an applied electric field. The net charge of a molecule along with its size and shape determine its rate of migration during electrophoresis. The technique works well for separating small molecules such as amino acids and nucleotides, but by far the most common application of electrophoresis is separating proteins or nucleic acid mixtures. In this case, the sample is placed on one end of an agarose or polyacrylamide gel, an inert matrix with a fixed pore size

Molecules are prevented or allowed to migrate when an electric field is applied. Protein mixtures are often exposed to SDS, a negatively charged detergent, before and during electrophoresis. The resulting SDS-coated protein migrates through the gel at a rate proportional to its molecular weight; the lower the molecular weight of the protein, the faster it will pass through the gel (~i~u2-15). When protein-binding dyes are applied to the gel, the separated proteins are visualized as distinct bands. A

Three slightly different but essentially similar methods of using gel electrophoresis are used to separate and detect specific DNA fragments, messenger RNA (mRNA), or proteins. Each of these methods involves three steps (Figure 2-16): I. Separation of the sample mixture by gel electrophoresis 2. Transfer of the separated bands to nitrocellulose or other types of polymer sheets, this The process is called blotting. 3. Leaves (or blots) are treated with "probes" that specifically react with the component of interest. The first of these developed methods, named Southern Blot after its inventor Edward Southern, was used to identify DNA fragments containing specific nucleotide sequences. Northern blots are used to detect specific mRNAs in the mRNA mixture. specific protein

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Fractions are collected; larger components pass faster Figure 2-14 Chromatography is a powerful technique for separating the components of a mixture in solution. (A) In paper chromatography, the sample is applied to one end of a piece of chromatography paper and dried. The paper is then placed in a solution containing two or more solvents that flow up through the paper by capillary action. Different components of the sample move at different speeds through the paper because they have different relative solubilities in the solvent mixture. After several hours, the paper was dried and stained to determine the location and relative amounts of isolated components. (B) In column chromatography, the sample is loaded onto the top of a column containing a permeable matrix of beads through which the solvent flows. The solvent is then slowly pumped through the column and collected in separate tubes (called fractions) as the water comes out of the bottom. The components of the sample migrate through the column in different fractions and are therefore classified into different fractions.

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Proteins Separated by Size

Figure 2-1 5 Gel electrophoresis separates the components of a mixture based on charge and mass. Proteins are usually separated by SDS-polyacrylamide gel electrophoresis as shown. (A) SDS, a negatively charged detergent, is added to the sample to coat proteins. (B) The samples are then placed into the wells of the polyacrylamide gel and an electric field is applied. Small proteins move along the gel more easily and faster than larger proteins. (C) Over time, the proteins in the mixture separate into bands composed of proteins of different sizes. These can be visualized using various protein staining reagents. [Adapted from Lodish et al., 1995.1

Physiological principles

Band corresponding to component A

electrophoretic transfer

electric current

Reagent specifications

Corresponding component B

Gel after electrophoresis

polymer thin film

Sonogram

Figure 2-16 Southern, Northern, and Western blots are similar methods for isolating and identifying specific DNA fragments, mRNA, or proteins in a mixture. For each method, the components of a sample are first separated by gel electrophoresis; individual bands are transferred to a polymer sheet, which is then flooded with a radiolabeled reagent specific for the component of interest. The presence and in some cases the amount of labeled components was determined by autoradiography. See Table 2-1 for details on each method.

is the deflection of ions from the standard path to the analyzer. The degree of deflection is detected by a series of detectors, which can then determine the presence and amount of gas in the gas sample introduced into the mass spectrometer. The various chemical composition measurement techniques described in this section are commonly used by physiologists, but many other techniques are also used in physiological research. To learn about other methods and more details on the methods discussed here, you can refer to chemistry and biochemistry textbooks.

Proteins in complex mixtures can be detected by western blotting (also known as immunoblotting). (So far, there are no Eastern, South-Western, etc. blotting, but this may only be a matter of time.) Table 2-1 summarizes what makes the three blotting methods unique. Many common methods for determining composition work for solutions but not for gases. However, mass spectrometers can distinguish the different gases that make up a gas mixture based on their mass and charge. This tool is most commonly used by animal physiologists to determine the composition of gases breathed by animals while they are resting or exercising in an experimental setting. Figure 2-17 shows the basic structure of a mass spectrometer. The gas sample is first ionized by intense heating and passed through an electron beam. The charged ions are then focused by an electric field and accelerated into the analyzer, where the ion beam is deflected by an applied magnetic field or by a tuning rod that emits a specific radio frequency, deflecting the ions. The lighter the mass of the ion, the smaller the charge and the smaller the will

Measuring Concentration: How Much?

Most instrumentation or analytical techniques used to determine the composition of liquid or gas mixtures also provide data on the concentrations of the components present. For example, the degree of color change in a colorimetric test depends on how much of the analyte is present in the sample. Likewise, the output signal of a mass spectrometer not only depends on

Table 2-1 Electrophoretic blotting methods - -

molecular discovery

Separation detection method*

DNA fragments produced by cleavage of DNA with recovery enzyme

Electrophoretic mixing of dsDNA fragments on agarose or polypropylene gels, denaturation of separated fragments in ssDNA and transfer of bands to polymer sheets, labeling of fragments of interest using radiolabeled ssDNA or RNA, detection of labeled by autoradiography Bands

northern blot

messenger RNA

Denature the sample mixture; electrophoresis on a polyacrylamide gel and transfer the separated bands to a polymer sheet; label the mRNA of interest with radiolabeled ssDNA. Identification of marker bands using autoradiography

western blot

protein

Electrophoresis of the sample mixture on an SDS-polyacrylamide gel and transfer of the separated bands to a polymeric film; radioactively labeled monoclonal antibodies are used to label the protein of interest. Identification of marker bands using autoradiography

southern blot

*double-stranded DNA

Double-stranded DNA, ssDNA = single-stranded DNA.

+ If no radiolabeled mAbs are available, bands contaminating antibody-protein complexes can be detected by adding a secondary antibody conjugated to each mAb. This secondary antibody is covalently linked to an enzyme, such as alkaline phosphatase, that catalyzes a colorimetric reaction. When the substrate is added, a colored product forms on the band containing the protein of interest, resulting in a visible colored spot in that area of the blot.

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Figure 2-17 The identity of gases in a mixture and their concentrations can be determined by mass spectrometry. (A) The fixed collector mass spectrometer detects the degree to which an ionized sample is deflected by an applied magnetic field and consists of four basic components. First, there is a well-designed inlet device (I) through which the sample is introduced into the system in a constant viscous flow. Second, there is an ionization chamber (Z) kept under vacuum and high temperature (approximately 190°C), where the sample is passed through an electron beam and accelerated by applying an electric field. Gas molecules leave the chamber as negatively charged ions. Third, it is an analytical tube in which the accelerated ion beam is exposed to a magnetic field that causes the ions to move

Flow in a curved path (3). Finally, the ion beam is collected by an ion collector located at the end of the analysis tube (4). The degree to which ions bend in an applied magnetic field depends on the strength of the field as well as the ion's mass, charge and size. and speed. Only those ion species that are bent parallel to the sides of the analysis tube reach the ion collector and are detected. (B) Under a constant magnetic field, the specific properties of the particles detected in the mass spectrometer depend on the strength of the ionization voltage, which can vary. The lower the ionization voltage, the heavier the detected particles. [Adapted from Fessenden and Fessenden, 1982.1

The types of gases present in the mixture, but the amount of each gas present. Therefore, the output signal produced by an analyzer (whether it is a transmission spectrophotometer, densitometer or mass spectrometer) is directly related to the concentration of the species producing the signal. Typically, analytical techniques for determining the concentration of a particular substance are performed on multiple samples of different known concentrations of the substance. The output signal is then plotted against concentration to give a standard curve. By comparison with this standard curve, the actual concentration of the test sample is determined from the output signal it produces.

This is difficult, if not impossible, to do by examining intact organs in situ; instead, experiments are performed on isolated organs surgically removed from animals and preserved in artificial environments. Two examples are intended to illustrate the power of this experimental approach. When the heart of nearly all vertebrates, including mammals, is isolated and placed in a saltwater bath, it continues to beat and perform work by pumping saltwater or other fluids supplied to it. An isolated vertebrate heart will continue to beat without neural input if kept at the proper temperature and perfused with an oxygenated solution that has the correct ionic composition and contains an energy source such as glucose. When the heart is isolated, physiologists can measure the effects of chemical stimulation of drugs and hormones or electrical stimulation of cardiac nerves on heart rate, amplitude, flow rate, and mechanical movement. Experiments on isolated hearts are critical to improving our understanding of the cardiovascular system. A second example is the vertebrate pineal gland, a small organ that sits on top of the vertebrate brain. The pineal gland, which plays a key role in regulating daily (circadian) rhythms in physiological processes, is sensitive to light.

Experiments with Isolated Organs and Organ Systems All animals have several different vital organ systems that must be coordinated and controlled to maintain homeostasis. As we will explore in later chapters, the function of these organ systems is largely regulated by neuronal and/or hormonal inputs. To understand mechanisms of physiological control, it is essential to characterize the primary control inputs and their sources. In many cases it is

Physiological principles

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It responds to relevant stimuli and releases varying amounts of regulatory chemicals into the bloodstream depending on the time of day. When the pineal gland is isolated and placed in an appropriate culture system, it continues to exhibit circadian rhythms. Direct experiments with this in vitro compound provide answers to specific questions about the regulation of physiological systems by the pineal gland.

Observing and Measuring Animal Behavior Scientists who study animals often supplement their experiments by observing animal behavior. However, meaningful behavioral experiments are difficult to perform because animals must be in an appropriate physiological state (e.g., to reproduce, raise pups, digest food, etc.). In addition, experiments must take advantage of the animals' natural behavioral tendencies. Despite these difficulties, experimental approaches to control and stimulate specific behavioral states can provide important insights into physiological processes that are not always amenable to direct physiological studies. A prerequisite for such experiments is a thorough understanding of the natural behavior of animals in their habitat. The Power of Behavioral Experiments

A study of the retrieval behavior of ground-nesting birds in the 1950s and 1960s shows how behavioral studies can contribute to physiological knowledge. K. Z. Lorenz and N. Tinbergen found that when geese were lying outside the nest, they could not only identify and retrieve their eggs, but also various objects (e.g. grapefruit, light bulbs, baseballs) near their nest. Tinbergen and his students then performed an ingenious experiment with seagulls, presenting them with two objects and recording which object was retrieved first. By using the Painvise comparison process, they were able to define the attributes that Seagull uses to select what to fetch. Although the birds retrieved many different objects, these experiments showed that real eggs were always preferred over unnatural objects. The relative size, color, and spotting of eggs have been found to independently affect the likelihood of eggs being retrieved. Taken together, these experiments demonstrate that eggs are a strong natural stimulus for gulls, triggering specific retrieval behaviors. Knowing the exact nature of the stimuli that lead to this behavior, physiologists are better able to perform physiological experiments on the nature of bird vision. Behavioral experiments typically analyze the total amount of time the animal under study spends performing each behavior as well as the timing of the behavior. These data, combined with information about other animals' behavior and important environmental variables, can often reveal how closely that behavior is related to the animal's internal state. The vast majority of animal behavior information collected in this way is related to reproduction

Feeding, two of the most important behaviors in animals; reproductive and feeding behaviors are both strongly influenced by the physiological state of the animal. Careful observation can often shed light on which patterns of behavior in one person affect another, and can shed light on why this is the case. In sticklebacks, for example, the male displays a red belly to signal to other males that he is defending the nest, and to females that he is interested in laying eggs. So the meaning of this signal depends on the gender of the recipient. Bullflies are formed by a physiological process triggered by the beginning of the breeding season. The coordination between behavior and physiology in this species has been studied using behavioral analysis as a guide for physiological studies. Behavioral Research Methods

Various instruments are used to record and analyze the physiological basis of certain behavioral movements. In some experiments, high-speed cameras are used in conjunction with electrophysiological detectors of neuronal or muscle activity to simultaneously capture behavior and its physiological underpinnings. Because the behavioral behaviors of interest are often rapid and transient, these events are often recorded at high speed and the tapes played back at slow speed to facilitate analysis. The interaction of bone components in certain behaviors (e.g. eating, walking on a treadmill) can be studied using an X-ray camera. As with many other aspects of physiology, the availability of cheap, fast computers with increasing data storage capacity has revolutionized the collection and analysis of data. Figure 2-18 shows how many techniques commonly used to study animal behavior and underlying physiological processes can be applied to a single behavior—the feeding attack of a venomous snake. To find out how such a punch occurs, the movement of the body and jaw must be related to the force exerted by the contraction of the jaw muscles. The rapid attack was recorded in two views, back and side, with the camera looking directly at the animal and through a mirror mounted 45 feet above the snake. A raster image in the background is integrated into the video image, allowing quantification of the animal's position. The snake was placed on a platform that recorded the forces applied along three orthogonal axes. By measuring these external parameters, the researchers could record the forces associated with the snake's movement on the surface. The force exerted by the snake's jaws was recorded by strain gauges attached to the head, and muscle activity was measured by electrodes in the four outer jaw muscles. All data is recorded on magnetic tape and in a computer using data acquisition hardware and software. Values of measured variables are usually displayed as a function of time and correlated with behavioral analysis recorded on videotape. Data from one such experiment shows how muscle contraction leads to positioning of the fangs and closing of the jaw around prey.

Exploring Experimental Methods in Physiology

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bipolar electrode

Strain gauges

Power Platform Oscilloscope

high speed camera

tape recorder

Figure 2-18 The feeding thrust of a venomous snake can be analyzed to determine the muscles used and the pattern of contraction. (A) To record the electrical potential of the mandibular muscles, thin bipolar wire electrodes were surgically inserted into the four lateral mandibular muscles during surgery under anesthesia. There is also a strain gauge attached to the top of the snake's head to measure the movement of the snake below.

skull. (B) Snakes are placed on a force recording table and filmed when encountering prey. The electrodes and strain gauge leads are connected to an electronic amplifier that amplifies its low-voltage signal. The amplified signal is displayed on oscilloscopes and chart recorders and stored on tape recorders and computers.

These experimental measurements can be used to test hypotheses about which structures and muscles are involved in strokes and how their temporal relationships change during behavior. The experiment also showed how many physiological systems contribute to the emergence of complex behaviors. When other variables are measured in repeated experiments performed under the same conditions, a more comprehensive functional analysis of this predation behavior and a better understanding of animal performance can be achieved. With this experimental setup, it was possible to measure differences in beating behavior according to the size and type of prey. Such measurements can also provide the basis for formulating hypotheses about the neural control of muscle activity, visual feedback to control behavior, and many other interesting topics.

Importance of Physiological State in Research

You will be instructed to act like taking a deep breath, running, or tensing your muscles during physical therapy. Some animals can be trained to perform feats (such as walking on a treadmill) required for a particular experiment.

Performing, being trained, or just being allowed to perform spontaneously?

Research at all physiological levels, from molecular to behavioral, must take into account the physiological state of the animal at the time of experimentation (or tissue sampling). Some physiological states are fairly obvious to researchers, such as when an animal is diving (holding its breath), actively moving, or hibernating. Other physiological states may be much more subtle but have equally large effects on physiological processes. Of course, the overt or subtle nature of the physiological condition depends on the animal. For example, a mouse curled up with eyes closed, breathing relatively evenly, and showing no motor activity is likely to be asleep. But what about relatively slow, motionless fish? Is it asleep or motionless? Physiological state can be strongly influenced by environmental variables such as season and time of day. For example, vagus nerve stimulation in temperate frogs studied on spring nights resulted in a much slower heart rate than in frogs studied on autumn afternoons. Therefore, the results of an experiment are strongly influenced by the time of day and the season when the experiment is conducted. To characterize the physiological state of an animal, one or more variables can be measured and their values compared when the animal is in different behavioral states. For example, blood pressure, pulse rate, and skeletal muscle activity can all be measured simultaneously while observing the animal in different states, such as when B. is sleeping, active, digesting food, or hibernating. Such

physical principles

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Measurement often does not allow for the identification of causal relationships between measured variables. However, conclusions can be drawn from these data and testable hypotheses can be formulated regarding the relationship between the measured variables. Because multiple physiological states can exist simultaneously (e.g., sleeping in winter, breath-holding during hibernation), experiments to determine physiological states are often complex and time-consuming. However, when such experiments are carefully designed to reveal the effects of physiological states on fundamental physiological processes in animals, they can greatly improve our knowledge of physiological systems. For example, a typical experiment might measure important physiological variables during intermittent hibernation in ground squirrels. Comparison of recorded body temperature and metabolic rate with observed behavioral activity over time showed that increased waking activity was associated with increased body temperature and metabolic rate. These correlations suggest that as animals become active, important physiological systems become active around the same time. Although it is intuitively clear that animals need more blood flow during physical activity, it is unclear from these data how this increase in blood flow is achieved or regulated. Do the physiological changes precede or result from the behavioral activity? Distinguishing these explanations from other possible explanations requires conducting experiments that focus on causal relationships between specific behaviors and the physiological systems that underlie changes in physiological state. As discussed here, observations of physiological-behavioral relationships often form the basis of such follow-up experiments. Perhaps just as importantly, they can provide a means of characterizing specific physiological states. For example, in a study investigating the causal relationship between blood flow and heart rate during hibernation, variables such as body temperature or metabolic rate can be used to ensure that animals are indeed hibernating during experimental testing.

Summary Physiological research should start with a well-structured, specific hypothesis that is relevant to a specific level of analysis and that can be tested experimentally. Hypothesis testing is made easier by applying the August-Krogh principle that a key issue in the design of physiological experiments is to analyze the level of each physiological question under study. The choice of level determines the appropriate method and test animal for measuring the physiological variable of interest. Technologies that detect or analyze events at the molecular level have brought enormous benefits to animal physiology. Radioactive isotopes can be incorporated into physiological processes.

important molecules or their precursors. After an animal is injected with a radiolabeled molecule, its motion can be determined by subsequently taking a tissue sample and measuring the particles emitted by the radioisotope using a Geiger or scintillation counter. The presence and location of radiolabeled molecules in thin tissue sections can be demonstrated by autoradiography. Monoclonal antibodies covalently labeled with fluorescent dyes or radioisotopes are another powerful tool for tracking the movement of specific proteins within physiological systems. Due to their high specificity, monoclonal antibodies allow the detection of a single protein (such as nerve growth factor or neurotransmitter), even if it is present in very low concentrations in the cells or tissues being examined. Genetic engineering, including recombinant DNA technology and gene cloning, is also revolutionizing animal physiology. Genes cloned into easy-to-grow bacterial cells can be used to produce large quantities of gene products such as human insulin and other hormones. Genetic engineering techniques also allow the production of transgenic animals (usually mice) that contain extra copies of the gene of interest. In knockout mice, the normal gene is replaced with a mutant form of the same gene, preventing the animal from producing a functional protein. Analyzing the effects of adding or deleting specific genes can provide insight into the mechanisms and regulation of physiological processes. Microelectrodes and micropipettes have multiple uses in cell physiology. The most common use of microelectrodes is to record electrical signals from neurons or muscle cells. With specially designed microelectrodes, the concentration of ions and certain gases as well as the pressure of fluids in cells or blood vessels can be determined. Micropipettes are used to inject materials (eg, dyes, radiolabeled compounds) into individual cells or fluid-filled tissue spaces. The structural analysis of cells and the physiological processes that arise from these cells rely heavily on microscopy. Light microscopy uses photons of visible or near-visible light to illuminate specially prepared tissue samples. The sample is first fixed (preserved), embedded in plastic or wax, and then cut into very thin sections (sections) using a microtome. Finally, sections are treated with organic dyes or fluorescently labeled antibodies that bind and stain different cellular components. Once the tissue is prepared, it is usually viewed using one of several types of light microscopes. The advent of electron microscopy, which uses electron imaging, has greatly improved the resolution of microscopic analysis and enabled the visualization of intracellular structural details that cannot be seen with light microscopy. In transmission electron microscopy, a beam of electrons is directed through ultrathin tissue sections stained with electron-dense heavy metals. In a scanning electron microscope, electrons bounce off the surface of a sample, creating a three-dimensional image of surface features of cells and other structures.

Exploring Experimental Methods in Physiology 35 ……………………………………………………………………………………… …

Cell culture, the growth of cells outside the body, allows the propagation of relatively short-lived cell lines as well as "immortal" cell lines that can grow indefinitely. Cultured cells are usually fairly homogeneous, making them useful in experiments aimed at studying the function, secretion, responses, and other properties of specific cell types. Such experiments rely on biochemical analysis to determine the composition of the sample mixture obtained from the cells, and the concentrations of the components present. The most commonly used techniques in biochemical analysis include colorimetry, transmission spectrophotometry, paper and column chromatography, electrophoresis, and mass spectrometry. With increasing levels of tissue architecture, in vitro preservation of isolated organs or whole organ systems allows the study of intact tissue function in artificially controlled environments. Key variables such as temperature, oxygen availability, and nutrient levels can be manipulated to model homeostasis or altered to test specific hypotheses. .Animal physiologists often supplement their experiments with observations of animal behavior. Experimental approaches to control and stimulate specific behaviors can provide important insights into physiological processes that are not always amenable to direct physiological studies. Furthermore, analysis of the total time spent performing each behavior and the timing of the behavior, coupled with information about other animal behaviors and important environmental variables, can reveal the close relationship between the behavior and the animal's internal physiological state. Finally, in all experimental approaches, from the simplest (molecular) level to the most complex (behavioural) level, the physiological state of the animal at the time of the experiment (or tissue sampling) is an important consideration. Physiological states may depend on internal regulators (sleep, hibernation, activity, etc.) or environmental influences. To characterize the physiological state of an animal, one or more variables can be measured and the values of these key variables correlated to various behavioral states.

Review Questions 1. What is the difference between a scientific problem, hypothesis, theory, and law? 2. Researchers conduct experiments on crickets, bullfrogs, and rattlesnakes, but test a single hypothesis related to a single physiological process. Explain how this investigator applied August Krogh's principles.

3. 4. 5.

What are radioisotopes and monoclonal antibodies? What common properties make them useful to physiologists? What is a clone and how is it made? If an interesting and useful mutation in a physiological system turns out to be lethal before the animal reaches the reproductive phase of its life cycle, how can it be maintained in the laboratory to allow repeated experiments to study it over the long term? Why do air bubbles in microelectrodes used to record neural action potentials interfere with recordings? What is the main difference between light microscope and electron microscope? What are the advantages and disadvantages of both? Describe the difference between experiments performed in vivo, in vitro, and in situ. What are the advantages and disadvantages of each experimental method? How can you tell if an animal's resting heart rate is influenced by circadian rhythms?

Recommended Reading Burggren, W. W. 1987. Invasive and noninvasive methods in physiological ecology: a plea for integration. In New Directions in Physiological Ecology, eds. ME Feder, AF Bennett, WW Burggren, and R Huey. New York: Cambridge University Press, pp. 251–272. (Description of the two principal methods of animal experimentation.) Burggren, W. W. and R. Fritsche. 1995. Cardiovascular measurements in animals in the milligram body weight range. Brazil. ]. Med. Biol. Res. 28:1291-1305. (Describing methods for extending cardiovascular technology to microanimals.) Cameron, J. N. 1986. Principles of Physiological Measurements. New York: Academic Press. (A brief but detailed introduction to several important physiological measures.) Hall, Z. 1992. Introduction to Molecular Neurobiology. Sunderland, MA: Sinauer Associates. (A comprehensive discussion of how molecular approaches can provide comprehensive insights into vital organ systems.) Lodish, H., et al. 1995. Molecular Cell Biology. 3D output. New York: American Science Books. (A very well written and very comprehensive text describing many techniques for molecular analysis of cells.) Lorenz, K. Z. 1970. Animal and Human Behavior Research. Volume 1, Cambridge, MA: Harvard University Press. (A collection of research papers, translated from the original German, describing the early research of Lorenz, who won the 1973 Nobel Prize in Physiology.)

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Organisms on our planet form a vast and varied diversity, from viruses, bacteria and protozoa to flowering plants, invertebrates and "higher" animals. Despite enormous diversity, all forms of life known to us are composed of the same chemical elements and share similar types of organic molecules. Furthermore, all life processes occur in aquatic environments and depend on the physicochemical properties of this extremely abundant and very specific solvent. All living organisms share a common biochemical identity, which is one of the strongest evidences for their evolutionary relatedness, a common thread running through all fields of biological research.

Source of key biochemical molecules

Biologists generally believe that life arose through accidental processes and natural selection under favorable environmental conditions on primitive Earth. Experiments first performed by Stanley Miller in 1953 showed that certain molecules (e.g., amino acids, peptides, nucleic acids) necessary for primitive life could be produced by exposing them to experimental atmospheres of methane, ammonia, and water in a manner similar to An electrical discharge in the form of lightning. This simple atmosphere is thought to be similar in composition to Earth's primordial atmosphere some 4 billion years ago. Earth's early atmosphere was altered over subsequent eons by photosynthetic plants, which added to the current abundance of oxygen and absorbed nitrogen compounds, incorporating them into nitrogen-containing biological compounds. Experimental formation of simple organic molecules under primordial atmospheric conditions suggests that these molecules may have accumulated in ancient shallow seas and formed an "organic soup" in which life may have undergone its first stages of evolutionary organization. The combination and recombination of these molecules ultimately gave rise to the simplest forms of life capable of generating and assembling more complex molecules into information.

Functional combinations, such as nucleic acids and enzymes. The key to forming a protocell-like organism is the formation of small liquid droplets surrounded by a membrane. Lipid molecules (fat molecules) spontaneously form a double layer of "molecular skin" around the microscopic droplets. As these membranes begin to bind other substances (simple nucleotides, etc.), the first steps in the formation of true membranes begin to form - the thin structural environments that envelop the contents of the cell and control the movement of molecules between the interior of the cell and the surrounding environment and provide Organizing its content provides possible structure. Many such additional steps define the pathways of today's extensive biodiversity of more than 35 phyla that exist on Earth today. This hypothetical scenario of the first stages of life's evolution raises many questions. How much did the origin of life depend on the "right" conditions? Would different kinds of life arise on Earth if the chemical and physical environments were very different? What if there are no carbon atoms? As we will soon see, the origin of life as we know it (and imagine it) depends largely on the chemical composition of Earth's environment. If some fundamental properties of matter in the early atmosphere were different, then life would either not exist, or at least be significantly different. There has been a bitter debate between vitalists, who do not believe that life depends on any particular "life" in the inanimate world, and mechanists, who claim that life can ultimately be explained in physical and chemical terms. Until the early 19th century, researchers in the natural sciences believed that the chemical composition of living matter was fundamentally different from that of non-living minerals. Vitalists believe that "organic" matter can only be produced by living organisms, mysteriously distinguishing them from inorganic matter. The concept came to an end in 1828, when Friedrich Wohler discovered that both cyanate and ammonia came from non-living mineral sources to synthesize the simple organic molecule urea:

The principles of chemical reactions apply to the assembly of macromolecules and more complex organelles that make up cells. His successful organic synthesis laid the foundation for modern chemical and physical research to elucidate the mechanisms of life processes. Modern biochemists can now simulate virtually all synthetic and metabolic reactions normally performed in cell-free systems isolated from living cells in vitro. The biochemical and physiological processes of living organisms are ultimately determined by the physical and chemical properties of the elements and compounds they contain. At first glance, the properties of living systems seem too fantastic and complex to be explained by mere mixtures of elements and compounds. But living systems are not simple chemical "soups"; rather, they are highly organized structures, often composed of very large and complex molecules called macromolecules. Many types of macromolecules are involved in regulating and controlling chemical activities in living cells. Organelles such as the plasma membrane, lysosomes, and mitochondria give structural organization to the cell, the fundamental unit of living systems, distinguishing it from its environment and dividing its interior into compartments and subcompartments. Organelles also hold molecules in functionally important spatial relationships with each other. Cells organize into tissues, tissues organize into organs, and these transform into interacting systems. Thus, organisms consist of a hierarchy of organizations, with each higher level adding more functional complexity to the whole (see Figure 1-1). In this chapter, we start at the most basic level—the chemistry level—to learn the simple principles

Atoms, Bonds, and Molecules All matter is made up of chemical elements that can be arranged in the well-known periodic table of natural elements, as well as dozens of fragile, synthetic elements created in the laboratory (Figure 3-1). Of all the chemical elements, only a small fraction occurs naturally in animal tissues. Table 3-1 compares the major components of the Earth's mineral crust and seawater with those found in the human body. About 99 percent of the human body is made up of just four elements: hydrogen, oxygen, nitrogen and carbon. This applies to all living things. Is the predominance of these elements in living systems merely an accident, or is there even a mechanistic explanation for their predominance in various organisms that have evolved over the past 3 billion years? Biologist George Wald, who contributed much to our understanding of the chemical basis of vision, argued that the biological predominance of hydrogen, oxygen, nitrogen, and carbon was no accident but the inevitable result of certain fundamental atoms Relationship properties of these elements - properties that make them particularly suitable for the chemistry of life. We will briefly review the factors that affect the chemical behavior of atoms, and then return to Wald's ideas. Atomic structure is far more complex and subtle than can be fully described here; for our purposes we need only consider a few fundamental features that affect the formation of chemical bonds between atoms and molecules. Basic

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Figure 3-1 In the periodic table of elements, each row corresponds to a different electron orbital layer. The elements in the colored alphabet are physiologically important in their sound form.

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\ Molecules, Energy and Biosynthesis

................................................... ................... Table 3-1 Comparison of the chemical composition of the human body with that of seawater and the earth's crust*.

human body

seawater

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They are important to animal physiology because they govern the interactions between elements central to organic life. In fact, the interaction between these three particles determines the attraction between the elements necessary for life itself. Each atom consists of a dense nucleus of protons and neutrons surrounded by a "cloud" of electrons equal in number to the number of protons in the nucleus. Atomic particles have the following charges and masses (in Daltons, Da): Protons: Neutrons:

+ 1; 1.672 sum 0; 1.674 sum

Electrons: -1; 0.001 and

all other

Is AH absorbing or releasing energy?

Under what conditions does an endothermic reaction occur? For a system in equilibrium, what is AG? How does ATP "donate" stored chemical energy to an energy-absorbing reaction? What does the term coupling reaction mean? How does increased temperature increase the rate of a chemical reaction? What factors affect the optimum temperature for an enzymatic reaction? How do catalysts increase the rate of a reaction? Why do organisms need catalysis? How do enzymes display substrate or binding specificity? How does pH affect enzyme activity? How has the theory of "spatial tuning" of active site specificity been proven correct? What factors affect the rate of an enzyme-catalyzed reaction? The Michaelis constant KM is equal to the substrate concentration at which a given reaction proceeds at half its maximum rate V. Do high K values indicate greater or lesser enzyme-substrate affinity? Why do high substrate concentrations reverse the effect of competitive inhibitors but have no effect on noncompetitive inhibitors? How does each inhibition affect the MichaelisMenten constant K? explain why. Why does aerobic metabolism provide more energy per glucose molecule than anaerobic metabolism? What is the advantage of a gradual decrease in electron pressure over a single large decrease in electron pressure in the electron transport chain? How is energy released in discrete quantities in the electron transport chain? How does the energy release mechanism of the citric acid cycle differ from that during glycolysis?

Recommended Reading Atkins, P. W. 1994. Physical Chemistry. New York: W. H. Freeman & Co. (A full undergraduate-level treatment of many of the fundamental concepts introduced in this chapter.) Lehninger, A.L., et al. 1993. Principles of Biochemistry. Second Edition New York: Worth it. (A short and clear book on principles of biochemistry.) Lodish, H.D., et al. 1995. Molecular Cell Biology. 3D output. New York: American Science Books. (A comprehensive textbook describing the many fundamental biochemical processes that occur in cells.) Stryer, L. 1995. biochemistry. 4th Edition, New York: W. H. Freeman and Co. (A readable reference for information on biochemical structures and mechanisms.)

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The complex chemical reactions that ultimately determine animal life can only occur under stable, constrained conditions. This stability is largely maintained in cells through the action of biofilms, which form a protective barrier that only allows certain substances to enter or leave the cell. Animal tissues contain a surprising amount of biofilms. For example, the cell membrane of a chimpanzee brain is estimated to be about 100,000 square meters, the size of three football fields. Although the cell membrane is a major component of all living matter and is essential to all life

Their existence was not questioned until the 1930s. At the time, there was little or no direct anatomical evidence for biofilms, so their presence could only be inferred from physiological studies. The first important observation of the diffusion-limiting properties of the cell surface was made in the mid-19th century by Karl Wilhelm von Nageli, who discovered that the cell surface acts as a barrier to the free diffusion of dyes from the extracellular fluid into the cell. cell. From these experiments, he concluded that a "plasma membrane" exists. He also discovered the osmotic behavior of cells, noting that cells swell in dilute solutions and contract in concentrated solutions. Structural evidence for the presence of specific cell membranes first arose after the development of the electron microscope (see Chapter 2). The surface of each cell type is a continuous bilayer membrane with a thickness of 6 to 23 nm (Figure 4-1). Understanding membrane structure and function is critical to the study of animal physiology. In this chapter, we discuss membrane structural features and their critical roles in maintaining cellular integrity and controlling cellular activities. In the next chapter, we will discuss the electrical behavior of cell membranes, which are responsible for the transmission of signals between cells, which in turn coordinate the behavior of animals.

membrane structure and organization

10 nanometers

Figure 4-1 As shown in this electron micrograph, the plasma membrane forms a barrier between the inside and outside of the cell. The cell interior (lower right) is separated from the cell exterior by a surface membrane bilayer, visible in cross-section as a ~10 nm thick dark light outline. The dark-light-dark sandwich-like appearance is due to differential staining of the "unit membrane" by electron-impermeable substances during tissue preparation. [Contributed by J.D. Robertson. ]

On their surface, cells are surrounded by a plasma membrane, an extremely thin, complex, lipid-based structure that surrounds the cytoplasm (including the cytoplasm and all organelles) and the nucleus. Internal organelles, such as the An-producing mitochondria we discovered in Chapter 3, have their own surface membranes. This sealing property of the plasma membrane is its most obvious and at the same time most critical function. By means of various metabolic mechanisms described later; the membrane regulates the traffic of molecules between the orderly interior of the cell and the more disordered, potentially destructive, external environment.

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Physiological principles

...................................................

membrane composition

Cell membranes are permeable by integrins. These proteins act as selective filters and active transporters responsible for bringing nutrients into cells and transporting cell products and wastes out of cells. Other proteins contained in the membrane sense external signals that control the cell's response to changes in the environment. The cell membrane maintains different concentrations of certain ions on its sides, resulting in a concentration gradient of multiple ion species across the membrane. Channel proteins contained in the cell membrane are actively involved in the transfer of substances between compartments and ultimately regulate the cytoplasmic concentration of dissolved ions and other molecules with great precision. This allows for the maintenance of the intracellular environment required for the cell's delicately balanced metabolic and synthetic chemical reactions. All biological membranes, including the inner membranes of eukaryotic organelles, have essentially the same structure: lipid and protein molecules held together by noncovalent interactions. Lipid molecules are arranged in a continuous bilayer called a lipid bilayer, and the passage of most water-soluble molecules is relatively impermeable. In 1925, Gorter and Grendel demonstrated for the first time that cell membranes are lipid bilayers through simple and elegant experiments. First, they dissolved lipids in red blood cell "ghosts," the empty membrane sacs left when red blood cells burst. The extracted membrane lipids are then spread in a trough above the water. Due to their asymmetry, lipid molecules arrange their own polarity in such a way

The bulk groups form hydrogen bonds with water, and their hydrophobic hydrocarbon chains protrude into the air. When the dispersed lipid molecular film is gently compressed into a continuous monolayer, it occupies approximately twice the surface area of the original red blood cell. Since the only membrane in mammalian erythrocytes is the plasma membrane, it was concluded that the lipid molecules in the membrane must be a continuous bilayer. As we can see in Figure 4-1, the bilayer has since been observed in cross-section using electron microscopy and freeze fracture, where the membrane is cut from the middle of the bilayer (see Chapter 2). The chemical properties of lipid molecules determine the structure of the membrane, which means that they associate spontaneously to form bilayers, even under artificial conditions (see Chapter 3). Membranes are very fluid structures in which most lipid and protein molecules "float" in the plane of the bilayer (Figure 4-2). The relative proportions of lipids and proteins present in the membrane depend on the type of cells or organelles that the membrane surrounds. Lipids are much smaller and simpler molecules than proteins, and they form the primary structure of membranes. Integral proteins embedded in membranes perform more specialized roles, such as transporting molecules across the membrane, catalyzing reactions, and transmitting chemical signals. Other proteins connect the membrane to the cytoskeleton or to neighboring cells. Some proteins are tightly associated with lipid molecules due to lipophilic groups exposed on the surface of protein molecules. Protein-lipid complexes are called lipoproteins.

Figure 4-2 The Singer-Nicolson fluid mosaic model for membranes is widely accepted. The globular integral proteins embedded in the lipid bilayer provide a mechanism for transmembrane transport. The inner mitochondrial membrane has a higher protein content, so the lipid bilayer is less than that depicted in this figure. carrying glycoprotein

Oligosaccharide side chains, essential for cellular recognition and communication. Cholesterol molecules are close to the heads of phospholipid molecules and they reduce membrane flexibility. The inner ends of the phospholipid tails are highly fluid and impart fluidity to the membrane.

Membranes, channels and transport

Lipid molecules are insoluble in water but soluble in organic solvents. They account for about half of the mass of the animal cell plasma membrane, and the rest are mainly composed of proteins. There are about 106 lipid molecules per square micron of membrane, which means that a typical small cell contains about 106 lipid molecules. The three main types of lipids in cell membranes are phosphoglycerides characterized by a glycerol backbone. Sphingolipids with backbones composed of sphingosine bases

* Sterols, like cholesterol, are non-polar and only slightly soluble in water

The first two lipid types are amphipathic, meaning they have hydrophilic (water-soluble) and hydrophobic (water-insoluble) ends (Figure 4-3). The dual nature of these amphiphilic membrane lipids and their hydrophilic heads

and hydrophobic tails are crucial for the organization of biofilms. Their polar heads seek water, and their non-polar tails seek each other (see Figure 3-14), attracting each other through van der Waals force. These molecules are therefore well suited to provide an interface between the non-aqueous lipid environment (phase) within the membrane itself and the aqueous intracellular and extracellular phases in contact with the inner and outer membrane surfaces. The same force causes the lipid bilayer to reseal when it ruptures, giving the cell the ability to repair itself. Differences in the length of the two fatty acid chains and their composition (see Figure 3-20) affect lipid packing and thus fluidity, resulting in subtle differences in the properties of lipid bilayers. The hydrophobic nature of the phospholipid hydrocarbon tails results in lower membrane permeability to polar substances (such as inorganic ions and polar non-electrolytes such as sucrose and inulin) and their corresponding greater permeability to nonpolar substances (such as steroid hormones). High permeability.

Figure 4-3 Phosphatidylcholine, a glycerol phosphate

extreme head

non-polar tail

Eride's allegations have given the leadership team a polar character. Note that the hydrocarbon chains on the left in the diagram are unsaturated. To differentiate unsaturated fatty acid chains from saturated fatty acid chains, in this figure (and in the figure below) unsaturated fatty acid chains are shown with distinct bends rather than small kinks. In fact, in unsaturated fatty acids, only the double bond is rigid. Both saturated and unsaturated fatty acid chains tend to align in parallel within each phospholipid monolayer because the individual carbon-carbon bonds in the remainder of the chain are free to rotate. [Story, 1988.1

Physiological principles

.....................................

The third class of membrane lipids, sterols, are primarily nonpolar and only slightly soluble in water (Figure 4-4). In aqueous solution, they form complexes with proteins that are more water soluble than sterols alone. Once the sterol molecule reaches the membrane, it fits snugly between the phospholipid and the hydrocarbon tail of the glycolipid (Figure 4-5), increasing the viscosity of the membrane hydrocarbon core. Fluid Chimeric Membranes The concept of a lipid bilayer membrane enveloping most cells gained wide acceptance in the early 1950s due to convincing evidence from various measurement techniques (Spotlight 4-1). Chemical stimulation and immunochemical studies of cell membranes confirmed that proteins are also important components of cell membranes. In addition, enzymatic properties of membranes, such as active transport and other metabolic functions, require the participation of proteins. An example is the protein complex described in Chapter 3, which is responsible for electron transport and oxidative phosphorylation. Despite these early advances in membrane characterization, it was not until 20 years later that researchers realized how fluid and heterogeneous membranes really are. Some protein molecules were found to freely diffuse laterally along the membrane, possibly due to the fluidity of the lipid matrix. In addition, labeling studies have shown that protein molecules or molecular parts facing one side of the membrane are distinct from those facing the other side, and that they do not typically migrate "backwards" across the membrane as previously suspected.

Non-polar hydrocarbons speak of pH, pH, and sphingolipids

Sterols Figure 4-5 Nonpolar sterols intercalate between the hydrocarbon tail and the polar headgroup of phospholipids in the membrane.

Furthermore, in many membranes the distribution of lipid species in the two lipid layers differs.

Fluid mosaic model Based on evidence that emerged in the 1950s and especially in the 1960s, Singer and Nicolson (1972) proposed a fluid mosaic model of membranes in which globular proteins are mixed with some protein molecules integrated into lipid bilayers, which penetrate the bilayer Molecular layers are fully penetrated, while others are only partially penetrated (see Figure 4-2). These integrins are considered to be amphipathic, with their nonpolar parts buried in the hydrocarbon core of the bilayer, while their polar parts protrude from the core, forming affinity for charged amino acid side groups in the aqueous phase. water surface. On the other hand, uncharged hydrophobic side groups are attached to the hydrocarbon bilayer (Fig. 4-6). The hydrophobicity of these side groups is important to prevent integrins from leaving the lipid bilayer. Evidence continues to support the model, which is now widely accepted a quarter of a century after it was first described. Membrane fluidity shows that lipid molecules in membranes rarely move from intact proteins using various techniques

CH3

- +

I HC-CH, I CH2 I CH2 Cholesterol

Hexachloromethane,

Figure 4-4 Cholesterol, a sterol, is an important constituent of LLPLD membranes [Aus Lehnlger, 19751

Figures 4-6 are cross-sectional views showing the complexity of the mosaic bilayer model. The charged hydrophilic amino acid side groups of the protein protrude into the aqueous phase, and the uncharged hydrophobic groups are buried in the lipid phase of the bilayer.

membrane,

channels and transport

................................................ Membrane 1 Side-to-side (about once a month), but adjacent molecules in the monolayer switch positions about 10 times per second. This rapid exchange of lipids within the membrane results in rapid migration along the membrane plane, but not across it. Membranes are highly dependent on their composition, and cholesterol plays an important role in controlling this membrane property. The plasma membranes of eukaryotes contain large amounts of cholesterol, at most one molecule per phospholipid. When cholesterol is present, it binds weakly to neighboring phospholipids, making the lipid molecule significantly less mobile but more efficient (Figures 4-7). However, incorporating too much cholesterol into cell membranes can cause the membranes to lose their elasticity. This is the underlying mechanism of "arteriosclerosis," a major cause of cardiovascular disease, in which the cell membranes of enchelic cells are damaged. The cells lining the arteries become abnormally stiff (and extra cholesterol plaque builds up inside the blood vessels as well). The lipid composition of biofilms varies by tissue type. While most membranes contain a large proportion of cholesterol (>IS%), other types of lipids may be present in higher or lower proportions. Lipids also differ in their head groups (see Figure 3-13), which in turn affects their interactions with proteins. In fact, some integrins only function when certain proportions of lipid types are present. Thus, during cell development, cells must regulate the distribution of lipid species across their membranes and rearrange lipid concentrations according to specific functional requirements.

……

Hetuogenei9 of Integral Membrane Proteins Integral proteins found in the plasma membrane (see Figure 4-2) have multiple functional forms, including ion channels, various carriers and membrane pumps, receptor molecules, and recognition molecules. The amount of intact protein varied, but in some membranes the protein content was so high that only about three lipid molecules separated the proteins at the closest point. Morphological evidence for a layered mosaic of globular proteins in the lipid bilayer can be seen in freeze-etched electron micrographs of the membrane surface (Figs. 4-8). when digested

Phospholipids

cholesterol

Figure 4-7 Cholesterol interacts weakly with neighboring phospholipids in the membrane and partially disrupts their fatty acyl chains. therefore. The amount of membrane is less fluid, but mechanistically, the amount of lesterol present in lipid bilayers varies greatly by cell type. In some cells, the cell membrane contains almost many cholesterol molecules (phospholipids), while in other cells the cell membrane contains almost no cholesterol. The structural formula of cholesterol is shown in Figure 4-4

Figure 4-8 Freeze etching method provides morphological results for mosaic membrane models. In these freeze-etched electron macrographs, the plasma membrane splits in the middle of the bilayer, revealing membrane-embedded particles 5–8 nm in diameter. Digestion with proteolytic enzymes leads to gradual loss of these particles, indicating that globular proteins are introduced into the liquid phase of the membrane (A). Control (B): 45% of the pellet was digested. (C) 70% digestion [courtesy of L. H. Engstrom and D. Branton. ]

4. When fixed with permanganate, the membrane appears as follows

Spotlight 4-1

Lipid case

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Three-layer profile: a slightly dyed central region sandwiched between two electron-dense outer layers (see Figure 4-I), with a total thickness of about 7.5 nm. 1955

J. David Robertson (1960) named this three-tier structure as

There is considerable accumulating evidence for the existence of lipid bilayer membranes:

equipment film. The unit membrane concept is consistent with a bimolecular lipid layer sandwiched between two protein layers.

1. The lipid content of the membrane is consistent with that of the dissection

5. The thickness of the lipid bilayer, calculated as twice the length of a single membrane lipid molecule, is roughly the same

Directed llpld molecules such as Gorter and

Dimensions of the unit membrane as seen in electron

Grendel in 1925 2. The ease with which non-electrolytes cross membranes

6. Freeze Etching Electron Microscopy to Reveal Membranes

This membrane is consistent with the presence of the membrane, enhancing the tendency of such molecules to leave the membrane

There is a preferred splitting plane in the middle, consistent with the separation of a bilayer into two

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7. Artificial lipid bilayer (see Spotlight 4-2), reconstituted

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Lipid bilayers with similar thickness and putative structure

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Bimolecular lipid cores of liquid-embedded membranes

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Model, Permeability and Electrical Properties

membrane.

Psychologically similar to a cell membrane. these are different

3. The capacity of the biofilm is generally 0.5 times that of the viscous layer.

The existence of Enzen can be traced to specialized channels and carriers present in natural membranes.

This size consists of two phosphorus molecules arranged next to each other (or 6.0–7.5 nm).

In the case of proteolytic (protein digesting) enzymes, the globular units visible in the membrane were gradually removed, proving that they were indeed proteins. Changes in Membrane Shape

Membrane composition varies widely between cell types. At one extreme is the metabolically inert myelin sheath that surrounds the axons of some neurons in which the lipid bilayer is essentially uninterrupted. At the other extreme are structures in which cell membranes contain non-lipid macromolecular repeating units that all but eliminate lipid bilayers. This membrane is designed for highly specialized purposes, such as signal transduction or enzymatic activity. For example, in visual receptor cells, the repeating macromolecular unit is a molecule of the visual pigment opsin. The mitochondrial membrane dedicated to enzymatic activity is composed almost entirely of repeating subunits of ordered enzyme aggregates. Between these two extremes is the plasma membrane and most intracellular membranes, whose bilayers are often interrupted by intact protein molecules. This drastically alters the basic structure of lipid bilayers with intact proteins required for functional specialization.

Transport in Membranes: An Overview Because of their structure, membranes can be very selective about which molecules can pass through them. Hi

The hydrophobic interior of the lipid bilayer makes the membrane extremely impermeable to most polar molecules. This prevents the water-soluble components of the cells from easily entering or escaping. However, such movement is sometimes necessary or desirable, so mechanisms have evolved in all cells to transfer these molecules across membranes. In addition, macromolecules such as proteins and large particles must be transported across the plasma membrane using specialized mechanisms. To understand these specific mechanisms of membrane transport in living cells, we will first investigate the physics of solute and solvent displacement in solution and across semipermeable membranes. This membrane is very similar to that of living cells, and the principles discussed here apply to many physiological situations. diffusion

The random thermal motion of suspended or dissolved molecules causes them to diffuse from regions of higher concentration to regions of lower concentration, a process known as diffusion. Diffusion is very slow at the tissue scale, not at the cellular scale. For example, copper sulfate crystals dissolve so slowly in unstirred water that it may take a full day to fully color a liter of water. However, at the microscopic scale of a cell, diffusion times are only fractions of a millisecond. The diffusion rate of a solute s can be defined by the Fick diffusion equation:

membrane,

channels and transport

................................................... ...................................translucent

where dQSld is the diffusion rate (ie, the amount of s diffused per unit time), D is the diffusion coefficient of s, A is the cross-sectional area of s diffusion, and dC,ldx is the concentration gradient of s (ie, the concentration varies with distance). The gradient factor dCs/dx is obviously very important as it determines the rate at which s diffuses along the gradient. D varies according to the nature and molecular weight of the substance and solvent, which in most physiological cases is water.

one-way flow

Bubble

When a solute is present on both sides of a membrane through which it can diffuse, it exhibits unidirectional flow in either direction (Figure 4-9A). The flux or diffusion rate is the amount of solute passing through a unit area of the membrane in one direction per second

net flow

where J usually has units of moles per square centimeter per second (M cm-2.s-l). Flow in one direction (eg, from extracellular to intracellular) is considered independent of flow in the opposite direction. Therefore, if the inflow and outflow are equal, the net flow is zero. When the unidirectional flux is greater in one direction, the net flux is the difference between the two unidirectional fluxes (Figure 4-9B). The permeability of a membrane to a substance is the rate at which the substance passively permeates the membrane under certain conditions. Greater permeability correlates with greater flux, all other factors being equal. If we assume that the membrane is a homogeneous barrier with a continuous concentration gradient of the non-electrolyte species between the high concentration (I) side and the low concentration (II) side, then - PIC, - C,)

dQs -

Among them, dQ and ldt are the quantity of substance s passing through the unit membrane area per unit time (for example, the number of moles per square centimeter per second), and C and C are the quantity of substances at two concentrations respectively (B.M~m - membrane On a side, P is the permeability constant of the substance, with dimensions in velocity (cm-s-I. Note that Equation 4-3 applies only to molecules that are not actively transporting or are not affected by forces other than simple diffusion. This does not apply to electrolytes, Because they are charged when they dissociate, their flow does not depend only on force

Figure 4-9 A solute can move through a membrane in either direction, depending on the prevailing physical and chemical conditions. (A) Arrows indicate the actual flux between compartments I and II. (B) Single arrows indicate the net flux generated by compartment Ito II.

Concentration gradients, but also electric gradients (i.e. potential differences across membranes). From Equation 4-3 it can be seen that the flux of the non-electrolyte should be a linear function of the concentration gradient (C, - C,). This linear relationship is characteristic of simple diffusion and can be used experimentally to distinguish passive diffusion of matter from any other mechanism. Permeability constants take into account factors inherent to all membranes and materials. These factors determine the likelihood that molecules of a given substance will pass through the membrane. This relationship can be formally expressed as:

where D is the diffusion coefficient of the species in the membrane, K is the partition coefficient of the species, and x is the thickness of the membrane. The more viscous the film or the larger the molecule, the lower the Dm value. The permeability constants of different substances vary greatly. For example, the permeability of red blood cells

100

Physiological principles

...................................................

Cells for different solutes range from 10-12 cm-s-I to cm s-l. Importantly, the permeability of certain membranes to certain substances can be greatly altered by the reaction of hormones and other molecules with receptor sites on the membrane, thereby affecting channel size or delivery mechanisms. For example, vasopressin increases the water permeability of mammalian renal collecting ducts by 10-fold. Likewise, neurotransmitters acting on specific integral membrane proteins in nerve and muscle cells lead to a dramatic increase in the permeability of Nac, KC, Ca2+ or C1- ions.

Osmosis

In 1748, AbbC Jean Antoine Nollet showed that if you put pure water on one side of an animal membrane (such as the bladder wall) and an aqueous solution containing electrolytes or other molecules on the other, the water would pass through the membrane into the solution. This movement of water along its concentration gradient is called osmosis (from the Greek osmos, "to squeeze"). We don't usually think of "water concentration", but actually water behaves like any other substance in that it diffuses along a concentration gradient. It was later discovered that osmosis creates a hydrostatic pressure gradient. Osmosis is a collective property that is critical to living systems. As can be seen in Figure 4-10, when water diffuses into the solution through a semipermeable membrane, the pressure difference causes the solution level to rise. The solution level continues to rise until the net rate of water movement (flux) through the membrane becomes zero. This occurs when the hydrostatic pressure of the solution in chamber I1 is sufficient to push water molecules back into membrane chamber I at the same rate that osmosis causes water molecules to diffuse from I to II. Reverse osmosis requires hydrostatic back pressure. The diffusion of water from compartment I to compartment I1 is called the osmotic pressure of the solution in compartment 11. In 1877, Wilhelm Pfeller made the first quantitative study of osmotic pressure. He deposited a "film" of copper ferrocyanide on the surface of the porous clay cup,

This creates a membrane through which water molecules can diffuse more freely than sucrose molecules. Thanks to the clay matrix, these artificial membranes are also strong enough to withstand relatively high pressures without rupturing. With these membranes, Pfeller was able to measure osmotic pressure directly for the first time. Some of his results are shown in Table 4-1. Note that the osmolarity in the table is proportional to the solute concentration. Osmosis is responsible for the net movement of water across cell membranes and epithelial cells. To understand this, imagine that 1.0 M sucrose in water is carefully layered under 0.01 M sucrose in water. There will be a net diffusion of water molecules from the lower sucrose concentration solution (0.01 M solution) into the 1.0 M sucrose solution, and there will be a net diffusion of sucrose in the opposite direction until equilibrium is reached. If the two solutions are separated by a water-permeable but sucrose-impermeable membrane, there will still be a net diffusion of water molecules from a solution with a higher concentration of H,O (0.01 M sucrose solution) to a 1.0 M sucrose solution showing a higher concentration of H 2 0 Low. Since sucrose is not permeable to the membrane, there is a net diffusion of water (permeate flow) from a solution with a lower solute concentration to a solution with a higher solute concentration through the membrane. Osmotic pressure is not only proportional to the solute concentration C (moles of solute particles per liter). Table 4-1 Osmotic pressure of different concentrations of sucrose* Sucrose

Osmotic pressure

Osmotic pressure ratio

(%I

(ATM)

as a percentage of sucrose

1 2 4 6

070 1,34 2 74 4,10

070 067 068 068

* Results obtained in experimental measurements by Pfeffer (1877)

semipermeable membrane

time

Figure 4-10 Osmotic flow of water through a semipermeable membrane creates hydrostatic pressure. Compartment I contains purified water; Compartment II contains water impermeable to solutes. Osmotic pressure forces water from chamber I into chamber II until the hydrostatic pressure difference equals the opposite osmotic pressure difference. When the pressures are equal, the flow is zero.

Membranes, Channels, and Transport 101 ................................................ ...

= osmotic pressure of the solvent), and its absolute temperature T: TT

Creative Academy

(4-5)

and

where K and K are constants of proportionality. Jacobus van't Hoff related these observations to the gas laws and showed that molecules dissolved in solution behave thermodynamically like gas molecules. So TT

Real Time Clock

where n is the number of molar equivalents of solute, R is the molar gas constant (0.082 L atm K-l mol-'), and V is the volume in liters. However, like the gas law, this osmotic pressure expression is only valid for dilute solutions and fully dissociated electrolytes. Large concentration gradients across cell membranes can generate surprisingly high osmotic pressures—on the order of several atmospheres. Such pressure, if left unchecked, can be severe enough to blow up a cell. Consequently, mechanisms have been developed to regulate osmotic homeostasis that minimize osmotic pressure gradients across cell membranes and tissues (see Chapter 14).

Osmotic Pressure and Tonicity Two solutions exerting the same osmotic pressure on a membrane that is only water permeable are said to be isotonic with respect to each other. If a solution exerts a lower osmotic pressure than another solution, it is hypotonic relative to the other solution; if it exerts a greater osmotic pressure, it is hypertonic. Osmotic pressure is therefore defined in terms of an ideal osmometer in which an osmotic membrane allows water to pass but completely blocks solutes. All solutions with the same number of dissolved particles per unit volume have the same osmotic pressure and are therefore defined as isotonic. In contrast to its osmotic pressure, the tonicity of a solution is defined by the response of cells or tissues immersed in the solution. A solution is said to be isotonic with respect to the given cells or tissues if the cells or tissues immersed in it neither shrink nor swell. When the tissue swells, the solution is said to have a hypotonic effect on the tissue; if it contracts, the solution says

is the constant of proportionality relative to 1 mole of ideal gas in the gas equation PV/T = R, with a value of 1.985 cal.molk'.K-'; P is in atmospheric pressure and V is in liters.

is hypertonic. These effects are due to the movement of water across the cell membrane in response to the osmotic pressure difference between the interior of the cell and the extracellular solution. If cells did behave like ideal osmometers, then tonicity and osmolarity would be equivalent, but that's often not the case. For example, sea urchin eggs maintain a constant volume in a NaCl solution that is isotonic with respect to seawater, but expand when immersed in a CaCl solution that is isotonic with respect to seawater. Therefore, NaCl solutions are isotonic with respect to sea urchin eggs, while CaCl2 solutions are hypotonic. The tonicity of the solution depends on the rate of intracellular accumulation of the solute in the relevant tissue and the concentration of the solution. The more easily a solute accumulates, the lower the tonicity of a solution for a given concentration or osmolarity. This is because water obeys the principle of osmosis and the cells expand as they gradually fill with solutes. Therefore, the terms isotonic, hypertonic and hypotonic have meaning only in relation to actual experimental measurements in living cells or tissues.

Electrical Effects on Ion Distribution Membrane permeability of charged particles depends on the membrane permeability constant and the potential across the membrane. Understanding how charged particles interact with membranes is extremely important to understanding how electrically excited cells work. Neurons are the most highly specialized cell of this class. Since neurons will be discussed in the next few chapters, only some important observations are summarized here. Two forces can act on charged atoms and molecules (e.g. Na+, K+, Clk, Ca2+, amino acids) resulting in a net passive diffusion of any species across the membrane:

A chemical gradient due to a difference in the concentration of a substance on either side of a membrane. electric field or potential difference across the membrane

Ions move away from regions of high concentration, and when that ion is positively charged, it also moves toward an increasing negative potential. The sum of the concentration gradient and the electrical gradient determines the net electrochemical gradient acting on the ion. When an ion is in equilibrium with respect to the membrane (that is, when there is no net transmembrane flux of that ion), there is a potential difference just sufficient to balance and cancel the chemical gradient acting on the ion. The potential at which ions are in electrochemical equilibrium is called the equilibrium potential, and the unit is volts (or millivolts). Several factors affect the value of the equilibrium potential, but the most important is the ratio of ion concentrations on opposite sides

102

Physiological principles

.....................................

membrane. For monovalent ions such as Na+ or K+, at 18°C, the equilibrium potential (in volts) is 0.058 x log, the ratio of the extracellular to intracellular concentrations of the ion. This gives a potential difference of 58 mV. The membrane has the same effect on the net diffusion of the ion as a 10:1 transmembrane concentration ratio. Thus, a seemingly paradoxical situation arises in which ionic species can passively diffuse (i.e. migrate "uphill") against their chemical concentration gradient. A region of higher concentration occurs when the electrical gradient (i.e., potential difference) across the membrane opposes and exceeds the concentration gradient. For example, when the negative charge inside the cell is greater than the equilibrium potential of K+, potassium ions diffuse into the cell even though the intracellular K+ concentration is much higher than the extracellular concentration. The distribution of ions across the membrane and the associated equilibrium potential is described by the Nernst relationship, which is discussed in detail in the next chapter. Electricity cannot act directly on uncharged molecules such as sugars. These substances are primarily affected by the concentration gradient to which they are exposed.

balance

start

+ time

Second

K A\

start

balance

+ time

brown scale

When a diffusible solute is separated by a membrane that is freely permeable to water and electrolytes but completely impermeable to one ionic species, the diffusible solute is unevenly distributed between the two compartments. This phenomenon was discovered in 1911 by Frederick Donnan, who was the first to describe how solutes are distributed, and the equilibrium state is named after him. To understand Donnan equilibrium, imagine starting with pure water in two chambers and adding some KC1 to one of the chambers (Figure 4-11). Dissolved salts (K+ and C1-) diffuse through the membrane until the system is in equilibrium, ie. H. until the K+ and C1- concentrations on both sides of the membrane are equal (Figure 4-11A). Now suppose you add the potassium salt of a non-diffusible anion (a large molecule A- with multiple negative charges) to the solution in compartment I. K+ and C1- are rapidly redistributed until a new equilibrium is established by the movement of some K+ and some C1- from compartment I to compartment I1 (Fig. 4-11B). Donnan equilibrium is characterized by the mutual distribution of anions and cations

Figure 4-1 1 Donnan equilibrium distribution across a semipermeable membrane. (A) When KC1 is placed in compartment I of a vessel separated by a permeable membrane, K+ and CI- diffuse across the membrane until the concentrations on both sides are equal. (B) When an anion-impermeable potassium salt is placed in compartment I, some K+ and C I diffuse into compartment II until electrochemical equilibrium is restored. It should be noted that these chambers (unlike living cells) are not stretchable.

We can understand this situation by considering the consequences of the following physical principles: 1 in two compartments. must have an electron property

At equilibrium, the diffusible cation K+ is more concentrated in the compartment where the non-diffusible anion A- resides than the other compartments, while the diffusible anion C1- is less concentrated in this compartment than the other compartments.

Exchange means that in each compartment, the total number of positive charges must equal the total number of negative charges. Therefore, in this example, [K+] = [Cl-] in compartment 11. Statistically, the diffusible ions K+ and C1 cross the membrane in pairs to maintain electrical neutrality. The probability of their crossing is the product of [K+] X [Cl-1. At equilibrium, the rate of diffusion of KCl across the membrane in one direction must be equal to the rate of diffusion of KCl in the opposite direction. Therefore, in equilibrium, the product [K+] x [Cl-] in one compartment must correspond to the product in the other compartment. If x, y, and z represent the ion concentrations in compartments I and II, as shown in Figure 4-12, we can represent the equilibrium

Figure 4-12 Donnan equilibrium can be described algebraically. Figure 4-11B shows the equilibrium state that occurs after the addition of a non-permeable anion salt to compartment 1.

Conditional (i.e. the product [K+] x [Cl-] is equal in both compartments) algebra:

Various ions and molecules are accessible, and there is almost never a single "non-diffusible anion" representing the various anionic side groups of proteins and other macromolecules. Although the physical and mathematical principles recognized by Donnan play a role in regulating the distribution of electrolytes in living cells, imbalance mechanisms must significantly alter the distribution of many substances across the cell membrane. In particular, the permeability of cell membranes to certain ions can change over time, changing conditions significantly. Thus, cells cannot be considered as passive "osmometers" and, except in certain circumstances, the distribution of substances across biofilms cannot be fully predicted by the principle of Donnan's equilibrium.

osmotic properties of cells

Of course, this equation also applies if A- does not exist. In this case, K+ and C1- are uniformly distributed with z = 0 and x = y. Rearranging Equations 4-8, we can see that in equilibrium, the diffuse ion distributions in the two compartments are mutual:

From this relationship it is clear that as the concentration of the non-diffusible anion z increases, the concentrations of the diffusible ions (x and y) become increasingly different. This uneven distribution of diffusible ions is a hallmark of Donnan equilibrium. In a Donnan equilibrium, the maldistribution of the osmotic pressure of the solute causes water to move to the compartment with higher osmotic pressure (compartment I in Figure 4-11). This difference in osmotic pressure plus the increase in hydrostatic pressure in this compartment is called osmotic pressure. This concept is important for understanding the balance of hydrostatic and osmotic pressures across specific biological barriers such as capillary walls. For simplicity, the interpretation of Donnan equilibrium depends on a set of ideal conditions. Of course, living cells and their surface membranes are much more complex. For example, cell membranes are somewhat permeable

[K+], = 140 [Ca2+],< 1 0

- ，。

[CI-1 = 3-4 [A-I = 140

We can now use the physical principles outlined above to analyze the properties of cell membranes that hold different concentrations of ions inside and outside the cell (Figure 4-13). Ultimately, the cell membrane must precisely regulate cell volume and thus intracellular osmotic pressure. ion homeostasis

Every cell maintains intracellular concentrations of inorganic solutes that differ from those outside the cell (Table 4-2). The most concentrated inorganic ion in the cytosol is K+, which is typically 10-30 times higher than in the extracellular fluid. Conversely, the internal concentrations of free Na+ and C1- are usually lower (by about a tenth or less) than the external concentrations. Another important generalization is that intracellular Ca2+ concentrations remain several orders of magnitude below extracellular concentrations. This difference is due in part to the active transport of Ca2+ across the cell membrane and in part to the sequestration of this ion in organelles such as mitochondria and the endoplasmic reticulum. Consequently, the concentration of Ca2+ M in the cytosol is usually significantly reduced. Cell membranes are typically about 30 times more permeable to K+ than to Na+. Membranes vary in their permeability to chloride ions. It is similar to K+ in some cells and lower in others. Cell membranes are less permeable to Na', but not high enough to prevent Na+ from stabilizing into the cell.

Figure 4-1 3 The concentrations of common ions inside and outside the vertebrate skeletal muscle cells vary greatly. Concentrations given are given in mmol/l. The concentration of intracellular Ca2+ applies to free, unbound and unbound ions in the sarcoplasm. The sum doesn't exactly match due to the incomplete list of ions. [A-1 represents the molar equivalent negative charges carried by various impermeable anions.

104

Physiological principles

.................................................. .....................................

Table 4-2 Internal and external concentrations of certain electrolytes in certain nerve and muscle tissues. internal concentration

External concentration (rnM)

(mM) Tissue

already

CI-

already

Potassium+

CI-

小鼠，~ns~von/out~von Na'

Potassium+

CI-

squid nerve

410

40-100

440

560

1/9

19/1

1/14-1/6

crab leg nerve

410

510

540

1/10

34/1

21.01

tailor muscle

140

120

2.5

120

1/12

56/1

1/30

Certain features of the cell membrane, particularly the different permeability of the membrane to different kinds of ions, suggest that under certain conditions Donnan equilibrium may hold. To understand when Donnan equilibrium can be used to determine the membrane properties of living cells, three related factors are important:

1. In cells, carboxyl groups and other anionic sites on impermeable peptide and protein molecules contribute most of the net negative charge. These charges must be balanced by positively charged counterions such as Na+, K+, Mg2+ and Ca2+. 2. These anionic sites trapped in the cell are similar to the artificial case given above (see Figure 4-1 1), where the Donnan equilibrium holds. In fact, if K+ and C1- were the only diffusible ions, the cell would be in equilibrium similar to that shown in Figure 4-11B. However, the cell membrane is permeable to Na+ and other inorganic ions, and if simply allowed to accumulate, the cell becomes saturated with these ions over time. This in turn causes water to seep into the cells, causing the cells to swell. 3. This osmotic disaster is avoided because the cell pumps Na+, Ca2+, and some other ions out at the same rate as it enters the cell, making the intracellular Na+ concentration about an order of magnitude lower than the extracellular concentration. This active pumping, which will be discussed later, corresponds to effective impermeability to Na+ and Ca2+. This prevents the concentrations of these ions from reaching equilibrium, and the cells actually behave as if they are in Donnan equilibrium. In fact, a non-uniform distribution of ions represents a steady state requiring a constant expenditure of energy (pumping ions), rather than a true equilibrium. Because K+ and C1- are by far the most concentrated and penetrating ions in tissue, their distribution resembles an ideal Donnan equilibrium. That is, the KC1 product [K+] of other ions present.

Figure 4-14 The KC1 product obeys Donnan equilibrium. The distribution of K+ and CI follows the Donnan equilibrium principle, provided the membrane is permeable to both K+ and CI-.

Cell Volume Plant and bacterial cells have a rigid cell wall secreted by a cell membrane. These walls limit the size of the cells and allow osmotic build-up of turgor pressure in these cells. Animal cells, by contrast, do not have rigid cell walls and therefore cannot withstand the buildup of high intracellular pressures. As a result, cells change in size when exposed to different concentrations of the impermeable substance dissolved in water. This contraction or expansion is due to the osmotic movement of water (Figure 4-15). The surface membrane can prevent cell osmotic swelling in two ways. One is that the water can be pumped out as fast as it can be pumped in. There is no evidence that this occurs, although the contractile vacuole of some protozoa would have a similar effect. Another mechanism, which appears to be the primary mechanism for regulating cell volume, is the pumping out of solutes entering the cell (Fig. 4-16). Therefore, at steady state, Na+, the main osmotic component outside the cell, is excreted. It is transported out of the cell by active transport as fast as it entered the cell. In fact, there is no net entrance. This situation corresponds osmotically to complete sodium impermeability, with a relatively fixed Na+ concentration in the cell. There is no compensatory osmotic water influx because Na+ is not allowed to continue to accumulate in the cell. Low intracellular (compared to extracellular) sodium concentrations are important to balance other osmotically active solutes in the cytoplasm. The importance of active transport in maintaining the sodium gradient as well as cell osmotic pressure and cell volume becomes apparent when cellular energy metabolism occurs

Na was pumped. passive sodium influx

@Na+ red blood cells

Active transport without Na+

Figure 4-15 Osmotic changes change the volume of red blood cells. (A) Isotonic solution: cell volume remains constant. (B) Hypotonic solution: Due to the higher osmotic pressure of the cytoplasm compared to the solution, water (arrow) enters the cell and causes swelling. (C) Hypertonic solution: In more concentrated media, water leaves the cells and causes contraction.

Cells are damaged by metabolic toxins (Figure 4-17). Without ATP to stimulate Naf to squeeze upward, sodium ions enter the cell along with their chloride counterions, and water permeates into the cell, causing the cell to swell.

Ton

Add hypertonic solution

cell burst

Figure 4-17. A metabolic inhibitor interferes with Na+ pumping, thereby affecting cell volume maintenance. Under normal conditions, Na+ levels inside and outside the cell are in balance: ions are passively entered into the cell and then pumped out of the cell. However, with the addition of metabolic inhibitors, the cells were no longer able to pump out the Na+ that was constantly permeating the cells. As a result, intracellular [Na+] increases and water percolates, causing the cell volume to increase beyond its original volume (dashed line). Eventually the cells burst due to massive swelling.

poorly permeable solutes

u time Figure 4-16 Hypertonic solutions with both impermeable and weakly impermeable solutes cause initial cell contraction. If the solute is completely impermeable, it will cause sustained cell contraction, since the solution is hypertonic in nature in this case. However, if the solute is only weakly impermeable, the solution is hypotonic and enters the cell slowly, followed by an osmotic flow. This process eventually causes swelling, even if the solution is hypertonic.

Passive transmembrane movement Molecules can cross the membrane in different ways without direct input of energy, ie passively. Note that while these processes do not directly require metabolic excitation, they ultimately depend on the concentration or electrical gradients across the cell membrane, requiring energy at some point in time to generate and maintain them. The energy stored in this gradient ultimately leads to the translocation of molecules across the membrane. Still, it makes sense and is appropriate to think of these processes as passive. There are three basic pathways for the passive transmembrane movement of molecules or ions (Figure 4-18). in the first case

106

Physiological principles

.................................................. .....................................

external

within

w.-

"garbage can

~two

Substrate molecule

Extracellular

carrier molecule

In order to leave the aqueous phase and enter the lipid phase, the solute must first break all of its hydrogen bonds with water. Each hydrogen bond requires approximately 5 kcal of kinetic energy. Furthermore, solute molecules that pass through the lipid phase of the membrane must be dissolved in the lipid bilayer. Therefore, its fat solubility will also play an important role in whether the body can penetrate the membrane. Thus, those molecules with the fewest hydrogen bonds to water penetrate the lipid bilayer most readily, whereas polar molecules such as inorganic ions are almost never dissolved in the bilayer. Many factors such as molecular weight and molecular shape affect the mobility of nonelectrolytes within membranes, but the empirically measured partition coefficient is the most important predictor of nonelectrolyte diffusion across lipid bilayers. To measure this property, the test substance is shaken in a closed tube filled with equal volumes of water and olive oil and the factor. Sufficient K is determined from the relative solubility in water and oil at equilibrium, using the equation concentration of solute in the concentration of solute lipid in water

K = molecule occupied

Figure 4-18 Substances cross membranes in three main ways. (A) Dissolved in the lipid phase. (B) Diffusion through unstable or fixed water channels. (C) Carrier-mediated transport (facilitated transport or active transport).

The molecules simply diffuse through the membrane. It leaves the aqueous phase from one side of the membrane, dissolves directly in the lipid layer of the membrane, diffuses through the thickness of the lipid or protein layer, and finally enters the aqueous phase on the other side of the membrane. In the second case, dissolved molecules remain in the aqueous phase and diffuse through water channels, water-filled pores, in the membrane. In the third mode, dissolved molecules bind to carrier molecules dissolved in the membrane. The carrier "mediates" or "facilitates" the movement of solute molecules across the membrane. Carriers can even "mask" polar solutes and, due to their lipid solubility, allow the solute to diffuse more easily across the membrane, reducing its concentration or electrochemical gradient. This is called carrier-mediated (or facilitated) transport and can take many forms. Let us now consider each of these three main paths in turn. Diffusion easily across the lipid bilayer

When a solute molecule comes into contact with the lipid layer of a membrane and its thermal energy is high enough, it can enter and pass through the lipid phase and eventually leave the other side of the membrane into the aqueous phase. arrive

(4-10)

Is the permeability of non-electrolyte membranes related to the lipid-water partition coefficient of solute? Collander (1937) systematically tested this idea in the macroalgae cell Chara by plotting the permeability coefficient (Equation 4-4) versus the partition coefficient (Equation 4-10). Lipid solubility is almost linearly related to the permeability of the substance, independent of molecular size (Figure 4-19). Non-electrolytes show a wide range of partition coefficients. For example, the value of polyurethane is 1000 times that of glycerin (see Figure 4-19). These differences depend on certain features of the molecular structure, such as

water,

Methanol,.

Harnstorff

Smallest numerator 0

Geria

next smaller

o next largest numerator

0,0001

0,001

0,01

0,1

Olive oil-water partition coefficient Figure 4-19 The non-electrolyte membrane permeability has a linear relationship with the oil-water partition coefficient. Note that the permeability of non-electrolytes is independent of molecular size.

Membranes, channels and transport

107

.................................................. ......................... H2C-OH

six six six

CHZ-OH

I I HO-CH I HC-OH I HC-OH I

Nurho

six six six

six six six six

H2C-OH

Hexanol

D-Mannitol

Figure 4-20 The structure of six carbon molecules determines their water solubility and fat solubility. Note the difference in the number of hydroxyl groups between hexanol and mannitol1. Hexanol is insoluble in water but easily soluble in lipids due to its weak hydrogen bonding ability. Due to its strong hydrogen bonding ability, mannitol is easily soluble in water, but hardly soluble in lipids.

This is illustrated in Figure 4-20, which compares two molecules with different solubilities. Hexanol and mannitol have similar structures, except that hexanol contains only one -OH group while mannitol contains six. These -OH groups promote hydrogen bonding with water, thus reducing fat solubility. In fact, each additional hydrogen bond results in a four-fold increase in the partition coefficient, which is reflected in a decrease in permeability (Figure 4-21). Therefore, hexanol diffuses more easily through the membrane than mannitol. The permeability of water through cell membranes is much higher than predicted by its partition coefficient (see Figure 4-19). This is partly because water can penetrate the lipid bilayer through selective permanent channels. Structural evidence for this can be found in certain epithelial cells whose water permeability depends on water channels in the plasma membrane. However, even in channel-free artificial lipid bilayers, water permeability is still many times higher than predicted from the solubility of water in long-chain hydrocarbons. One possible explanation is that small, uncharged water molecules can pass through temporary channels between lipid molecules. Other small, uncharged polar molecules such as CO, , NO,

extracellular space

Cytoplasmic B

solute

carrier

Extracellular

Figure 4-21 Hydrogen bonding significantly reduces solubility, thereby reducing membrane permeability.

and CO also have relatively high permeability through man-made and natural membranes. However, it is not known whether this is due to dedicated channels or lack of selective ones. Simple diffusion across a lipid bilayer exhibits unsaturated kinetics (see Figure 4-1 8A), meaning that the rate of influx increases proportional to the concentration of solute in the extracellular fluid. This is because the net influx rate is determined only by the difference in the number of solute molecules on either side of the cell membrane. This ratio of external concentration and influx rate over a wide concentration range distinguishes simple diffusion from channel permeation or carrier-mediated transport mechanisms (Fig. 4-18B and C). Diffusion through membrane channels

Charged molecules can diffuse across the membrane through specific water-filled channels. Since inorganic ions such as Na+, Kf, Ca2+, and C1- cannot diffuse through lipid-walled cells, specialized protein molecules have evolved that span cell membranes and act as pores. When these pores are open, certain solutes can pass through them (Figure 4-22A).

channel protein

lipid bilayer

Number of hydrogen bonds

Figure 4-22 Membrane transport proteins. . Act as a carrier or form channels in membranes. (A) Channel proteins form a water-filled pore in the bilayer through which certain ions can diffuse. (B) In contrast, the carrier protein switches between two conformations, allowing solute-binding sites to sequentially contact one side of the bilayer and the other.

“

……

Spotlight 4-2

artificial double layer

The principle of bilayer formation is shown in the figure (part B). The most stable configuration consists of two layers of lipid molecules that are hydrophobic and lipophilic

Many of our ideas about how molecules and ions move across membranes come from experiments and observations of artificial bilayers, similar to bimolecular sheets

The carbon tails are loosely bound together to form a liquid-lipid phase between the hydrophilic polar ends of the molecules, which face outward toward the aqueous medium. The thickness of the lipid film can be easily determined from inside

Makes up the base of the cell membrane. Artificial bilayers are very useful in studying osmotic mechanisms because

The interference color of light reflected from the two surfaces of the film. The film thickness is about 7 nm (black).

They can be made from chemically defined lipid mixtures. selenium

interference colors) are the most commonly used. these membranes

Selected substances can be added to test their effect on permeability. Channel forming substances, such as antibiotics

Has conductivity (ion permeability) and capacity consistent with its thickness and lipid composition. Al

Ionophores (molecules that facilitate the diffusion of ions across membranes) and excitable membrane channel components

Although they are much less permeable to ions than cell membranes, the addition of certain ionophores increases its

Tissues have been built into artificial bilayers, making it possible

Numerical characterization of cell membranes.

Their properties must be studied individually under the strictly controlled conditions shown in the accompanying drawings.

Double-filled 1mm orifice

Lipid bilayer formation can be induced through a 1 mm opening between two fluid-filled chambers. (A) By placing test samples with different electrolyte concentrations in each chamber, the permeability of the bilayer to the electrolyte in the chambers can be measured electrically. Lutonen (B) fills the opening with a small amount of it to form a double layer

l i p ~ in a solvent such as hexane. When a bilayer forms, its interference color is initially gray (left). When the film adopts a more stable bilayer structure (right), the interference color changes to black. [From Kotyk and Janacek, 1970.1

The workings of membrane channels can be demonstrated directly in artificial lipid bilayer membranes, which are highly impermeable to even the smallest charged molecules (Spotlight 4-2). Addition of small amounts of channel proteins extracted from cell membranes resulted in a dramatic increase in ion permeability. This increase was measured as discrete pulses of current carried by the ions from one side of the membrane to the other, just as they are measured in biological membranes. These uniform flows are due to the sudden opening of individual channels.

Channels that allow thousands of ions to flow down their gradients and across the membrane per second. Studies of the permeability of cell membranes to other polar substances have given an estimated equivalent pore size of 0.7 nm - the equivalent pore size. Interpret the rate of diffusion through the membrane. Consequently, membrane channels are thought to have diameters less than 1.0 nm, which is close to the practical resolution limit of modern electron microscopy and fixation methods.

For example, rod-shaped molecules of the antibiotic nystatin applied to either side of an artificial or natural membrane aggregate into channels. These pores allow the passage of water, urea and chloride, all of which are smaller than 0.4 nm in diameter. Larger molecules cannot penetrate the channel. Cations are also excluded, presumably because of fixed positive sites along the channel walls. Incorporation of nystatin into artificial membranes resulted in a negligible (0.001–0.01%) increase in membrane area occupied by immobilized channels, but resulted in a 100,000-fold increase in membrane permeability to chloride ions. This means that very little membrane area needs to be dedicated to channels to accommodate the ion permeability of natural membranes. This conclusion is supported by the fact that the capacitance of cell membranes remains relatively constant, while larger changes in permeability occur during excitation of some membranes. (This phenomenon is discussed further in Chapter 5.) Facilitates transmembrane transport

The membrane is permeable to various polar molecules, such as sugars, amino acids, nucleotides, and certain cellular metabolites, which can only cross the lipid bilayer very slowly by diffusion. This is due to facilitated transport, in which molecules move across the membrane through the action of membrane transport proteins (see Figure 4-22B). Unlike active transport, discussed later, facilitative transport does not require energy in the form of ATP. Membrane transporters exist in multiple forms in all membrane types and are extremely selective in the types of molecules they transport. Carrier proteins that transport a single solute from one side of the membrane to the other are called monotransporters, while carrier proteins that transport one solute and a second solute simultaneously or sequentially are called coupled transporters. Coupled transporters that carry two solutes in the same direction are called symporters, while coupled transporters that carry solutes in opposite directions are called antiporters (Figure 4-23). These terms also apply to active transport systems.

combined transport aircraft

Forwarder

Extracellular

antiporter

Figure 4-23 Membrane carrier proteins can be configured as uniporters, symporters, or antiporters. Uniporters transport one type of ion across the membrane in one direction, while symporters transport two different ions in the same direction simultaneously. Antiporters also transport two ions, but cause ion exchange by moving the two ions across the membrane in opposite directions. ~

5 and e,

Facilitated Diffusion

rice

passive communication

External Glucose Concentration (mM)

kilometer

Figure 4-24 The kinetics of simple diffusion are different from those of supported (facilitated) diffusion. In this example of glucose movement, the rate of simple diffusion is always proportional to the glucose concentration. However, when the glucose carrier protein is saturated, the carrier-mediated glucose diffusion rate reaches a maximum (V,,,). When transport reaches half of its maximum value, the carrier's binding constant for glucose (K i ), which corresponds to the enzyme's K m for its solute substrate, is measured. [Adapted from Lodish et al., 1995.1

The existence of this transporter was originally inferred from kinetic studies of the transfer of molecules across membranes (Fig. 4-24). For some solutes, the measured influx rate will reach a plateau beyond which an increase in solute concentration will not result in a further increase. This suggests that osmosis must have a rate-limiting step. Experiments aimed at elucidating this permeation kinetics concluded that transport occurs through the formation of carrier-substrate complexes that are conceptually similar to enzyme-substrate complexes. Each carrier protein has a characteristic binding constant for its solute, which corresponds to the concentration of the solute at which the transport rate is half of its maximum value (see Figure 4-24). As with enzymatic reactions, solute binding can be blocked by specific competitive and noncompetitive inhibitors. The carrier and solute molecules form complexes transiently based on binding, stearin specificity, or both. The specificity of these transporters was first demonstrated in studies in which single-gene mutations abolished the bacteria's ability to transport certain sugars across cell membranes. Similar mutations are now found in many conditions, including human genetic disorders that affect the transport of certain solutes through the kidneys, gut or lungs. In cystic fibrosis, for example, a defect in the chloride ion transporter (CFTR) appears to be responsible for the fluid imbalance in the lungs.

Active transport All channel proteins and most carrier proteins allow solutes to pass through the membrane passively at no energetic cost (other than the initial cost of generation).

110

Physiological principles

...................................................

Potential energy in the form of different solute concentrations on either side of the membrane, as previously described). Concentration gradients determine the direction of passive transport. As diffusion continues, the solute concentrations in the two compartments approach equilibrium, at which point net diffusion no longer occurs. For charged molecules, transport is affected by both concentration gradients and transmembrane electrical (i.e., electrochemical) gradients. All plasma membranes have a potential difference, which is negative on the inside compared to the outside of the cell. This favors entry of positively charged ions and prevents entry of negatively charged ions. In this case, the passive process continues as above until the membrane is in equilibrium. The distribution of ions across cell membranes is only in true equilibrium in dead cells. All living cells continually expend chemical energy to maintain transmembrane concentrations of solutes away from equilibrium. This energy is usually provided in the form of ATP. Mechanisms that actively transport substances against a gradient are collectively referred to as diaphragm pumps. When the source of power to such pumps is cut off, active uphill transport ceases and passive diffusion regulates the distribution of substances. The concentrations of these substances are gradually distributed to an equilibrium state. The Na+/K+ pump is a paradigm of active transport

Displaying many features of active transport, this system maintains steep concentration gradients for Na+ and KC in the cell. The concentration of K+ inside the cell is about 10-20 times higher than outside the cell, while the opposite is true for Na+ (see Figure 4-13). These concentration differences are maintained by Na+/K+ pumps found in the plasma membrane of nearly all animal cells. The pump is an ATPase with binding sites for Na+ and ATP on its cytoplasmic surface and a binding site for K+ on its outer surface. At steady state, the number of Na+ ions pumped or transported out of the cell is equal to the number of Na+ ions coming in. Even with continuous exchange of Na+ (and other ionic species) across the cell membrane, the net Na+ flux is zero for any period of time. Two factors determine the size of any Na+ concentration gradient established between the inside of the cell and the outside of the cell: the rate at which Na+ is actively transported, and the rate at which Na+ leaks (i.e., passively diffuses) back into the cell. Of course, the membrane allows Na+ to flow back into the cell. Rate determines the rate at which the Na+ pump must work to maintain a given ratio of extracellular to intracellular Na+. There is evidence that increasing the intracellular Na+ concentration leads to an increase in the rate at which the pump expels Na+ (this may simply be a mass action effect due to the increased availability of intracellular Na+ to carrier molecules in the membrane). .

Some important features of active transport should be noted:

1. Transport may occur under significant concentration gradients. The most commonly studied membrane pump is the pump that transports Na+ from the inside of the cell to the outside of the cell under a 10:1 Naf concentration gradient. 2. Active transport systems usually exhibit a high degree of selectivity. For example, Na+ pumps cannot transport lithium ions, which have ionic properties very similar to sodium ions. 3. Requires ATP or other chemical energy sources. Active transport is halted by metabolic toxins that prevent ATP production. 4. Some diaphragm pumps exchange one molecule or ion on one side of the diaphragm with another molecule or ion on the other side. NaC/K+ antiporter is characterized by the active outward transport of Na+ and the simultaneous inward transport of K+ through the sodium potassium pump. This process involves the forced exchange of two potassium ions outside the cell for three sodium ions inside the cell (Figure 4-25). In the absence of external K+, Na+ ions, which would normally be exchanged for K+ ions, are no longer pumped out. 5. Some pumps do electrical work by producing a net charge flow. For example, the Naf/K+ exchange pump just mentioned produces a net outward movement of positive charge in the form of three Na+ exchanges for only two K+ per cycle. Ion pumps that produce a net movement of charge are considered rheological because they generate an electrical current across the membrane. If the current has a measurable effect on the transmembrane voltage, then it can also be said to be an electrogenic pump. 6. Active transport can be selectively inhibited by specific blockers. The cardiac glycoside ouabain, applied to the extracellular surface of the cell membrane, blocks the potassium-dependent active excretion of Na+ from the cell. This occurs by competition for the K+ binding sites of the Na+/K+ pump on the outer surface of the membrane. 7. The energy for active transport is released by the hydrolysis of ATP by enzymes (ATPases) present in the membrane. Active transport shows Michaelis-Menten kinetics and competitive inhibition of similar molecules. Both behaviors are characteristic of enzymatic reactions. Calcium-activated ATPase is associated with calcium pump membranes. Associated with the Na+/K+ pump are Na+ and K+-activated ATPases isolated from erythrocyte membranes and other tissues. These enzymes catalyze the hydrolysis of ATP to ADP and inorganic phosphate only in the presence of Na+ and K+ and bind the specific Na+ pump inhibitor ouabain. Facts about ouabain combined with memory

membrane,

channel,

and transport

111

................................................... ................... K+ and ouabain

electrochemical potassium gradient

1 ATP

Na+ binding site

AD P + Pi

Figure 4-25 Na+/KCATPase actively pumps Na+ from K+ into the cell against their respective electrochemical gradients. (A) For each ATP molecule that is directly hydrolyzed to drive transmembrane transport, three Na+ ions are pumped out and two K+ ions are pumped in. The specific pump inhibitors ouabain and K+ compete for the same site on the outside of the ATPase. (B) Schematic model of the Na+/K+ATPase showing the movement of Na+ and K+ through a single protein. Binding of Na+ (step 1) and subsequent phosphorylation of the ATPase cytoplasmic surface by ATP (step 2) induces a conformational change in the protein that results in the transmembrane transfer of Na+ (step 3). Na+ is transported out of the cell and K+ is bound (step 4). Subsequent dephosphorylation of the protein (step 5) causes the protein to return to its original conformation, followed by Kt transmembrane transfer (step 6) and release into the cytosol (step 7).

K+ beam board

Membrane and blockade of Na+/Kt pumps are evidence for the involvement of these ATPases in active Na+ and K+ transport. The function of Ka'/KyATPasc is thought to depend on a series of conformational changes in the transporter that allow the co-transport of K+ and Naf across the cell membrane (see Figure 4-25). The actual process of metabolically active transport occurs across the cell membrane and pumps molecules into or out of the cell. However, the cellular organization in the epithelial layer enables the active transport of substances from one side of the epithelial layer to the other because the cell surfaces on both sides are asymmetrical in their transport properties. One side of a cell may have a preference for importing material, while the other side tends to export it, resulting in transfer of material to the opposite side of the cell. This property enables the skin and bladder epithelium of amphibians, the gills of fish, the cornea of vertebrates, kidney tubules, intestines, and many other tissues to transport salt and other substances through the tissue.

Shipping type?

Ionic gradients as a source of cellular energy

Electrochemical gradients across biomembranes represent an important source of energy immediately available to cells. This energy can be used to drive passive or secondary active transport, as well as to store or transmit information along the surface of the cell membrane (see Chapter 5). The amount of free energy stored in the electrochemical gradient depends on the ratio of ion concentrations—or, more precisely, the ratio of chemical activity of the ionic species—on both sides of the membrane. Energy release occurs when ions are allowed to flow through the membrane along its gradient. Three important cellular processes utilize free energy

112

Physiological principles

...................................................

Biogradients: Generation of electrical signals, chemiosmotic energy transfer, and uphill transport of other molecules. Generation of Electrical Signals Electrochemical energy is stored in the membrane mainly in the form of Na+ and Ca2+ gradients. This electrical energy is released through "controlled" channels. These channels are normally closed, but in response to specific chemical or electrical signals, they switch to an open state in which they exhibit selective permeability to specific ions. These ions then flow passively across the membrane along their electrochemical gradient. Due to the charge it carries, when an ionic species crosses the membrane, it generates a current and changes the potential difference that exists across the membrane. This electrical activity is the functional basis of the nervous system (the topic of Chapter 5).

Intima. membrane

matrix

space

H+ translocation during electron transport

Phosphorus transporter

oh-

concentrate on

Cherniosmotic energy transduction The energy released from food metabolism eventually leads to the transfer of electrons down the respiratory chain in the mitochondria. This in turn releases their energy, which is stored in the electrochemical proton gradient on the inner mitochondrial membrane (see Chapter 3). This novel energy storage mechanism, independent of traditional high-energy chemical intermediates, baffled cell biologists for many years until Peter Mitchell proposed the chemiosmotic coupling hypothesis. Chemiosmosis refers to the direct link between chemistry ("chemistry") and transport processes ("osmosis"). Two ideas are at the heart of the chemiosmotic theory: oxidoreductases align within the inner membrane of the mitochondria, so that the electron transport system of the respiratory chain pumps hydrogen ions from within the mitochondrial matrix across the inner membrane into the intermembrane space (Fig. 4-26) .The inner mitochondrial membrane has low intrinsic permeability to H+, so this active pumping produces excess OH- in the mitochondrial matrix (and thus high pH) and excess H+ in the intermembrane space (and low pH ). Thus, the high-energy H+ gradient established across the inner membrane provides the free energy to remove HOH from ADP+PI as required to produce ATP:ADP

+ p

forward

Adenosine triphosphate

+hydrogen

+7,3 kcal-mol-I

This reaction also requires the targeting of the ATPase complex to the inner membrane of the mitochondria1 to utilize the transmembrane separation of H+ and OH. Enzymatically removed H+

inner mitochondrial membrane

Figure 4-26 The phosphate and ATP-ADP transport systems that generate ATP are located in the inner mitochondrial membrane. Phosphate transporters couple the uptake of HPOZ (inorganic phosphate) with the outward movement of OH anions. At the same time, the ATPADP antiporter exchanges incoming ADP3- for ATP4 exported from the matrix. The exported OH is linked to the translocated H+. This results in a net absorption of ADP 3 and outward absorption through respiration. HPO: in exchange for ATP4-. This process is driven by the outward translocation of H+ during electron transport. For every 4 H+ transferred outward, 3 are used to synthesize ATP molecules and 1 is used to export ATP in exchange for ADP and P1. [Adapted from Lodish et al., 1995.1

ADP is believed to be "siphoned" into the OH-rich interior of mitochondria to form HOH (Figure 4-27). The OH removed from the inorganic phosphate molecule comes from the mitochondria, where it reacts with excess H+ to form HOH. Thus, the H+/OH gradient provides the energy required to remove water during phosphorylation. After dehydration, a phosphate bond is formed at the active site of the ATPase without further energy input. ADP

+ p

Adenosine triphosphate

Chemiosmotic energy transduction, similar to that proposed by oxidative phosphorylation in mitochondria, has been proposed as a mechanism of energy transduction during photosynthesis in chloroplasts and photosynthetic bacteria. In addition, there is evidence that the Na+/K+ pump, which normally uses ATP to generate a Na+ gradient, can operate in reverse under special circumstances, such that movement of Na+ along its gradient causes the pump to extract ATP from ADP to synthesize P, .

inner mitochondrial membrane

H+ membrane space

H+

two

Mitochondrial 1-substrate OH-

time

Ministry of Health

H+ Figure 4-27 The second stage of Mitchell's chemiosmotic theory explains energy transduction in mitochondria. With the catalytic help of F, ATPase, ADP and P, H+ and OH- located in the inner mitochondrial membrane are removed by the high OH level in the mitochondrial matrix and the relatively high concentration of H+ in the intermembrane space, respectively. This process allows P to condense with ADP to form ATP.

coupled transport

The movement of some molecules down a concentration gradient is driven by the movement of another substance down its concentration gradient. Thus, the ubiquitous Na+ gradient serves to transport certain sugars and amino acids across the membrane via a symport mechanism and to drive Ca2+ out of the cell via an antiport mechanism. In the next section, this coupled transport will be considered in detail. Symporter A Symporter is a form of coupled active transport system that operates using energy stored in an ion gradient. An example is the transport of the amino acid alanine coupled to Na+ (Fig. 4-28). In the presence of Na+, amino acids are taken up by cells until the internal concentration is 7 to 10 times the external concentration. In the absence of Na+, the intracellular concentration of alanine only approaches the extracellular concentration. In both cases, the influx rates showed saturation kinetics, suggesting a carrier mechanism. The role of extracellular Na+ is to increase the activity of the alanine carrier. Increasing the intracellular Na+ concentration by blocking the Na+ pump with ouabain has the same effect as decreasing the extracellular Na+ concentration. Thus, it appears that the Na+ gradient is important for inward alanine transport, not just the presence of Na+ in the extracellular fluid. The transport of amino acids and sugars is associated with the inward escape of Na+ through common carriers. The carrier molecule must contain Na+ and organic subunits

l l Alanine concentration Figure 4-28 Cell uptake of amino acids (such as alanine) depends on Na+ concentration. (A) Intracellular concentration of alanine, an amino acid, over time with and without extracellular Na+. The dashed line indicates the extracellular concentration of alanine. (B) Lineweaver Burk diagram showing alanine influx with and without extracellular Na+. The abscissa is the reciprocal of the extracellular alanine concentration. The public intercept shows that the transport rate is independent of [Na+] at infinite alanine concentrations. [From Schultz and Curran, 1969.1

Strat molecule before it can be transported (Figure 4-29). The tendency of Na+ to diffuse along its concentration gradient drives this carrier system. Anything that reduces the Na+ concentration gradient (low extracellular Na+ or increased intracellular Na+) reduces the inward drive and thus the coupled transport of amino acids and sugars into the cell. Experimentally reversing the direction of the Na+ gradient can reverse the transport direction of these molecules. In this case, carrier-mediated Na+ transport also depends on the presence of amino acids and sugars. In the absence of amino acids and sugars, the ability of the co-carrier to transport Na+ is very weak, reducing the endoleak of Na+. Common carriers appear to be passively shuttled across the membrane without directly using metabolic energy. The coupled uphill transport of organic molecules gains energy from the downhill diffusion of Na+. However, the potential energy stored in the Na+ gradient ultimately comes from the metabolic energy driving the Na+ pump. Therefore, the Na+ concentration gradient is an intermediate form of energy that can exist

114

Physiological principles

................................................ .. .......... ..........Sodium+

glucose

Extracellular

carrier protein

Yeast Sol

Figure 4-29 Carbohydrates and amino acids can be transported by co-transport involving sodium. Carrier proteins must bind Na+ and organic substrates to transport ether. net transaction

Due to the Na+ gradient, the port faces inward, as indicated by the arrow. Note that glucose moves against the gradient

Used to power several energy-intensive processes in the membrane.

Resulting from the Na+ concentration gradient entering the cell from the lumen. The Na+/K+ pump, located in the membrane on the other side of the cell, facing the plasma and blood, maintains this gradient by removing Na+ from the cell.

The antiporter Na+ concentration gradient also plays a role in maintaining very low intracellular Ca2+ concentrations in certain cells through the Na+/Ca2+ antiporter system. In most, if not all cells, intracellular Ca2+ concentrations are orders of magnitude lower than extracellular concentrations, and many cellular functions are less regulated by changes in intracellular Ca2+ concentrations when extracellular Na+ is removed way lower because Ca2+ is expelled from the cell in exchange for invading Na+. However, competition for Ca2+ inside the cell is more successful than at the extracellular surface, resulting in a net efflux of Ca2+. Likewise, the immediate source of energy is the Na+ gradient, which ultimately depends on ATP-activated Na+ active transport. Ca2+ is also transported independently of the Na+ gradient by the ATP-fed Ca2+ pump, which is the major source of Ca2+ efflux under normal conditions. The Na+/H+ antiporter in the proximal tubule of the mammalian kidney is another example of reverse cotransport (see Chapter 14). Here, H+ is excreted from the cells lining the tubules into the urine containing the tubules, while Na+ is taken up into the cells at a 1:1 stoichiometry. That is, for every H+ expelled, one Na+ enters the cell. The benefits of this are (1) avoiding the energy cost of doing electrical work by exchanging two equivalent positive charges, and (2) enabling the kidneys to recycle Na+ from the urine and excrete excess protons. Unlike Na+/K+ pumps, Na+/H+ exchangers are designed in such a way that Na+ is delivered from the lumen into the cell. Also in contrast to the Na+/K+ pump, this mechanism is not an example of primary active transport where ATP is the immediate source of energy. In contrast, Na+/H+ exchangers are an example of secondary active transport, where the energy source is an electrochemical gradient of one or two exchanged ions. In this case, the energy driving the exchange

Membrane Selectivity The utility of cell membranes lies in their selectivity—their ability to only allow certain types of molecules to pass through. This selectivity is important because nonselective membranes cannot protect cell contents from unwanted chemicals. Each type of membrane transport system also exhibits selectivity for a given membrane of different transport systems. For example, when the Na+ in the saline solution used to bathe nerve cells is replaced by lithium ions, the Li+ readily passes through the Na+ channels that open when the nerve cell membrane is electrically excited. The other alkali metal cations K+, Rb+ and Cs+ are essentially unable to pass through these channels. On the other hand, Na+-pumping ATPases in the same membrane are highly specific for intracellular Na+ and are not activated by Li+. Therefore, lithium ions flowing through Na+ channels gradually accumulate in the battery until electrochemical equilibrium is reached. This is an example of electrolyte selectivity through a transport system rather than through membrane channels. We will now investigate how this selectivity of electrolytes and non-electrolytes can be achieved. Electrolyte selectivity

How do channels differentiate between different ions? While enzymes recognize substrates based on different shapes or chemical structures, membranes can distinguish between ions that are essentially the same shape and size. For example, Na+ and K+ are nearly identical in shape and size (K+ is slightly larger), but the membranes of resting neurons are about 30 times more permeable to K+ than to Na+. At first glance, we might conclude that the ions vary according to their hydrated size, and that K+ can freely flow through channels that are too small for Na+. Size could explain why K+ channels exclude Cs+ or Rb+ (Table 4-3) but not Na+.

................................... Table 4-3

channel

Ionic radius and hydration energy of alkali metal cations. Cat~on L

l o n ~ crad~us(A)

Hydrate free energy (kcal mol)

one)

-122

Sodium+

0 95

-98

Potassium+

1 33

- 80

Rubidium+

1 48

-75

CS+

1,69

-67 lipid bile

Especially considering that Na+ permeability can vary significantly. For example, during excitation of a nerve or muscle membrane, the Na+ permeability of the membrane increases about 300-fold to reach values about 10-fold higher than the K+ permeability at rest. If during excitation the membrane suddenly forms channels that allow the passage of Na+ ions simply because of their size, then the permeability of K+ through the same channels should increase at the same time because they are of comparable size. Since this increase does not occur, the selectivity of the membrane must be based on size rather than size. In fact, the estimated pore sizes of different membrane channels suggest that size alone cannot be a determinant of membrane selectivity. In addition to size, two interesting features appear to be important for membrane pore selectivity: ease of dehydration and interaction with pore charges. For ions to enter the pores, they must dissociate from the water molecules. Ease of dehydration appears to be an important factor in selectivity, especially when the charge in the pores is weak. Since large ions are more easily dehydrated than small ions (see Table 4-3), pores with less polar sites along the pore are more likely to accommodate large ions than small ions. In channels with highly charged sites, the interaction of dehydrated ions with these sites is more important in conferring specificity than the ease of dehydration. Thus, channels lined with predominantly positively charged residues selectively repel positively charged ions but allow negatively charged ions to pass through (Fig. 4-30). In this case, smaller ions can be closer to the poles and thus interact more than larger ions, increasing the effect. Selectivity of nonelectrolytes Almost all nonelectrolytes cross the membrane by dissolving and simply diffusing through the lipid bilayer. Because the relationship between permeability and partition coefficient K is essentially linear (see Figure 4-19), selectivity is entirely determined by the molecular properties that determine the partition coefficient. The few non-electrolytes that deviated from the linear relationship between partition coefficient and permeability all had larger than expected permeabilities. Some of these substances cross the membrane by carrier-mediated transport. choose,

Figure 4-30 Positive charges lining the membrane channel allow the passage of anions but hinder the diffusion of cations through the channel, as shown in a hypothetical cross-section through a highly simplified membrane channel.

Small molecules such as ethanol, methanol, and urea can pass through lipid layers and water-filled channels. All of these variant molecules are small and water-soluble, regardless of their relative solubility in water relative to lipids (i.e., their partition coefficients). It is important to note that the mechanisms for precisely controlling non-electrolyte transmembrane entry have not yet evolved, leaving cells vulnerable to the penetration of these molecules. Drugs applied to human skin, such as B. Anti-nausea drugs delivered through a skin patch behind the ear can enter the body through this route.

Endocytosis and exocytosis The above-mentioned polar small molecule transmembrane transport process cannot transport large molecules such as proteins, polynucleotides or polysaccharides. Nevertheless, cells manage to take up and sequester large molecules through different mechanisms than small solutes and ions. The transmembrane movement of macromolecules is achieved through the sequential formation and fusion of membrane-bound vesicles. The process by which substances enter cells is often referred to as endocytosis. The process is more specifically known as pinocytosis when liquids are ingested and pinocytosis when solids are ingested. The secretion of macromolecules from cells is called exocytosis. Both exocytosis and endocytosis involve the fusion of distinct regions of lipids

116

Physiological principles

.................................................. .....................................

The formation of the bilayer proceeds in at least two steps: the bilayers come closer together and then fuse. Both processes are believed to be controlled by specialized proteins. endocytic mechanism

and subsequent vesicle budding from the surface membrane. Once the coated vesicle enters the cytoplasm, it is believed to fuse with other organelles such as lysosomes and release its contents. Clathrin and receptors are recycled into the surface membrane.

The transfer of macromolecules across membranes by endocytosis requires specific control mechanisms. Receptor-mediated endocytosis depends on the presence of receptor molecules embedded in the cell membrane (Figure 4-31A). They bind specific ligand molecules or particles, including plasma proteins, hormones, viruses, toxins, immunoglobulins and certain other substances that cannot pass through membrane channels. Receptors are free to diffuse laterally within the membrane plane, but after ligand binding, receptor-ligand complexes tend to accumulate in pits of the membrane, called coated pits. Coating pits internalize the ligand. One theory of how this occurs is based on the formation of vesicles that pinch into the cytoplasm, as shown (Figure 4-31B). This is called a coated vesicle because a layer of the protein clathrin covers the cytoplasmic surface of the vesicle membrane. Clathrin organizes into pentagonal or hexagonal lattice arrays on membrane surfaces and appears to serve multiple functions. This includes the binding of receptor molecules occupied by the ligand

The release of chemicals from cell membranes through exocytosis plays a crucial role in the endocrine and nervous systems. For example, the presynaptic terminals of neurons contain many membrane-bound internal vesicles, approximately 50 nm in diameter, that contain neurotransmitters. These vesicles fuse with the surface membrane of the nerve terminal and release their contents outside the cell, a typical method of exocytosis. This activity is more likely to occur when a nerve impulse enters a terminal and is used to release synaptic transmitters that interact with the postsynaptic membrane. Hormone secretion also involves a similar mechanism. An important feature of exocytosis (like endocytosis) is that secreted or ingested macromolecules are tethered in vesicles and thus do not mix with macromolecules or organelles in the cell. Since vesicles can interact with

plasma membrane

Figure 4-31: Formation of coated vesicles during receptor-mediated endocytosis. (A) The process involves six main steps: (1) binding of ligand molecules to surface receptor molecules located in coated pores formed by clathrin molecules bound to the surface membrane; (2) invagination of the coated pits; (3) coated vesicles form; (4) coated vesicles fuse with existing vacuoles and exclude clathrin molecules; (5) fusion complexes are further processed according to their contents, while (6) clathrin and receptor molecules are recycled for reuse in the plasma membrane. (B) Electron micrographs of coated pits (top) and coated vesicles (bottom). These two stages recorded from a chicken oocyte show a dense envelope on the cytoplasmic surface of the cell membrane. It can be seen how the surface membrane pinches the vesicles. [Pearse, Part A, 1980; Part B, Bretscher, 1985.1

Exocytosis mechanism

Since they only have a specific membrane, they ensure a directed transfer of their contents into the cell. Once the vesicle membrane is integrated into the surface membrane during exocytosis, the released contents—hormones, neurotransmitters, and accessory molecules—diffuse into the interstitial space. Exocytosis requires a method to restore the relatively large number of secretory vesicle membranes that initially surround the expelled macromolecule. If this newly bound membrane is not removed, the surface area of the plasma membrane will continue to grow. However, endocytosis is believed to be responsible for the eventual repair of this excess membrane by reorganizing it into new secretory vesicles. Evidence for this membrane recycling by endocytosis comes from experiments in which electron-impermeable protein molecules such as horseradish peroxidase were introduced into the extracellular fluid and their movement into the cell was determined using electron microscopy . In these experiments, horseradish peroxidase was present intracellularly, but only in vesicles. Since the large size of the horseradish peroxidase molecule prevents its entry by passing directly through the biomembrane, it must be taken up in large quantities during the endocytic formation of vesicles from the plasma membrane into the cytoplasm. Calcium ions are responsible for the secretion of neurotransmitter substances by nerve cells and hormones by endocrine cells. Although the exact role of Ca2+ in triggering secretion is unknown, increased intracellular Ca2+ appears to increase the likelihood of exocytotic activity, possibly by allowing vesicles to fuse to the inner surface of the membrane. The membrane regulates exocytotic activity by regulating the accumulation of intracellular Ca2+. The rate of exocytosis increases because increased calcium influx allows for increased Ca2+ levels. The vesicle membrane itself can be actively involved in the first step leading to exocytosis. The secretory granules (or vesicles) of the adrenal medulla have been found to be rich in an unusual phospholipid, lyso-lecithin, which promotes membrane fusion and thus facilitates the fusion of the vesicle membrane with the surface membrane. Secretory granules must be in contact with the plasma membrane before the two membranes can fuse. Release of secretory products from secretory gland cells can be blocked by colchicine, an antimitotic agent that causes microtubule disassembly, or cytochalasin, an agent that disrupts microfilaments. This pharmacological evidence led to the hypothesis that microtubules or microfilaments are involved in the movement of secretory granules to sites of exocytic release on the inner surface membrane.

Connections Between Cells Animal cells are organized into cooperative units called tissues. In certain tissues, including epithelial cells, smooth muscle, cardiac muscle, central nervous tissue and

In many embryonic tissues, adjacent cells are joined by specialized adaptations at their junctions. These special surfaces fall into two broad categories: gap junctions and tight junctions. Gap junctions improve intercellular communication by connecting neighboring cells through tiny, water-filled channels, while tight junctions "sewn" cells involved in transepithelial transport into layers. gap junction

Gap junctions provide communication between cells by allowing inorganic ions and small water-soluble molecules to pass directly from the cytoplasm of one cell to the cytoplasm of another cell. These connections couple cells electrically and metabolically, which have important functional implications for the tissue. The distance between the two membranes of a gap junction is only 2 nm (Fig. 4-32). Two adjacent membranes each contain clusters of hexagonal arrays of six subunits that span the narrow space between the two membranes (Fig. 4-33A). The subunit arrays are approximately 5 nm in diameter and resemble miniature donuts, with hollow centers forming channels between the interiors of adjacent cells (Fig. 4-33B). Cellular continuity across gap junctions is assessed by injecting fluorescent dyes such as fluorescein (MW 332) and procion yellow (MW 500) into cells, which then diffuse into neighboring cells (Figure 4-34). This continuity of direct exchange of ions was confirmed by noting that when gap junctions are present, current flows easily from one cell directly to another. The intercellular channels in these compounds pass through molecules with a molecular weight of at least 500, so small molecules, such as ions,

gap junction

50nm

Figure 4-32 The gap junction distance (2 nm) between adjacent cells is at the lower limit of electron microscope resolution. Electron micrographs reveal gap junctions between the membranes of two adjacent mouse liver cells. [Contributed by D. Goodenough. ]

118

Physiological principles

.................................................. .....................................

Figure 4-33 Gap junctions allow the passage of molecules between adjacent cells. (A) Both membranes of adjacent coupled cells contain a series of hexagonal subunits, each connected to a matching subunit in the opposing membrane. The central channel penetrates the two subunits and provides a communication path between them

connected cells. (B) Detail of the canal complex. Molecules smaller than about 2 nm can pass through the channel between coupled cells. Molecules larger than 2 nm, such as proteins and nucleic acids, are too large to pass through the channel. [Part A, adapted from Staehelin, 1974; Part B based on Bretscher, 1985.1

Amino acids, sugars, and nucleotides can be easily exchanged between cells (see Figure 4-33B). This exchange of small molecules is responsible for gap junction-mediated intercellular communication. Gap junctions are unstable and close rapidly (within seconds) in response to any treatment that increases intracellular Ca2+ or H+ concentrations. Uncoupling of cells from their neighbors can be achieved by infusion of Ca2+ or H+

Access to coupled cells by lowering the temperature or using toxins that inhibit energy metabolism. The subsequent loss of electrical transmission between the cells confirmed the decoupling. Thus, gap junctions remain intact only if surface membrane metabolic activity maintains sufficiently low intracellular free Ca2+ and H+ concentrations. The mechanism by which the gap junction tube closes is unknown, but depending on the relative positions of the tube's six subunits, the tube appears to be either open or closed.

Fluorescein

The Brate nervous system allows the exchange of many types of cytoplasmic substances between neighboring cells. How does the combination of cells linked by gap junctions and free exchange of ions, amino acids, sugars and nucleotides change the concept of cells? What is the functional distinction between a single cell, a collection of cells connected by gap junctions, and tissues?

Figure 4-34 Gap junctions between coupled cells can be demonstrated by following the flow of fluorescent dye injected into a set of coupled epithelial cells. Subsequent diffusion of the dye into neighboring cells without loss to the extracellular space indicated the existence of a direct pathway from the cytoplasm of one cell to that of neighboring cells.

Tight junctions Tight junctions seal cells in layers of epithelial cells so that even small molecules cannot pass from one side of the layer to the other. Two opposing cell membranes are in close contact and completely enclose the extracellular space in between. Tight junctions are most commonly found in Ep-

restricted area

Figure 4-35 Adjacent epithelial cells, such as those lining the mammalian small intestine, are connected by intercellular junctions. In this reconstruction of cell-to-cell transitions, membranes and associated structures are drawn out of scale.

Zonula adherens (intermediate connection)

macular stick

intercellular space

Itelial tissue acts as a zonula occludens, a thin band of protein molecules that surrounds cells like a gasket. The occlusive zonules are in close contact with those of the surrounding cells, forming a water-tight seal that prevents the transfer of material from one side of the epithelium to the other by leakage from both sides of the cells (Fig. 4-35). As a whole, the zonules are conceptually like a continuous layer of rubber penetrated only by the ends of the epithelium. 3 Substances can pass through the ends of cells (transcellular pathway) but not around cells (paracellular pathway). In tissues such as the mammalian small intestine, gallbladder, and proximal tubules of the nephron, these zonules are not completely continuous and therefore not truly "dense". These tissues are so permeable that they do not generate transepithelial potential differences, although their cells contain ion pumps that generate transepithelial ion flux. Unlike gap junctions, tight junctions do not appear to have dedicated channels for cell-to-cell communication. Figure 4-35 shows two other types of cell junctions: zonular adhesions and desmosomes are mainly used to help structurally bind adjacent cells.

Epithelial Cell Transport Epithelial cell layers line animal body cavities and exposed surfaces and provide a barrier for the movement of water, solutes, and cells from one body cavity to another. Every organ or compartment in an animal has such a layer of surface cells. Some of these sheets only act as passive barriers between the cubicles and other cubicles

Do not preferentially transport dissolved substances and water. In other cases, they participate in active transport and perform regulatory functions. In animals, for example, osmoregulatory activity is carried out by active transport of epithelial cells in various specialized tissues and organs (see Chapter 15). Epithelial cells share several similarities. First, they occur on surfaces that separate the interior of an organism from its environment. This also includes lining up deeply invaginated surfaces, such as in the intestinal lumen, but still including the external space. Second, the cells that make up the outermost layer of the epithelium are usually connected by tight junctions, and in different epithelial cells, paracellular pathways between the serosal (inner) and mucosal (outer) sides of the epithelium are eliminated to varying degrees (Fig. 4 ) -36). .In epithelial cells such as capillary walls, leaky junctions allow water and solute molecules to pass through the epithelial layer by diffusion within intercellular channels. This diffusion through the paracellular pathway is independent of metabolically activated transport mechanisms, so such channels allow only passive movement of water and ions. Substances actively transported across epithelial cells must follow transcellular pathways involving the cell membrane. These substances must pass through one side of the cell membrane before passing through the other side of the cell. As discussed in the next section, the functional properties of epithelial cell surface membranes differ in some respects between their serous and mucosal surfaces. This asymmetry is important for epithelial active transport.

120

Physiological principles

.................................................. .....................................

electric meter

Lateral, luminal or mucosal side

I,/

variable current

transcellular pathway

acellular basement membrane paracellular pathway

Figure 4-36 Substances pass through the epithelial layer in two ways: Absolutely cellular and transcellularly. Active transport occurs only at the cell membrane, suggesting that all actively transported molecules follow a transcellular pathway

Active salt transport across the epithelium

The energy-intensive transport of ions from one side of epithelial cells to the other has been demonstrated in a range of epithelial tissues, including the skin and bladder of amphibians, the gills of fish and aquatic invertebrates, and the gut of insects and vertebrates tract, and the renal tubules and gallbladder of vertebrates. Much of the initial work on epithelial active transport was done on frog skin. In amphibians, the skin functions as an important osmoregulatory organ. Salt is actively transported from the mucosal side (i.e., the side facing the pond water) to the serous side of the skin to counteract salt leakage from the skin into the freshwater surrounding the frog. Similar absorption occurs in the gut. Due to the osmotic gradient between the hypotonic pond water and the more concentrated internal fluid, the water that penetrates the skin is excreted in the form of largely dilute urine that is hypotonic relative to body fluids (see Chapter 14) . German physiologist Ernst Huf and Danish physiologist Hans Ussing first used frog skin to study epithelial transport in the 1930s and 1940s. In their procedure, a few square centimeters of abdominal skin was removed from an anesthetized and decapitated frog and placed between the two halves of the Ussing chamber (Fig. 4-37). Dissection is very simple, as the frog's skin is mostly exposed in the extensive lymphatic space. Once the skin is lightly sandwiched between the two half-chambers, a test solution—such as frog Ringer's solution (a solution that mimics the ionic composition of frog plasma)—is introduced, with the frog skin acting as a barrier between the two. board compartment. The compartment facing the mucocutaneous side may be referred to as the outer compartment, and the compartment facing the serosa side as the inner compartment. Air is passed through the two solutions to keep them well oxygenated.

Between the two halves Figure 4-37 The Afrog skin separates the two halves of this Ussing chamber. Each half is filled with saline or other test solution. Adjust the current source so that the potential difference across the skin is zero. Under these conditions, the current flowing through the circuit (and thus the skin) corresponds to the rate of charge transferred across the skin by the active movement of sodium ions.

In 1947, Ussing reported the first experiments using two isotopes of the same ion to measure bidirectional fluxes (that is, these ion species move simultaneously and in opposite directions through epithelial cells). The Ringer's solution in the outer chamber is made of the isotope 12Na+ and the Ringer's solution in the inner chamber is made of 24Na+. The appearance of each of the two isotopes on the other side of the skin is tracked over time. The two isotopes were swapped in otherwise experiments of the same nature to rule out effects due to possible (but unlikely) differences in transmission rates inherent in the isotopes themselves. In all experiments, Na+ was found to show a net movement across the skin from the outer lumen to the inner lumen. The fact that Na+ flux is the result of active transport suggests that it occurs without a concentration gradient, or even against an electrochemical gradient. Inhibited by general metabolic inhibitors such as cyanide and iodoacetic acid and specific transport inhibitors such as ouabain. exhibit a strong temperature dependence. Shows saturation kinetics. Show chemical specificity. For example, Na+ is transported, but lithium ions, which have a very similar structure, are not. How is active movement of ions generated through cell layers in epithelial cells? Neighboring cells of the transport epithelium are tightly connected by tight junctions. For simplicity, assume that this proximity eliminates all extracellular channels

Membranes, Channels, and Transport 121 ................................................ ...

Diffusion of ions between the two sides of the epithelium. This will force all material passing through the epithelium to cross the epithelial cell membrane twice, first through the membrane on one side of the cell and then out through the membrane on the other side. Active transport through this pathway requires differentiation of the surface membrane of each epithelial cell such that the portion of the cell membrane facing the serous side of the epithelium differs in functional properties from the portion facing the mucosal side. Experiments on frog skin in the Ussing chamber provided several lines of evidence in support of the differentiated membrane hypothesis. For example,

Ouabain blocks Na+/K+ pumps and inhibits transepithelial sodium transport only when applied to the inner (serosa) side of epithelial cells. It is not effective on the outer (mucosal) side. In contrast, the drug amiloride is a potent passive carrier-facilitated transport inhibitor that prevents Na+ from passing through the skin only when applied externally. For active Na+ transport to occur, K+ must be present in solution inside the chamber, but not outside. The transport of Na+ shows saturation kinetics as a function of the Na+ concentration in the outer solution; it is not affected by the Na+ concentration in the inner solution.

These findings led to the epithelial Na+ transport model shown in Figure 4-38. According to this model, Na+/K+ exchange pumps are present in the membrane on the serous side of epithelial cells (as well as Na+/H+ and Na+/NH,+ exchange pumps in intact animals). This membrane behaves as is typical of many cell membranes: it pumps out Na+ in exchange for K+, thereby ensuring high intracellular K+ concentrations and low intracellular Na+ concentrations. K+ diffuses outward across the membrane on this side of the cell, producing a very negative resting potential. With mucous membranes, the situation must be different. The cell membrane on this side of the cell is relatively impermeable to K+. Furthermore, the net inward diffusion of Na+ through this membrane (apparently facilitated by carriers or channels in the membrane) displaces the Na+ pumped out of the cell from the serosal side. This model explains why Na+ pump inhibitors act only from the serosal side of epithelial cells and why changes in K+ concentration affect Na+ transport rates only on this side. Therefore, due to the functional asymmetry of the membranes on both sides, Na+ will flow from the mucosal side to the serosal side through the frog skin. The driving force is the usual Na+ active transport. on the cell membranes of all tissues. Frog skin as a model system for the general question of epithelial salt transport. Although the details

mucosal side (outward)

Epithelial cells of facial skin

Serous side (inward)

Figure 4-38 Transepithelial sodium transport depends on a combination of diffusion and active transport. In this model of isolated frog skin bathed in Ringer's solution, sodium passively diffuses into cells through a concentration gradient of the mucosal solution. K+ diffuses from the cell into the serous cavity when K+ is displaced by the influx of Na+. In the face of these leaks, Na+/K+ exchange pumps in the plasma membrane maintain high internal K+ and low internal Na+ concentrations. [From Koefoed-Johnsen and Using, 1958.1

Although differences may vary between each epithelial tissue type, the key features listed below are likely to be common to all transporting epithelia.

To varying degrees, tight junctions disrupt paracellular pathways. Therefore, transport via transcellular pathways is very important in epithelial trafficking. The mucosal and serosal fractions of cell membranes display functional differences and are asymmetric in pump activity and membrane permeability. Active transport of cations across epithelial cells is often accompanied by transport of anions in the same direction (either passively or actively) or by exchanging different cation species, thereby minimizing potential buildup. The opposite is true for actively transported anions. Epithelial transport is not limited to the pumping of Na+ and C1- ions. Various epithelial cells are known to transport H+, HCO3-, K+ and other ions.

water transport

To function properly, the right end of animal tissue always needs the right amount of water. This is accomplished by regulating water across the epithelial layers. Many epithelial cells absorb or excrete fluid. For example, the stomach secretes gastric juice, the choroid plexus secretes cerebrospinal fluid, the gallbladder and intestine transport water, and the kidney tubules of birds and mammals absorb water from glomerular filtrate. In some of these tissues, water passes through the epithelium without or against the osmotic gradient that exists between the bulk solution on either side of the epithelium. Some possible explanations for the uphill motion

122

Physiological principles

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . …… . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Water has been stated, but all of these assumptions can be grouped into one of two broad categories:

mucous membrane

Serosa

Water is transported by a specific water transport mechanism driven by metabolic energy. Water is transported secondary due to solute transport. The latter includes classical osmosis, where water undergoes net diffusion in one direction due to concentration gradients created by solute transport. So far, there is no convincing evidence that water is actively delivered by the main onboard water pump. The osmotic hypothesis of water transport was reinforced by Curran, who showed that an osmotic gradient generated by active salt transport from one subcompartment of the epithelium to another could theoretically result in a net flux of water across the epithelium (Fig. 4). -39). The biological relevance of the Curran model was later found in epithelial cells of the mammalian gallbladder. This finding led to the standing gradient hypothesis for split water transport proposed by Diamond and Bossert (1967). Figure 4-40 shows a simplified version of the schematic. Two anatomical features are very important. First, tight junctions near the luminal (mucosal) surface block extracellular pathways through the epithelium. Second, the transverse intercellular space, or intercellular space, between adjacent cells is bounded by tight junctions at the luminal ends and freely open at the basal ends. The static gradient hypothesis is based on active salt transport through the fraction of the epithelial cell membrane facing the intercellular space. The membranes bordering the transverse gap have been shown to be particularly active in pumping Na+ out of the cell. It is believed that as salt is transported from the cell into these long, narrow slits, the salt concentration creates an osmotic gradient

active solute

water follows

Figure 4-39. The Curran model for solute-dependent water transport depends on the active transport of solutes across a water-permeable membrane. A solute (such as Nat) is pumped from chamber I to chamber II through barrier A. Half-barrier B slows down the diffusion of solutes in compartment III, thereby keeping the osmotic pressure in compartment II high. A change in osmotic pressure in compartment II causes water to be pumped from I to II. At steady state, water and solutes diffuse into compartment III at the same rate as they emerge in compartment II. Compartment III is much larger than II, as evidenced by cracks in the compartment walls. [From Curran, 1965.1

Figure 4-40 Curran's model of water transport by solute has a biological counterpart. Compartments corresponding to those in Figure 4-39 are numbered I, II, and III. Salts that are actively transported into the intercellular space generate hyperosmotic pressure in the space. Water permeates into the crevices of cells, bulk solution flows through the freely permeable basement membrane and into the bulk fluid of the interstitium. Barriers A and B are similar to A and B in Figure 4-39. [From Diamond and Tormey, 1966.1

Between the extracellular spaces on either side of the tight junctions that connect epithelial cells. There may also be a permeability gradient within the fracture, with the salt concentration highest near the closed end of the fracture and decreasing towards the open end of the fracture, where it is in equilibrium with the main phase. Due to the high extracellular osmotic pressure in the fissures, water permeates into the fissures through "not so tight" tight junctions or possibly from inside the cell across the cell membrane into the intercellular space. The water leaving the cells must be replaced by the water penetrating into the cells at the mucosal surface. Water penetrating into the crevices gradually enters the main phase together with the solute. In this way, the salt is steadily and actively extruded through the cell surface membrane, resulting in an increased concentration in the narrow intercellular space. This in turn results in a steady permeation of water from one side of the epithelium to the other. Ultrastructural studies support the general applicability of the standing gradient mechanism of solute-coupled water transport, suggesting that the necessary cellular geometry—that is, the narrow intercellular space sealed at the ends of the lumen by tight junctions—is present in all water-transporting epithelia have been studied in cells. Also important in this context are the deep basolateral fissures and folds that are typical of epithelial trafficking (see Figure 4-40). These spaces are fixed and enlarged in the epithelium under conditions that affect water transport. In fixed epithelial cells without water transport, intercellular fissures largely disappeared.

Summary Lipid bilayer membranes are the basic structures for the formation of various organelles and surface membranes. Their roles include (1) cellular and subcellular compartmentalization, (2) maintenance of the intracellular environment using selective osmotic and transport mechanisms, and (3) regulation of intracellular metabolism by maintaining concentrations of intracellular enzyme cofactors and substrates , (4) metabolic activities performed by enzyme molecules present in ordered arrays in or on the membrane, (5) extracellular chemical signals sensed and transduced by surface receptor molecules and regulatory molecules in the membrane, ( 6) Propagation of information-carrying electrical signals regulating transport of substances within cell membranes or both, (7) Exocytosis and exocytosis of bulk materials. The basic structure of the membrane is a lipid bilayer in which the hydrophilic heads of the phospholipid molecules point outward and the lipophilic tails point inward at the center of the bilayer. The most widely accepted model of membrane structure proposes chimeras of globular proteins, including enzymes, that invade the bilayer. Due to the uneven distribution of solutes inside and outside the cell, water will flow from the area of lower osmotic pressure to the area of higher osmotic pressure after entering the cell. Osmotic pressure is the hydrostatic pressure required to balance osmotic flow (the movement of water through a semipermeable membrane) along a concentration gradient at equilibrium. The concept of tonicity describes the osmotic effect of a solution on a given tissue, while osmolarity describes the number of dissolved particles per volume of solvent and how the solution behaves in an ideal osmometer. Permeability is a measure of how easily a substance can pass through a membrane. Substances can pass through the membrane in a number of ways. Nonpolar molecules can readily diffuse through the lipid phase of the membrane. Water and some small polar molecules diffuse through transient water channels created by thermal motion. There is good evidence for the existence of immobilized channels that are more or less specific for particular ions and molecules. Transmembrane diffusion of certain substances can occur through carrier molecules that form complexes with the substance and facilitate its transmembrane transport by transporting the substance within the lipid phase of the membrane. In addition to these passive mechanisms, there are several active transport systems that transport substances across membranes. Active transport of substances occurs via carriers and requires metabolic energy, usually provided by ATP. It is responsible for moving substances across the membrane against a concentration gradient. The best-known active transport system is the sodium-potassium pump, which keeps the intracellular NaC concentration lower than the extracellular concentration. stored energy

As an extracellular-intracellular Na+ concentration gradient, it is used to drive upward movement of a range of other substances such as calcium ions, amino acids, and sugars by exchange diffusion and coupled transport. Naf and Kt gradients are also important for the generation of electrical signals such as nerve impulses. Another important function of active transport is to compensate for the tendency of certain substances (such as Na+) to enter the cell, causing an uncontrolled increase in osmotic pressure and subsequent cell swelling. Therefore, the continuous removal of NaC by the Na+/K+ pump is an important factor in cell volume control. Transepithelial transport depends on the asymmetry of permeability and pumping activity of the mucosal and serosal fractions of the epithelial cell membrane. On the serous side of the cell, ions are actively transported across the membrane against the electrochemical gradient; on the mucosal side, ions are transported across the membrane by diffusion or facilitated transport. Diffusion of ions back through the epithelial layers is slow because the spaces between cells are limited by tight junctions. Water is osmotically absorbed across some epithelial cells by a steady salt concentration gradient established by active salt transport within the epithelial cell interior and between the intercellular space. There is no evidence for true active water transport.

Review Questions What is the physiological function of membranes? What is the evidence for the existence of membranes as true physical barriers? What is the evidence for the lipid bilayer model of membranes? What is the evidence for globular protein chimeras anchored in membrane lipid bilayers? Explain what isotonic and isotonic mean. How can one solution be isotonic and not isotonic with another solution? What factors determine the permeability of a membrane to a particular electrolyte? Non-electrolyte? Describe the possible mechanism by which water and other small (less than 1 nm in diameter) polar molecules move across membranes? Why do non-polar substances diffuse more easily through membranes than polar substances? There is no convincing evidence of direct active water transport. Explain one way water moves through epithelial cells against a concentration gradient, ie. H. From a concentrated salt solution to a more dilute salt solution. How does facilitation differ from simple diffusion? What factors affect the rate at which facilitated ions are transported across membranes?

Physiological principles

.......... How does active transport differ from facilitated transport? Why can a sodium ion concentration gradient be considered a common cellular energy currency? By what parameters does the membrane discriminate between ions of the same charge? Explain the osmotic consequences of cellular metabolic intoxication. How do cells maintain a higher K+ concentration inside the cell than in the extracellular fluid? What are the morphological and functional differences between gap junctions and tight junctions? A given cell is 40 times more permeable to K+ and C1 than to all other ions. If the input-to-output ratio of K+ is 25, what is the approximate input-to-output ratio of C1-? Explain how substances are transported through cells because cell membranes can only transport substances in and out of the cell. Describe the first experiments demonstrating the active transport of Na+ across epithelial cells. What evidence is there that active transport of Na+ and K+ occurs only on the serosa of epithelial cells?

Recommended Reading Bretscher, M. S. 1985. cell membrane molecules. science. yes. 253:100–108. (This well-illustrated article explores the complex, heterogeneous nature of cell membranes.) Goodsell, D. S. 1991. inside living cells. Trends in Biochemistry. science. 16:203-206. (This article takes the reader on a tour of the amazing structures and processes in living cells.) Lodish, H. et al. 1995. Molecular Cell Biology. 3D output. New York: W.H. Freeman. (This comprehensive textbook describes many of the fundamental biochemical processes that occur in cells.) Singer, S.J., and G.L. Nicolson. 1972. A liquid mosaic model of cell membrane structure. Science 175720-731. (This is an original work proposing a fluid mosaic model of cell membrane structure.) Verkrnan, A. S. 1992. Water channels in the cell membrane. install. Physiology Pastor. 54:97-108. (This review focuses specifically on channels involved in transmembrane movement.) Yeagle, P.L. 1993. cell membrane. (This book discusses the morphology and molecular physiology of plasma and organelle membranes.)

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physical process

.................................... There are many specific adjustments at the interface between an animal and its environment to Nerve cells that receive information; other specialized nerve cells monitor the body. These cells have properties that make them particularly good at gathering information, are called sensory neurons, and are discussed in Chapter 7. A second important system involved in the coordination of processes in animals is the endocrine system. The cells of this system are grouped into organs called endocrine glands, and their signals are molecules that are released into the animal's bloodstream, a process called secretion. Other glands, the exocrine glands, produce chemicals that are secreted at specific sites. Chapter 8 discusses the properties of endocrine and exocrine glands. Signaling molecules from the endocrine glands, called hormones, can affect a wide range of different parts of the body simultaneously as they are transported throughout the body in the circulatory system. Hormones act on their target cells through specific receptor molecules, and the effects of hormones on their target cells depend on the nature of the receptor molecules and their effects on internal processes

target cells. Chapter 9 discusses the mechanisms that control hormone release and the mechanisms by which hormones act on their targets. The externally visible behavior of animals, as well as most activities in the body, depend on the contraction of muscle cells. In Chapter 10, we discussed the cellular properties of muscles that allow them to move the body or change the shape of internal organs. We then turn to how to coordinate muscle movement to produce efficient behavior. Finally, in Chapter 11, we'll look at some examples of how certain behaviors are actually triggered. Intensive experimental studies have elucidated the details of how specific behaviors are initiated and shaped by tracking information, from sensory input to processing in the nervous system, to what enables an animal to find food or a mate, or to flee a potential predator in front of the animal. Generation of motion. Part I1 focuses on the properties of individual cells that allow them to perform their individual tasks and work together efficiently and harmoniously. Another focus is on the mechanisms that coordinate cellular functions into larger-level tissues to enhance the overall health of animals.

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Linear Velocity The speed at which AP propagates depends in part on the forward distance that the flow created by the Na+ influx can travel at any given time. This distance depends on the relationship between the longitudinal resistance (inside the axon) and the transverse resistance (across the axonal membrane) experienced by the current flowing in the axon per unit length (Equation 6-2). Lateral resistance R ,,

a unit of length, i,

increases proportionally to the radius of the axon. As the radius increases, h increases. Since the propagation velocity depends on the rate of depolarization at any point in front of the AP, the membrane capacitance cannot be ignored. Note that the time constant (RmX C ),

The unit length of the axonal membrane remains constant

When the axon diameter changes, because the capacity (C,)

Increase

is proportional to the surface area, while the resistance (R,) is proportional to the increased membrane area. A-

The radius r of the axonal membrane is inversely proportional to

Increased h correlates with increased axon diameter

axon, since the area As of a cylinder per unit length is equal to 2rrl. The series resistance R per unit length

occurs without changing the time constant of the membrane. Therefore, an increase in diameter leads to larger externalities.

The axoplasm is inversely proportional to the cross-sectional area,

Membrane flow at distance x without membrane expansion

Axe, axon. because of a

r r 2 , the resistance R, is inversely proportional to

time constant and increased depolarization rate brings

Proportional to the square of the radius. next is for everyone

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As the radius increases, the drop in R will be greater than

Increase line speed.

R.,

Length constant A = dR,/(R,

+ R),

(Equation 6-2), so

Second

I electrode 1f

I electrode 27

injured part

time

Myelin is thick. This reduction in C means that less capacitive current is required to change Vm, allowing more charge to flow through the axon to depolarize the next segment. Changes in resistance and capacitance significantly increased the length constant A of the myelin-covered axon membrane, thereby increasing the efficiency of longitudinal current propagation. However, if this insulation completely covers the conducted current, this effect will not occur because the resistance will eventually decrease to zero with distance. In contrast, the length of myelinated segments is typically approximately 100 times the outer diameter of the axon, ranging from 200 μm to 2 mm. These segments are interrupted by short unmyelinated gaps called nodes of Ranvier, approximately 22 μm of which lie where excitable axons are exposed to extracellular fluid (Fig. 6-8B). The segments of the axon that lie beneath the myelin sheath are called internodes. During development, myelin is deposited around the axons of the vertebrate peripheral and central tracts by two types of glial cells:

These are the central nervous system, nerves, and peripheral glands. Between Ranvier nodes, the sheath is very close to the axonal membrane, almost excluding the extracellular space surrounding the axonal membrane. Furthermore, the intersegmental axon membrane was found to lack voltage-gated Na+ channels. Thus, when a local current flows in front of the AP, it exits the axon almost entirely through the Ranvier node. As mentioned previously, very little current is consumed when the membrane capacitance is discharged along the internode due to the low capacitance of the thick myelin sheath. APs initiated at one node depolarize the membrane with electrical tension at the next node; thus, APs in myelinated axons do not diffuse continuously along the axonal membrane as they do in unmyelinated nerve fibers. In contrast, APs are produced only in small regions of the membrane exposed at Ranvier nodes. The result is a salt line, a series of discontinuous regenerative depolarizations that occurs only at the Ranvier nodes, as shown in Figure 6-9. The speed of signal transmission is significantly increased due to the occurrence of electronic propagation of local circuit currents

Direction of propagation

1-1

I ^\

1-1 i\

--d * 4

T -

1-6

+-i\4

Faster than the internodal segment. Myelinated fibers conduct at speeds ranging from a few millimeters per second to over 100 m.s-l, while unmyelinated fibers of similar diameter conduct at fractions of a meter per second (see Table 6-1). The evolution of the salt line and the resulting increase in the rate of AP propagation may be critical for the successful coordination of large muscle activity in vertebrates. Myelination allows APs to move rapidly among the many axons within the compact nerve trunk. The importance of specialized segregation of myelin for coordinating neuronal information is especially evident in demyelinating diseases such as multiple sclerosis. In this disease, the myelin sheath on some axons is reduced or disappears, resulting in large differences in the rate of neuronal transmission between neurons, which significantly impairs the control of coordinated movements.

Information transfer between neurons: synapses All information processing in neurons relies on the transmission of signals from one neuron to another in structures called synapses. In an electrical synapse, the presynaptic neuron is electrically coupled to the postsynaptic neuron through specific proteins in the membrane. Transmission across electrical synapses is similar to signal transmission along a single axon. However, electrical synapses are relatively rare. Most signaling between neurons occurs at chemical synapses. At the chemical synapse, AP in the presynaptic neuron triggers the release of neurotransmitter molecules, which diffuse through a narrow space (approximately 20 nm wide) called the synaptic cleft, which separates the presynaptic and synaptic clefts. The membrane of the post-synaptic neuron separates. As recently as the 1970s, only a handful of chemicals were considered synaptic transmitters. They are all thought to work in a similar way, consistent with findings obtained in studies of synaptic transmission between motor neurons and the skeletal muscle they control, called the neuromuscular junction (NMJ). 50+ today

*-,

Figure 6-9 In beating conduction, actions are carried from one node to another. (A) Current propagates longitudinally between nodes. The large red arrows indicate Na+ influx through activated Na+ channels at the nodes. Smaller red arrows indicate Kt later efflux through activated K+ channels. Points (B) represent V values at a single point in time for each node^ shown in this section. ~t s it i.e., the core membrane is in the descending phase of AP; at position 2, the membrane is in the growing phase. At positions 3, 4 and 5, the membrane is in the following phases of AP

Neurotransmitters have been and are being discovered in a variety of studied animals, and we now know they work in very different ways. Initially, it was thought that neurotransmitters could only function by changing the voltage on the postsynaptic cell, that is, by hyperpolarizing or depolarizing the cell. However; neurotransmitters can also increase or decrease the number of ion channels introduced into the postsynaptic cell membrane, alter the excitability of postsynaptic cells by altering the rate at which ion channels open and close, or alter their sensitivity to activating signals. The discovery of these multiple modes of action has greatly improved our understanding of the role of synapses in neuronal communication. Synaptic transmission has long been a controversial topic. In the early 20th century, the great histologist Santiago Ramon y Cajal used light microscopy and a silver-based staining technique developed by the neuroanatomist Camillo Golgi to show that neurons are colored as discrete units. Despite such observations, many anatomists still believe that the nervous system is a continuous network rather than a collection of morphologically independent neurons. It wasn't until the development of electron microscopy in the 1940s that definitive evidence was found to support the idea that neurons were indeed separated from each other and that certain regions of neurons were specialized for intercellular communication. But in 1897, long before the ultrastructural basis of neuron-neuron interactions had been determined, the functional connection between two neurons was named synapse (from the Greek, meaning "synapse") by Sir Charles Sherrington. Buckle up"), which he is widely believed to be). Founder of modern neurophysiology. He concluded: "...the neuron itself is clearly a continuum from one end to the other, but the continuum of neuron-to-neuron encounters at synapses cannot be demonstrated. Another kind of propagation can occur " (Sherrington, 1906). Although Sherrington had no direct information on the microstructure or microphysiology of these specialized regions of interaction between excitable cells, he had extraordinary insights from which he skillfully leveraged resources

Experiments with spinal reflexes in animals, most of which are mammals, are designed. Among other things, he concluded that some synapses are excitatory, increasing the likelihood of AP appearing in postsynaptic cells, while others are inhibitory, reducing AP appearing in postsynaptic cells possibility. In this section, we first consider synaptic transmission through electrical synapses, which are analogous to signaling along axons. We then turn to the topic of chemical synapses, first studying transmission at the neuromuscular junction and then other types of chemical synapses that have only recently been discovered.

Synapse Structure and Function: Electrical Synapses Electrical synapses transmit information between cells by direct ionic coupling. At an electrical synapse, the plasma membranes of the presynaptic and postsynaptic cells are in close proximity and connected by protein structures called gap junctions (Figure 6-10A), allowing electrical current to flow directly from one cell to the other cells (Figure 6). -10B; see also Chapter 4). When current flows through a gap junction, the electrical signal in the presynaptic cell generates a similar, albeit somewhat attenuated, signal in the postsynaptic cell by simple electrical conduction across the junction (Figure 6-10C). Thus, at electrical synapses, information transmission is purely electrical, without the intervention of chemical transmitters, and a key characteristic of electrical synaptic transmission is its speed. As we will soon see, signaling between chemical synapses is always slower than purely electrical signaling. Electrical transport can be demonstrated experimentally by injecting current into the cell and measuring the effect in a connected cell (see Figure 6-10C). A subthreshold current pulse injected into cell A triggers a transient change in the membrane potential of that cell. If most of the current injected into battery A propagates to battery B through the gap junction, this will also cause a significant change in the V of battery B. The change in potential cell B transmembrane recording is always smaller than the change recorded in cell A due to the potential drop as the current crosses the gap junction. Gap junctions, where current flows from one cell to the other, are usually, but not always, resistively symmetrical to the current path—that is, when current flows in either direction between the two cells, it typically encounters same resistance. However, at some specialized synapses, current transmission between two coupled cells occurs smoothly in one direction but not in the other (Fig. 6-10D). This association is considered corrective. Transmission of APs across electrical synapses is fundamentally not different from propagation within cells, as both phenomena rely on passive propagation of local circuits beyond APs to depolarize and excite the area in front of them. Because of the safety factor——

AP (the ratio of the change in V1 during AP to the change in Vm required to bring the cell to threshold) is typically about 5, and the attenuation of the change in V1 from one cell to the next must be achieved if depolarization of the postsynaptic cell is to achieve Threshold and trigger impulse, it should not be greater than the safety factor. Thus, a single presynaptic action potential may not deliver sufficient local circuit current across the electrical synapse to initiate an action potential in the postsynaptic cell. This may be an evolutionary reason why electrical synapses are less common than chemical synapses. However, when fast signal transmission is important, the fact that electrical synapses transmit signals much faster than chemical synapses gives them a distinct advantage. Electrical transmission between excitable cells was first discovered in 1959 by Edwin J. Furshpan and David D. Potter while they were studying the nervous system of crayfish. They found that electrical synapses between the cancer's lateral giant nerve fibers and large motor axons have the unusual property of preferentially conducting current in one direction (see Figure 6-10D). Since their earlier work, electrical transmission between cells in the vertebrate central nervous system and in the vertebrate retina, between smooth muscle fibers, between cardiac fibers, between receptor cells and between axons has been discovered. The speed at which electricity flows through an electrical synapse. making this type of information transfer particularly effective at synchronizing electrical activity within populations of cells. It can also be used to rapidly transmit information at a series of intercellular junctions -- for example, in the giant nerve fiber of an earthworm, which consists of many segmental axons connected in series along the worm's body, and in the heart muscle of the earthworm's vertebrates The heart, where signals are passed between muscle cells. At certain synapses, transmission occurs both electrically and chemically. Such associative synapses were originally discovered in the ciliary ganglion cells of birds, they are also in circuits that control the escape response in fish, in the synapses some neurons form with interneurons in the lamprey spine, and in Motor synapses were found in spinal cord neurons of frogs. But intriguingly, associative synapses are an unusual phenomenon.

Synaptic structure and function: chemical synapses A common mode of synaptic transmission is the so-called fast chemical synaptic transmission, found at many synapses in the central nervous system and at the neuromuscular junction. (Although this transmission is called "fast," it's actually much slower than transmission across electrical synapses.) Neurons and muscles. The sequence of events at these nerve endings is

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Electricity flows between cells through gap junctions

Man

current source

for. 50 mV

current source

Ant ~ dromisch

25 mV

Figure 6-10 Presynaptic and postsynaptic cells have electrical continuity at the electrical synapse, allowing rapid transmission of signals between cells. (A) Electron micrograph of dense gap junctions in the membrane. Each "donut" in this photomicrograph is a protein complex that forms a pore that allows ions and small molecules to move between coupled cells. When cells are coupled via gap junctions, each cell's membrane contains these protein complexes that align with each other to form a continuous channel between the cytoplasmic compartments of the two cells. (B) Gap junctions connecting the presynaptic and postsynaptic membranes allow ionic currents to flow between cells. (C) In galvanically coupled cells, current injection into one cell triggers a change in the potential of both cells. Typically, electrical coupling at electrical synapses is symmetric, so injecting current into one of the cells changes V in both cells, although V changes more in the injected cell than in the cell to which it is coupled. However, there are exceptions, as shown in Section D. (D) Giant electrical synapses of crayfish illustrating the relationship between pre- and postsynaptic signals in electrical synapses with asymmetric electrical coupling. (1eft) AP in the presynaptic axon is transmitted across the electrical connection, causing the postsynaptic cell to threshold and trigger the AP with only a slight delay. This recording is a typical example of signal transmission across electrical synapses. (Right) However, in this asymmetric electrical synapse, APs in axons that are postsynaptic in the left trace do not cause significant changes in neurons that are presynaptic in the left trace change in potential. Injecting current pulses into one cell and then the other revealed that there was preferential current flow in one direction between the two neurons, an arrangement that is unusual in electrical synapses. [Part A courtesy of N. Gilula; Part D, adapted from Furshpan and Potter, 1959.1

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It is summarized in Figure 6-11. Briefly, as AP travels down the axon and diffuses to the axon terminal, neurotransmitter molecules stored in terminal membrane-enclosed spheres called synaptic vesicles are released into the synaptic cleft, containing Fluid-filled spatial separation of presynaptic and postsynaptic cells. Released neurotransmitter molecules bind to specific protein receptor molecules in the postsynaptic membrane, which include ligand-gated ion channels, as shown in Figures 6-11 Show. When a neurotransmitter molecule binds to a receptor protein, a brief flow of ions occurs across the membrane of the postsynaptic cell. This mechanism underlies synaptic transmission in all animals. Chemical transport and the presence of carrier substances were the subject of intense scientific debate in the first 60 years of the 20th century. Otto Loewi received the first direct evidence of a chemical transmitter substance in 1921. In his experiments, he isolated frog hearts with attached vagus nerves. When he electrically stimulated the vagus nerve, the heart rate slowed, but he also found that when the stimulated vagus nerve slowed the heartbeat, a substance was released into the surrounding saline solution that slowed the second frog's heartbeat ,also. Loewi's discovery led to the subsequent discovery that acetylcholine is a transmitter substance released by postganglionic neurons of the parasympathetic nervous system in response to vagal stimulation (see Chapter 11) and by motor neurons innervating vertebrate skeletal muscle.

sleep terminal

B AP Arrival Vessel

presynaptic axon

Fusion with terminal membranes, leading to transmitter exocytosis

For decades, it was assumed that all synaptic transmission occurs through mechanisms very similar to transmission at neuromuscular junctions. However, this perception has changed. It is now known that, in addition to fast chemical synapses, most species also have synapses affecting slow chemical synaptic transmission, in which communication between presynaptic and postsynaptic cells is slower than at the neuromuscular junction and via different The postsynaptic mechanism takes place. Although physiologists have assumed for decades that each synaptic terminal can contain only one neurotransmitter, it has recently been found that many neurons synthesize and release more than one transmitter substance; in such neurons, a substance can cause Fast transmission, while another substance can cause slow transmission. In many respects, slow synaptic transmission resembles fast chemical transmission (Figure 6-12). Neurotransmitter molecules are packaged into vesicles at the presynaptic terminal and released through AP-induced exocytosis. However, there are significant differences between these two synaptic mechanisms. In slow synaptic transmission, neurotransmitters are usually synthesized from one or more amino acids, called biogenic amines when they consist of a single amino acid, or neuropeptides when they consist of multiple amino acid residues. As the name suggests, postsynaptic responses start more slowly (hundreds of milliseconds) and can last longer (from seconds to hours). The vesicles used in the fast system are synthesized and packaged at the nerve terminal, while the vesicles used in the slow system are larger and then usually synthesized in the cell body

Transmitter binds to postsynaptic receptor protein; ion channel opens

D Transrnltter removed from column; melt film recycled

receptor protein

periosteum

Figure 6-11 In fast chemical synaptic transmission, the signals in the presynaptic and postsynaptic cells are connected by chemical neurotransmitters. The presynaptic and postsynaptic cells are not electrically coupled, there is no direct current between them. Ionic current flows through the postsynaptic membrane only when ligand-gated ion channels in the postsynaptic membrane open. (A) At rest, transmitter molecules are packaged into membrane-bound vesicles within axon terminals. (B) When AP enters a presynaptic junction, it causes voltage-gated Ca2+ channels in the membrane to open, allowing Ca2+ ions to flow into the junction. An increase in intracellular free Ca2+ causes synaptic vesicles to fuse with the presynaptic membrane and release neurotransmitters into the membrane

Exocytosis ruptures the synapse. (C) Neurotransmitter molecules, driven by their concentration gradient, diffuse across the synaptic cleft and bind to receptor proteins in the postsynaptic membrane, thereby opening ligand-gated ion channels. In this case, Na' flows into the presynaptic cell through open channels. The vesicle membrane remains fused with the terminal membrane, but moves to the sides of the terminal. (D) Transmitter molecules are removed from the cleft, the postsynaptic ion channel closes, and the membrane added to the presynaptic terminal when the synaptic vesicle fuses eventually returns to the terminal (small arrows) and can be reused for more vesicles.

Second

fast chemical transport

slow chemical transport

large vesicle

receptor

Figure 6-12 Fast and slow synaptic chemical transmission operates through different postsynaptic mechanisms (A). During rapid chemical transmission, neurotransmitters are synthesized and stored in signaling. These messengers are usually small molecules in small, transparent blood vessels. The blood vessels are located near the plasma membrane, and the messengers are released through exocytosis in the synapses, which are released through specialized slits in the membrane. Once released, these neurotransmitters act on Igand-gated channels. In postsynaptic channels, the transmitter is usually membrane (B). In slow synaptic transmission, larger molecules such as peptides come into play, resulting in many ammunition interactions

These transmitters are stored in large, prominent vesicles and released from sites that lack morphological specialization and are far from sites of rapid neurotransmitter release. In postsynaptic cells, these neurotransmitters often act indirectly through G protein-bound receptors to modify channels and other intracellular processes. A single neuron can affect both types of transmission, and a single neurotransmitter can affect postsynaptic neurons through ligand-gated channels and G protein-coupled receptors.

From there they are transported to nerve endings. Vesicles that mediate slow synaptic transmission can release their transmitter molecules at many sites in the presynaptic terminal, and often affect postsynaptic cells not through ligand-gated channels, but through intermediates called G proteins. Molecules alter the concentration of second messengers inside cells. Physiological and anatomical evidence suggest that individual presynaptic neurons may be involved in two types of neurotransmission. Release of neurotransmitters into the synaptic cleft is controlled by mechanisms common to both fast and slow synaptic transmission. When AP reaches the axon terminal, it activates voltage-gated Ca2+ channels in the axon terminal membrane, allowing Ca2+ to enter the terminal (see Figure 6-11B). The increased Ca2+ concentration within the terminal triggers exocytosis of the vesicles containing the transmitter material, allowing neurotransmitter molecules to enter the synaptic cleft where they diffuse from the presynaptic terminal. During fast synaptic transmission, synaptic vesicles containing neurotransmitters fuse with the plasma membrane at specific sites called active zones. After crossing the synaptic cleft, some neurotransmitter molecules bind to receptor molecules in the postsynaptic membrane. When transmitter molecules bind to these receptor molecules, they alter the flow of ions through channels connected to the receptor molecules so that permeating ions carry electrochemical gradient-driven postsynaptic currents. In slow transmission, neurotransmitters affect postsynaptic cells through G protein intermediates, thereby altering the activity of intracellular second messengers, which in turn affect ion channels or other intracellular processes (see Figure 6-12).

Postsynaptic currents generated by neurotransmitters result in changes in the postsynaptic cell membrane potential. A postsynaptic cell initiates an AP when the sum of potential changes caused by many such synaptic events is sufficient to exceed the postsynaptic cell's threshold potential. In fact, currents generated in a postsynaptic cell can increase or decrease the likelihood of an AP appearing in that cell; that is, synaptic effects can be either excitatory or inhibitory. What constitutes a synaptic signal is explored later in this chapter.

fast chemical synapse

Synaptic transmission is most extensively studied at the neuromuscular junction (also known as the motor junction or motor endplate) of vertebrate skeletal muscle, where acetylcholine has been shown to be a neurotransmitter. We will use the neuromuscular junction as the main example because it is well studied. This is a good example because the fast chemical synaptic transmission between neurons in the CNS is very similar to that in the CNS

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................................................... ................................... Neuromuscular junctions, although in many cases the transmitters are different.

Structural features The frog motor endplate (Fig. 6-13) includes structural specialization of presynaptic terminals, postsynaptic membranes, and associated Schwann cells. Axonal bifurcations at presynaptic motoneuron terminals, each branch approximately 2 µm in diameter sits in a longitudinal groove along the surface of the muscle fiber. Every 1:00 to 2:00 p.m., the muscular membrane lining the cavity is thrown into laterally connected folds. Immediately above these folds within the nerve terminal is the active zone—a mildly thickened lateral region in the presynaptic membrane where many synaptic vesicles gather. Vesicles are released along the active zone by exocytosis (Figure 6-14). There

The presynaptic terminal contains thousands of vesicles, each approximately 50 nm in diameter. For example, the nerve terminal branches innervating a single frog muscle fiber typically contain about 10" of total synaptic vesicles. When the vesicles fuse with the plasma membrane and release transmitter molecules into the releasing synaptic cleft, the transmitter molecules diffuse through their The concentration gradient reaches the postsynaptic membrane. The cleft itself is filled with mucopolysaccharides that "glue" together the presynaptic and postsynaptic membranes, both of which typically show some thickening at the synapse. Unlike the plasma membrane The fused end is incorporated into the end, which can be recycled (see Figure 6-11D). When acetylcholine (ACh) is released into the synaptic cleft, it can attach to the ACh-specific receptor molecule on the postsynaptic membrane bound to the endplate The ion channel that causes the reaction to be selective for Naf and K+ is momentarily opened.

Mvelinierte axon

nerve endings

, synaptic vesicle

, the synaptic cleft

Myelin B

Schwann cells

fold

link fold

'Figure 6-13 Structural specialization was found in pre- and postsynaptic cells of the frog neuromuscular junction. (A) Diagram illustrating the innervation pattern of frog muscles. Each neuron innervates several muscle fibers. (B) Diagram of the neuromuscular junction. Nerve endings lie in longitudinal grooves on the surface of muscle fibers. The depression contains transverse connecting folds extending into the muscle fibers. Above each connective fold of a neuron is an active zone rich in synaptic vesicles. Schwann cells cover the terminals. (C) Electron micrograph of the neuromuscular junction (compare part B). Muscle (see cells appearing below and contain striated muscle fibers, Chapter 10). The membrane of muscle fibers is thrown into many connecting folds. Axon terminals, visible in the upper longitudinal section, contain bundles of light-colored synaptic vesicles above a region where the presynaptic membrane is slightly thicker than usual and forms the active zone. Covering the active zone are denser granules and mitochondria. The synaptic cleft is filled with amorphous mucopolysaccharides. [Electron micrographs of McMahan et al., 1972.1

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Figure 6-14. The presynaptic terminal at the neuromuscular junction contains thousands of vesicles. Cross-section of a frozen-etched specimen of the electrical organs of an irradiated torpedo. Synaptic vesicles can be seen at the terminal. Two vesicles (black arrows) fuse to the presynaptic membrane after tissue fixation, illustrating the process of exocytosis. Calibration bar = 0.2 p m. [From Nickel and Potter, 1970.1

However, in the synaptic cleft, ACh is hydrolyzed by acetylcholinesterase (AChE). This enzyme can be detected histochemically and is present in junctional folds. Removing neurotransmitter molecules from the synaptic cleft is important because it limits the amount of time the neurotransmitter can be active. In cholinergic synapses, hydrolysis of ACh inactivates the transmitter and shuts down synaptic transmission. While many neurotransmitters are inactivated by enzymatic action, others are taken up by presynaptic terminals carried by specialized transport molecules. Synaptic Potential In 1942, Stephen W. Kuffler recorded the potential of individual fibers in frog muscles and found that depolarization was closely related to the motor endplate. These depolarizations occur in response to APs in motor neurons and precede APs generated in muscle cells. Potential changes for extracellular electrical recordings

The amplitude of the electrodes is greatest at the endplate and gradually decreases with distance, hence they are called endplate potentials (epps) or more generally postsynaptic potentials (psps). Kuffler correctly concluded that the arrival of APs at presynaptic terminals could lead to localized depolarization of the postsynaptic membrane, thereby initiating the propagation of APs through muscle. The development of glass capillary microelectrodes in the late 1940s made it possible to record potentials generated in smaller volumes of tissue, allowing more accurate identification of the source of endplate potentials. Many intracellular studies of synaptic transmission at the frog neuromuscular junction, mainly in the laboratory of Bernard Katz in the UK, have provided a very complete picture of the electrical events at this synapse. Like neurons, muscle fibers have a resting potential across the membrane (see Chapter 10). When muscle fibers are punctured by microelectrodes a few millimeters from the motor endplate, the microelectrodes record not only resting potentials, but also all or no muscle AP-innervated motor axons that appear a few milliseconds after the AP reaches the terminal end. Every time a motor axon is stimulated, muscle AP is recorded and the muscle fiber twitches. To understand the nature of neuromuscular synapses, Katz and others used drugs to interfere with their biochemical responses. For example, if South American curare (D-tubocurarine, Spotlight 6-3) is applied to the neuromuscular preparation of frogs, and the concentration of curare is gradually increased, "Allor-no" will suddenly appear at a certain concentration. Failure of muscle AP and consequent failure of muscle contraction. However, AP in the motor axon was not affected, nor was the ability of the muscle fiber to generate AP and contract when electrical stimulation was applied directly to the fiber. Since presynaptic and postsynaptic APs are not affected by venom, curare must directly interfere with synaptic transmission at the neuromuscular junction. Determining the mode of action of curare provides insight into the process of synaptic transmission. For example, in an experiment designed to demonstrate how curare affects synaptic transmission, microelectrodes were inserted very close to the endplate region (i.e., less than 0.1 mm away) (Figure 6-15), and curare was added to preparation. What do the following results tell us about the nature of synaptic transmission? As the concentration of curare was gradually increased, an increase in muscle AP was observed starting from a depolarization that was significantly slower in time and lower in magnitude than normal, and the initial slope of the rising phase was not as abrupt as in normal muscle AP ( See Figure 6-15B). The initial slow rise of V,,, is the endplate or postsynaptic potential. Increasing the concentration of curare further decreased the amplitude of the endplate potential.

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Uh-huh

muscle fiber

10 T ~ m e(ms)

Figure 6-15 Action potentials in muscle are generated from graded endplate potentials. (A) "All-or-nothing" muscle AP recorded in muscle fibers distal to the endplate region. (B) Recording close to the endplate showing AP originating from the endplate potential. (C) Endplate potentials can be recorded without superimposed APs if the magnitude of the endplate potentials can be reduced to such an extent that the muscle fibers no longer reach threshold. Curare, a drug that blocks receptor channels in postsynaptic membranes, provides a means to reduce the amplitude of endplate potentials. When the preparation was soaked in curare-containing saline, the membrane distal to the endplate (Lef?record) maintained its resting potential when the motoneuron fired, while the graded endplate potential near the endplate was recorded.

The synaptic potential must reach a minimum level (threshold potential) to trigger muscle AP. Thus, if an increase in curare concentration causes the amplitude of the endplate potential to drop below a threshold, abrupt breakdown of the AP will occur. These results suggest that curare disrupts synaptic transmission by blocking endplate potential in proportion to its concentration. When the concentration of curare was sufficient to reduce the amplitude of the synaptic potential in the muscle to just below threshold, the AP was eliminated and the synaptic potential became visible without superimposed AP (see Figure 6-15C). If the recording electrode is now repeatedly reinserted into the muscle fiber at increasingly greater distances from the motor endplate, the amplitude of the measured postsynaptic potential decreases exponentially with distance from the endplate (Fig. 6-16). Unlike AP, it is in

End plate distance (rnm)

Figure 6-16. The amplitude of the endplate potential decreases exponentially with distance from the motor endplate. (A) Endplate potentials were recorded using microelectrodes inserted sequentially into partially curarized frog muscle fibers at distances of 0.0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mm from the endplate. (B) Endplate potential recordings at each site. Each shot gives the distance in millimeters from the end plate. (C) Peak potential plot for each recording showing that the amplitude of the endplate potential decreases exponentially with distance from the endplate. [Adapted from Fatt and Katz, 1951. ]

Because it is regenerative, synaptic potential propagation is not impaired and thus decreases with distance. In experiments like this one, curare allows physiologists to distinguish elements of synaptic responses in vertebrate muscle fibers.

Synaptic Currents As discussed in Chapter 5, changes in the permeability of a membrane to one or more ions (that is, opening or closing populations of membrane channels through which those ions selectively pass) often shift the membrane potential to a new level. This change in V occurs because when

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Texln from larger snakes also inhibits the release of the transmitter, causing fatal paralysis. The evolution of these toxins created them

Pharmacological Substances for Synapse Research

They have a high potency to create a vacuum (i.e. very small quantities are required), so they must be handled with great care in the laboratory

postsynaptic receptor toxin

The discovery and application of natural toxins greatly facilitated the study of axonal and synaptic transmission

Agonists and antagonists of receptor subtypes have contributed significantly to defining the roles of these receptors

Animals, plants or fungi - selectively destroying or partially imitating certain steps in the dispersal process. toxin

neural processing. Gamma-aminobutyric acid (GABA), primarily

They have been found to interact with ion channels, receptors and enzymes important for nervous system function. Some commonly used drugs useful in the study of synaptic transmission are described here.

An inhibitory neurotransmitter that has been extensively studied using two chemicals, one an agonist and the other an antagonist of GABA. The agonist muscimol is obtained from the mushroom Amanita muscaria. It specifically activates CI-type GABA channels. Bicuculline, produced from the plant Dicentra cucullaria, is its competitive antagonist

Channel Toxins Several toxins are specific to certain types of ion channels.

channel. A large number of reagents exist for ACh receptors. mu

Tetrodotoxin (TTX) from puffer fish (Sphoeroides sp.) bound

Carine and other active ingredients, including pilocarpine, activate muscles.

to sites on voltage-gated Na+ channels and blocks Na+ current

Basic ACh receptors. Muscarinic ACh receptors in vertebrates

Transmembrane. Likewise, saxitoxin (STX) from Di-

Most commonly found in visceral tissues innervated by cholinergic axons of the parasympathetic nervous system. atropine

Noflagellates block voltage-gated Na+ channels, although by a slightly different mechanism. Potassium channels can be blocked by a variety of drugs. For example, tetraethylammonium (TEA) is a synthetic organic compound that blocks most types of

Potassium+

Forming channels inside or outside the membrane, 4-aminopyridine can block a variety of substances. Calcium channels can

(Belladonna) is a plant alkaloid that blocks muscarinic synaptic transmission. Another plant alkaloid, nicotine, and certain other compounds, such as carbachol, act as agonists at the nicotinic ACh receptor. D-tubocurarine is the active ingredient in curare, a South American poisonous curare made from cartilaginous plants.

Blocked by one of several W conotoxins from the piscivorous cone snail (Conus geographus). this

mentosum. The molecule blocks postsynaptic transmission

Different isoforms of this toxin block different classes of Ca2+ channels.

By competing with ACh for the ACh binding site of the nicotinic receptor. It competitively binds to these sites without opening it

Glutamate-gated channel toxins have proven invaluable in this regard

channels, thereby interfering with production

The difference between different channel types. Kainic Acid,

Postsynaptic currents. Likewise, a-bungarotoxin (a-BuTX) is an iso

from red algae (Digeneasimplex) is on the one hand a potent agonist

Extracted from the venom of the krait, a member of the cobra family.

Subtype of glutamate receptors. Quisqualic acid, extracted from the seeds of the Quisqualis indica plant, is the second most potent agonist

very happy. This protein molecule binds to nicotinic ACh receptors irreversibly and with very high specificity. by using radioactive

Selective for another subtype. An important antagonist is conatokine from Conic spirochetes, which is a noncompetitive antagonist of a third class of glutamate receptors called NMDA receptors.

By using activity-tagged a-BuTX, it is possible to determine the number of ACh receptors in the membrane and to isolate and purify the receptor protein.

receptors for N-methyl-D-aspartate, activating them.

Eserine (physostigmine) is an anticholinesterase agent; that is, it blocks the action of acetylcholinesterase. Use of this alkaloid has

Presynaptic Toxins Several toxins act on presynaptic terminals and inhibit transmitters

Allows physiologists to measure the amount of acetylcholine released by synapses by preventing rapid enzymatic destruction

release. I-bungarotoxin, derived from cobra venom, inhibits transmitter release by permeabilizing nerve endings. No-

Transmitter molecules. Fractionated administration enhances the postsynaptic potential at cholinergic synapses.

When the channels are open, they allow the flow of ions, which transfer charge from one side of the membrane to the other, which in turn causes a change in the measured transmembrane voltage. In chemical synaptic transmission, when a neurotransmitter binds to a receptor protein, postsynaptic channels in the membrane open, and synaptic current can then flow through these newly opened postsynaptic channels. (In some cases, the postsynaptic channel closes, reducing the flow

ions across the membrane. ) The direction and strength of the synaptic current, controlled by the magnitude of the conductance through the open channel as well as the electrochemical driving force and the charge of the permeating ions, determine the polarity and magnitude of the postsynaptic potential. Because neurotransmitter activation has selective ion permeability Sexual channels that confer specificity on synaptic signaling by allowing only certain ions

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................................................... ..................................... the manner in which specific neurotransmitters are traversed across the postsynaptic membrane. The ionic currents that generate the postsynaptic potential can be recorded by voltage clamping the postsynaptic membrane, which keeps the postsynaptic potential constant (see Spotlight 5-3). In neuromuscular anatomy, this procedure must be performed near the motor endplate (Figure 6-17A). The motor nerve (presynaptic element) is stimulated and at the same time V, the postsynaptic membrane is voltage clamped to a predetermined value. Following the release of transmitters from presynaptic nerve terminals, synaptic currents are rapidly generated as ions flow down the electrochemical gradient through open channels in the postsynaptic membrane (Figure 6-17B). The ions responsible for the transport of synaptic currents at specific synapses were identified experimentally in which the extracellular concentration of specific ions was varied and the effect on synaptic current generation was measured. These measurements suggest that depolarizing synaptic currents in vertebrate neurons

Stimulation line

stage

recording electrode

-V,,

electrode

undamped synaptic membrane

Second

1、

k ...,.Iy n a p t i c current .:=:i

Motor axon stimulated for 50 ms Figure 6-17 By applying voltage to the postsynaptic membrane, synaptic currents can be measured. (A) Muscle membrane tension clamp setup maintaining the postsynaptic potential constant while recording ionic currents flowing through neurotransmitter-opened channels across the postsynaptic membrane (see Spotlight 5-3). (6) The top trace shows the potential of the endplate when the neuron is stimulated and the muscle is not tensed. The lower trace shows the synaptic current when the muscle fiber is tension-clamped under the same conditions. Synaptic currents decay much faster than endplate potentials.

The muscle transition consists of an influx of Na+ partially offset by a concomitant smaller influx; an efflux of Kf. At this synapse, both Naf and K+ ions pass through the same postsynaptic ACh-activated channels, suggesting that these channels have broader ion selectivity than the highly selective voltage-gated Na+ and K+ channels that underlie APs (see Figure 5 -26). The duration of synaptic current is much shorter than that of synaptic potential (see Figure 6-17B). Acetylcholine-activated channels open only temporarily as the transmitter is rapidly removed from the cleft by enzymatic destruction at the neuromuscular junction. Thereafter, the channel closes and synaptic current stops flowing. The postsynaptic potential lasts longer than the synaptic current because its time course depends on the time constant of the membrane and the duration of the synaptic current. The reversal potential of any fast chemical synapse; one (or more) ionic species (or species) carries a current across the postsynaptic membrane, and the change in V caused by this current determines whether the synapse is excitatory or inhibitory. Measuring the properties of the synaptic current provided the experimenter with clues about the identity of the ion carrying the synaptic current. These measurements are made by injecting current into the postsynaptic cell, adjusting the membrane potential to different values, and then observing the sign and magnitude of the postsynaptic potential generated by the synaptic input (Fig. 6-18A and B). The magnitude and sign of the postsynaptic potential depend on the transmembrane voltage and the nature of the ions carrying the current. Remember that activation of a membrane channel that selects for a particular ion species X causes V to shift closer to the equilibrium potential Ex for that ion (see Chapter 5). Consider the synaptic experiment shown in Figure 6-18, in which only one ion, X, carries the synaptic current. If the membrane potential V,, moves towards the equilibrium potential Ex, the driving force on X(V, - Ex) decreases. When V,,, = Ex, even though the channel is open, no current flows through the membrane because there is no driving force for the ions. In the experiment, if V,,, is placed on the other side of Ex, the current will flow again, because V,,,-Ex will be non-zero again, but the sign has changed, indicating that the driving force is in the opposite direction. As a result, X flows through the open channel in the opposite direction to its previous flow, and the sign of the postsynaptic potential is opposite to that of its previous value (Fig. 6-18B and C). Since the direction of the ionic current and the sign of the postsynaptic potential are opposite when V,,, flows through Ex, Ex is called the reversal potential Ere. When a synaptic channel opens, the synaptic current causes V,,, to shift in the direction of the current, wherever V,,, is experimentally adjusted to before the synapse is activated. The reversal potential has been shown to be a useful property of synaptic currents because it can indicate which ions are carrying the current. actually earlier

184

P H Y S l O L O G l K A L process

................................... Figure 6-18 Synaptic reversal potential changes measured by Membrane potential and recorded postsynaptic potential. (A) Method for determining reversal potentials (E,,) at synapses. Inject continuous current into the postsynaptic cell using electrodes to set V. At , endplate potentials with different values were generated. Any value of V is achieved by stimulating the presynaptic nerve. Endplate potentials were recorded with a second electrode in the postsynaptic muscle fiber. (B) At, IS is set to a more negative value than the V-rium potential Ex, the equilibrium potential (lower panel) of the ion carrying the synaptic current. When V is equal to Ex, no synaptic current flows and the amplitude of the postsynaptic potential is zero even though the postsynaptic ion channel is open. When V is set to a value more positive than Ex, the driving force for ions carrying the synaptic current is in the opposite direction to that when V is more negative than Ex. This results in a larger V. When V is more positive than Ex, ions flow through the synaptic channel in the opposite direction to its direction, and the sign of the end-plate potential is reversed. (C) The results of such experiments can be plotted to show the amplitude of the endplate potential as a function of Vm value. The line connecting the experimental points crosses the abscissa at E,". In this case E,ev = 0 mV.

Reversal Potential -

disaster

t nerve stimulation

time

- 100 films

Potential (mV)

When introduced into patch recordings, measuring the reversal potential of the current is the primary method for distinguishing the ion species responsible for a particular postsynaptic potential, although it is not meaningful in itself. When a single ion carries a synaptic current, the reversal potential Ere can be calculated using the Nernst equation for that ion species (see Nernst equation in Chapter 1).

Section 5). However, when a synaptic channel is permeable to multiple ions, as in the case of acetylcholine channels, Ere depends on the concentration and relative permeability of all ions involved. Ere can be predicted using the Goldman equation (see Goldman's Equation in Chapter S) instead of the Nernst equation if the concentrations and permeability of the different ion species are known. Alternatively, if the current is carried by only two ion species, Ere can be calculated for both ion species using Ohm's law (focus 6-4). An example of this is the acetylcholine-activated channel at the neuromuscular junction in vertebrates. When these channels are open, they are permeable to both Na+ and Kt. In this case, the reversal potential of the current, Ere, lies between the equilibrium potentials of the two permeating ions (Figure 6-19). In Figure 6-19, V,,, are electronically fixed at different values, and then the synapse is activated. When V, is fixed at ENa (trace a), the driving force on Na+ is zero (V, - ENa = O), but there is a large driving force on K+ (V, - E,). Thus, the synaptic current at EN is entirely carried by the outflowing K+, making V more negative. On the other hand, when Vmis is set to E (trace e), there is no driving force on K+ but a large driving force on Na+. In this case, all the current through the ACh-activated channel is carried by the inflowing Na+ and Vm becomes correct. So somewhere between ENa and E there must be a value of V where the Na+ and K+ currents through the channel are equal and opposite to each other such that although both ions flow through the channel there is no net current (trace c ). The Vm value is the reversal potential of the ACh activation current. In core endplate channels, the conductivities of the two permeant ions, Na+ and K+, are approximately the same. Note that no matter how many channels are activated, the synaptic current V cannot exceed Elev. When V, reaches Ere, the net driving force of the penetrating ions decreases to zero and V,,, cannot change anymore. This sets the maximum change in E

communication between neurons

185

.................................................. ...................................K+

~n shutdown

Vm=EINS,

time ~ me

v m = Ere”

Figure 6-19. Synaptic currents at the vertebrate neuromuscular junction are carried by sodium and potassium ions (A). Sodium and potassium currents originate from EN at different membrane potentials through activated acetylcholine (ACh) channels, and ACh-activated channels are approximately equally permeable to Na' and K+, so the magnitude of I and I depends on the driving force for each ion . The relative magnitudes of Na' and K+ currents are indicated by the length of the arrows (B). The magnitude and time course of the net current through ACh-activated channels are plotted over time. For combined traffic, the net traffic through the channel is zero

-t AC h release

In Vm it can be produced by activation of synaptic channels (or indeed any ion channel). Reversal potentials are also of particular functional importance at synapses, as the relationship between EreV and firing thresholds in postsynaptic cells determines how synaptic events affect postsynaptic cells. Postsynaptic Excitation and Inhibition Any synaptic event that increases the likelihood of AP initiation in the postsynaptic cell is called an excitatory postsynaptic potential (epsp). Conversely, any synaptic event that reduces the likelihood of an AP in a postsynaptic cell is an inhibitory postsynaptic potential (ipsp). A synapse is excitatory when the reversal potential (Ere) of the synaptic current is more positive than the postsynaptic cell threshold (Figures 6-20A and 6-21A). If Ere is more negative than the threshold, the synapse is inhibitory. In fast chemical synapses, excitatory currents are usually conducted through channels that conduct Na+ or Ca2+. These channels may also be K+ permeable, like ACh channels at the vertebrate neuromuscular junction, but K+ currents themselves do not contribute to synaptic excitability (see Figure 6-19). Inhibitory synaptic currents are usually carried by K+ or C1-permeable channels. The reversal potential E ,, , of K+ or C 1 is usually close to V , i.e. more negative than the threshold

old. When E,,, is more negative than Vre for inhibitory channels in the postsynaptic cell, the synaptic current causes Vm to become more negative than V and the cell hyperpolarizes towards Ere (see Figure 6-20A). Hyperpolarizing synaptic currents contribute to depolarizing synaptic currents, thereby reducing the net amount of depolarization in the postsynaptic cell. Although all excitatory synapses generate depolarizing postsynaptic currents, inhibitory synapses are a special case. For example, if Ere" of the synaptic current is exactly equal to Vrest (Vm-Ere" = 0), no net synaptic current flows even though the postsynaptic channel is open. The net current is zero because the driving force of one or more ions that can pass through the channel is zero. In this case, Vm does not change when the synaptic channel is open. In some cases, Erevis was more positive than V, but more negative than Threshold (Fig. 6-21B). In this case, the postsynaptic potential depolarizes but remains inhibitory because it increases the difficulty of getting Vmup to threshold. In both special cases, synapses are inhibitory because activation of these channels counteracts simultaneous activation of excitatory channels (Fig. 6-21C). In fact, the opening of the inhibitory postsynaptic channel causes a "short circuit" of the excitatory current, since the positive charge delivered into the cell by the excitatory current can leave the cell

186

physical process

.................................................. .....................................

Figure 6-20 Synaptic currents can be excitatory or inhibitory (A) Transmembrane D Induces excitatory depolarization.

----------

- -

----

- - - - - - - -

a, 0

----- ------

- -

--- ----

It increases the postsynaptic potential because it increases ionic conductivity and creates a net inward current, adding a positive charge to the interior of the cell. Emitter D, for example, can increase permeability to Na+. Transmitter H produces an inhibitory hyperpolarizing synaptic potential because ~t increases the conductance of the ion, resulting in a net loss of positive cell charge. For example, emitter H can increase the permeability of K+ or CI-. (B) The direction of positive current flowing through the channel opened by emitter D is opposite to the direction of positive current flowing through the channel opened by emitter H

exist

sender

Sender H

Cells prevent positive charges from bringing Vm to threshold by inhibiting the channel. Note that specific transmitter substances neither stimulate nor inhibit. Russell; The properties of the channels opened by transmitters and the properties of the ions flowing through those channels determine how transmitters affect postsynaptic cells. For example, ACh is an excitatory transmitter at the neuromuscular junction in vertebrates, where it opens channels that allow Na+ and K+ to cross the postsynaptic membrane. In contrast, ACh is inhibitory at the terminals of parasympathetic neurons innervating the vertebrate heart, which affects Kc-selective channels. It follows from this description that if the ionic gradient across the postsynaptic membrane is altered, it can excite inhibitory transmitters. The experimental manipulation was performed on mammalian spinal cord neurons and snail neurons (Fig. 6-22). ACh increases postsynaptic g in some cochlear neurons. In a group of these cells (termed H cells or hyperpolarized cells), intracellular C1 concentrations are relatively low, resulting in E,,

More negative than V,,. When ACh acts on H cells, it opens C1 channels, allowing C1 to flow into the cell through its electrochemical gradient. The result is a shift from Vm to Ec, causing the cell to become hyperpolarized (see Figure 6-22A). When all extracellular C1 is replaced by SO:- which cannot pass through chloride channels, the application of ACh results in the efflux of C1- because it now has an outward electrochemical gradient. This outflow of negative charge results in depolarization and an increase in the frequency of the action potential (see Figure 6-22B). Thus, ACh is normally inhibitory to these cells but can induce excitation when the electrochemical gradient of C1- is reversed. In fact, there are other brain cells in this snail (called D cells or depolarized cells) that naturally maintain high intracellular C1 concentrations by actively accumulating Clk. In these cells, as in H cells, acetylcholine causes an increase in GC. In D cells, however, the net effect is depolarization, since the electrochemical gradient of C1 is normally outward. Thus, in this example, excitation and inhibition depend primarily on the properties of the ionic gradient rather than the properties of the signaling molecule.

Second

Action potential

+ C--

critical point

yes

- - - - - - E r etI,,,,

critical point

perhaps

187

communication between neurons

.....................................................

------

~nhib

inhibition

resting potential

exciting

- - - - - - - - - - Rehv, exciting

Figure 6-21 Excitatory and inhibitory synaptic signals interact in the postsynaptic cell. (A) Excitatory postsynaptic potentials generate action potentials when the postsynaptic potential raises the membrane potential V above threshold. (B) If Ere is more negative than the pulse generation threshold, the postsynaptic potential is inhibitory even though it depolarizes V. (C) Inhibitory transmitters (as in part B) can sufficiently reduce the depolarization produced by excitatory transmitters (as in part A) to prevent postsynaptic potentials from reaching threshold.

critical point

excitation and inhibition

Presynaptic inhibition Experiments in the 1960s on mammalian spinal neurons and on the neuromuscular junction of crustaceans revealed additional inhibitory mechanisms at certain synapses. In this mechanism, known as presynaptic inhibition, inhibitory transmitters are released from terminals that terminate at the presynaptic terminal of excitatory axons (Figure 6-23). In this case, the presynaptic terminal of the excitatory axon is itself a postsynaptic element. During presynaptic inhibition, the amount of transmitter released from excitatory terminals decreases, thereby reducing synaptic excitation of the postsynaptic cell to the excitatory neuron (see Figure 6-23B). In some cases, presynaptic inhibitory transmitters increase g or g at the presynaptic terminal of an excitatory axon, thereby reducing the amplitude of any APs entering the excitatory terminal, thereby reducing the amount

The transmitter is detached from the terminal. In other examples of presynaptic inhibition, inhibitory transmitters alter certain properties of Ca2+ channels in the presynaptic membrane, making them less responsive to depolarization. This is because the release of the transmitter molecule depends on Ca2+ access to the terminal (see next section). Chapter) Reduction of Ca2+ entry reduces transmitter release. Regardless of the mechanism, the net effect of presynaptic inhibition is that the postsynaptic cell receives less transmitter and therefore generates fewer postsynaptic potentials. Postsynaptic and presynaptic inhibition have very different effects on postsynaptic cells. Postsynaptic inhibition normally reduces the excitability of the postsynaptic cell, making it less responsive to all excitatory inputs. In contrast, presynaptic inhibition acts only on certain inputs to the cell, allowing them to retain

normal length

C Ringer

rice

5 seconds

Second

besides,? - Ringer

Then switch to clinger-free

ACh (about 5M)

Figure 6-22 Experimentally changing the ionic gradient across the postsynaptic cell membrane can change the sign of the synaptic superpolar region because CI introduces negative charges into the cell as it moves along its electrochemical gradient (B). When the extracellular CI ions are completely replaced by SO, the CI remains.

In batteries, the electrochemical gradient of CI is reversed. Reversal of the electrochemical gradient results in a reversal of the direction of the synaptic current. As a result, the postsynaptic potential depolarizes and the synapse becomes excited. Cell electrical activity before, during and after synaptic activation is shown on the right [adapted from Kerkut and Thomas, 1964 1].

If g in this equation is greater than gNa, then V must be e

Spotlight 6-4

, not EN, and vice versa. Solving Equation 4 to get closer to E

calculated from

V,,, = Ere" gives

REVERSAL POTENTIAL The value of the reversal potential of the ion current caused by

From Equation 5 it can be seen that Ere, not just al-

Stimuli or neurotransmitters depend on the relative

Common sum of EN and E, but will be in between

The conductance of the carrier ions and their

,, both, depending on the ratio gN,lgK. so if g

Equilibrium potential. Assuming that only Na+ and K+ conduct current in response to stimulation, the reversal potential can be related to the conductivity of these ions using Equation 5-10, where the values g and gNa denote the respective transition values.

and g, become

equal to each other (e.g., when endplate channels in frog muscle are activated by ACh), the membrane potential shifts toward an inversion potential midway between g,= and g,:

Seven variations of two conductivities.

1、

if =

g, X (Fm- EK)

(1)

9a,

(2)

(Wm-ENa)

At a reversal potential, I and INa must be equal and in opposite directions, regardless of relative conductivity, because the net current must be zero. So if V ,

yes

At the reversal potential, Ere,,

(3)

-IK=lNa

Substituting from Equations 1 and 2, at the reversal potential we

For frog muscle, E is about -100 mV and EN is about +60 mV. Therefore, we can predict that during synaptic activation in frog muscles, Ere = (-100

+ 60) = -20

mV Measured Reverse

Salt potential of frog neuromuscular junction currents,

- 10 mV, slightly more positive than that, maybe even slightly larger than g,.

lead to gram,

In summary, the reversal potential of membrane currents varies depending on the ionic species involved, the equilibrium potential of those ions, and the relative conductivities

have

Every ion involved in the current.

- gKWm -

i)=

gNa(Vm - ENa)

(4)

Usually in response to other input. Thus, presynaptic inhibition provides a mechanism for targeted and subtle control of synaptic efficacy (efficacy of a).

(a presynaptic impulse that produces a change in the postsynaptic potential) among the many synaptic connections to a given neuron.

suppression terminal

Muscle Figure 6-23 Neurons that cause inhibition at the crustacean neuromuscular junction also inhibit presynaptic excitatory motor neurons. (A) Morphological arrangement of excitatory and inhibitory terminals showing the location of inhibitory synapses producing presynaptic inhibition and the arrangement of experiments presented in Part B. (B) Intracellular recordings from muscle fibers innervated by excitatory and inhibitory motor neurons. (1) Stimulation of an excitatory axon (marked E in trace) produced an excitatory postsynaptic voltage of 2 mV.

-t20 rns potential (epsp). (2) Inhibitory axon stimulation (labeled I in the curve) produced approximately 0.2 mL of depolarizing inhibitory postsynaptic potential (ipsp)! (3) When inhibitory neurons are stimulated milliseconds after excitatory neurons, postsynaptic excitatory potentials are unaffected. (4) However, when inhibitory neurons are stimulated milliseconds before excitatory neurons, excitatory postsynaptic potentials are all but abolished. [Adapted from Dudel and Kuffler, 1961.1

Presynaptic release of neurotransmitters. Attributes

The presynaptic terminal determines the effectiveness of synaptic transmission, as the number of transmitted molecules affects the size of the postsynaptic transmission. Therefore, understanding transmitter release is critical to understanding synaptic transmission and its normal role in neuronal communication. In addition to its importance in physiology, the history of transmitter release experiments provides classic examples of scientific methods and experimental strategies. A particularly striking example is the demonstration by Sir Bernard Katz and his collaborators that neurotransmitters are usually released in small packets called quanta. Recent experiments have shown that synaptic release is closely related to other forms of exocytosis that cells, such as glandular cells, use to release chemicals (see Chapter 9). Preserving this mechanism enables experiments aimed at understanding the details of all cellular exocytosis. Quantum release of neurotransmitters

In their studies of neuromuscular transmission, Paul Fatt and Bernard Katz (1952) found spontaneous "miniature" depolarizations (a

indirect neurotransmission

Biogenic amines represent an important class of neurotransmitters (Figure 6-33) that act through second messengers and cause slow synaptic transmission. This class of neurotransmitters includes: epinephrine, norepinephrine, and dopamine, which are classified as catecholamines based on their chemical structure. Serotonin (serotonin or 5-HT), indoleamine, histamine, imidazole. These substances can be detected visually in individual neurons because they fluoresce under ultraviolet light after fixing the tissue with formaldehyde. They act as neurotransmitters in some invertebrate neurons and in the central and autonomic nervous systems of vertebrates (see Table 6-2). Norepinephrine (also known as norepinephrine) is the major excitatory messenger released by the postganglionic cells of the vertebrate sympathetic nervous system (see Chapter 11). It is also released by chromaffin cells of the vertebrate adrenal medulla (see Chapter 8). Chromaffin cells are embryologically derived from postganglionic neurons and secrete epinephrine (epinephrine) and norepinephrine. Epinephrine and norepinephrine are very similar in structure (see Figure 6-33) and have similar pharmacological effects. Neurons that use epinephrine or norepinephrine as transmitters are adrenergic neurons. Epinephrine has an excitatory effect on some synapses; for others, it has an inhibitory effect. Its action depends on the properties of the postsynaptic membrane. Norepinephrine is synthesized from the amino acid phenylalanine (~6-34A) and is inactivated in several ways. It is taken up into synaptic neurons, part of it is repackaged into synaptic vesicles for re-release, and part of it is inactivated

196

physical process

.................................................. .....................................

epinephrine (epinephrine)

Catecholamines

Norepinephrine (Norepinephrine)

dopamine

and mescaline

Serotonin (5-Hydroxytryptamine)

histamine

Figure 6-33 Several neurotransmitters are monoamines. These transmitters are all synthesized from individual amino acid molecules and are classified according to their molecular structure. Epinephrine, norepinephrine, and dopamine form a group of substances called catecholamines. Mescaline is a hallucinogen that shares structural features with catecholamines and appears to mediate its effects by interacting with catecholamine receptors in the central nervous system. Serotonin (serotonin) is an indoleamine, and histamine is an ~imidazole. These transmitters are found in the nervous system of vertebrates and many invertebrates.

monoamine oxidase. Furthermore, it is inactivated by methylation in the synaptic cleft (Fig. 6-34B). Several psychoactive drugs have molecular structures similar to biogenic amines, enabling them to act on synapses where these transmitters are used. e.g. Mescal

Bloodline (see Figure 6-33) is a psychoactive drug extracted from the cactus that induces hallucinations, apparently by interfering with the analogue norepinephrine in central nervous system synapses. Both amphetamine and cocaine work by interacting with adrenergic neurotransmission—amphetamine works by mimicking norepinephrine, and cocaine works by interfering with norepinephrine inactivation. In addition to the relatively small "classical" transmitter molecules, there is a growing number (now more than 40) of peptide molecules produced and released in the vertebrate central nervous system. Many of these molecules, or very similar analogs, are also found in the nervous system of invertebrates. Some of these peptides act as transmitters; others act as modulators affecting synaptic transmission. Interestingly, many of these neuropeptides are produced in many tissues, not just neurons. Thus, single molecular species can be released from enteroendocrine cells, autonomic neurons, various sensory neurons, and various parts of the central nervous system. In fact, some neuropeptides were first discovered in gut tissues and only later in neurons. The gastrointestinal hormones glucagon, gastrin, and cholecystokinin (see Chapter 15) are prime examples. It is unclear how many peptide neurotransmitters there are. We know that some neuropeptides are neurosecreted; that is, they are released into circulation and carried to their targets by the blood, rather than being released into the confined space of the synaptic cleft. Hypothalamic-pituitary hormone-releasing factor acts via neurosecretion (see Chapter 9). There is evidence that a neuropeptide can be released from some neurons as a transmitter, from others as a neurosecretory, and as a hormone from non-neuronal tissues. This variety of functions is not really new. It has long been known that norepinephrine (and its close relative epinephrine) act as a hormone when released from the adrenal medulla and as a messenger when released at synapses. Recently, however, to the astonishment of neurophysiologists, it became apparent that nerve endings can release a neuropeptide as a cotransmitter that also releases a more familiar transmitter such as acetylcholine, serotonin, or norepinephrine . Several combinations of classical transmitters and paired cotransmitters have been identified in the mammalian brain (Table 6-3). In 1931, Euler and John H. Gaddum in the United States discovered the first neuropeptide when they analyzed acetylcholine in rabbit brain and intestinal extracts. The extract stimulated contractions of the isolated gut similar to ACh, but the resulting contractions were not blocked by ACh antagonists. This observation led Euler and Gaddum to discover that the contractions were caused in response to a polypeptide the researchers dubbed substance I. Since then, substance P and a growing number of other neuropeptides have been found in various parts of the central, peripheral, and autonomic nervous systems.

communication between neurons

197

.................................................. ........................................

Phenylalanine

OH Tyrosine

neuronal cycle

Ammonia nitrogen,

OH 3,4-Dihydroxyphenylalanine (Dopa)

Figure 6-34 Epinephrine is synthesized from phenylalanine with dopamine and norepinephrine as intermediates and inactivated by reuptake or methylation. (A) Biosynthetic pathway leading to epinephrine. Each of the bottom three molecules is used as a neurotransmitter by some neurons. (B) Norepinephrine is synthesized from the amino acid phenylalanine through conversion to tyrosine and stored in synaptic vesicles. After release into the synaptic cleft, some norepinephrine is reabsorbed into the presynaptic cleft, and some norepinephrine is inactivated by methylation and carried away with the blood. Cytoplasmic norepinephrine is either repackaged in synaptic vessels or degraded by monoamine oxidase (MAO). [Part A adapted from E~duson, 1974, part B adapted from Mountcastle and Baldessar~n~, 1968]

OH 3,4-Dihydroxyphenethylamine (dopamine)

OH norepinephrine

oh adrenaline

The Vous system of vertebrates and the nervous system of many invertebrates. To study the localization of these molecules, researchers typically perform immunolabelling with fluorescent antibodies that recognize specific neuropeptides. This labeling can be detected with fluorescence microscopy in tissue sections and provides information about the distribution of specific peptides in the nervous system.

temperature. Some well-known neuropeptides are vasopressin (see Chapter 14), hypothalamic releasing hormone (see Chapter 9), and various gastric hormones (see Chapter 15). Unlike small neurotransmitters that can be synthesized at synaptic terminals, neuropeptides are produced in the cell body and transported along the axon to the terminal. Neuropeptides are often synthesized as part of larger proteins called propeptides, which may contain the sequence of many biologically active molecules. Certain enzymes cleave the propeptide into individual peptide molecules. This method of production can limit the amount of peptide neurotransmitters available at synapses compared to locally synthesized neurotransmitters. However, peptides are more potent than small neurotransmitters for three reasons. First, they bind to receptors at much lower concentrations than other neurotransmitters (approximately low concentrations for typical neurotransmitters), so very small amounts of neuropeptides can be effective. Second, they act through intracellular pathways that result in dramatic amplification. So even a small amount can have a big impact. Third, the mechanism by which they terminate their action is slower than other neurotransmitters, so they act on receptors for longer. Recent research has focused on two groups of naturally occurring neuropeptides, called endorphins and enkephalins, that reduce pain perception and induce euphoria, similar to exogenous opiates such as opium and heroin. Endorphin and enkephalin levels

198

physiological process

...................................................

Table 6-3 Examples of small and large neurotransmitter molecules found together in neurons. small neurotransmitter acetylcholine

Peptides in the same neurons CGRP Enkephalln Galanin GnRH Neurotens~n

Somatostatin Substance P VIP Dopamine

CCK Enke ~ Harlem

adrenaline

Enkephalin Y Neurotensin Substance P

Norepinephrine

Enkephalln Neuropeptide Y Neurotens~n Somatostatin~n

Vasopressin GABA

CCK Enkephalin Somatostatin Neuropeptide Y Substanz P VIP

Most data are based on immunocytochemistry, and the precise chemical nature of the immunoreactive peptides has not yet been determined. Abbreviations: CCK, cholecystokinin; CGRP, calcitonin-generating peptide; GnRH, gonadotropin-releasing hormone; VIP, vasoactive intestinal peptide. Source: Adapted from Hall, 1992

The molecule has been found to surge in the brain during eating, listening to pleasant music and other activities generally considered pleasurable. Because of their properties, and because these neuropeptides bind to the same receptors in the nervous system that opioids bind to, they are called endogenous opioids. Before the discovery of these endogenous neuropeptides, it was difficult to understand how alkaloids of plant origin, such as opium, morphine, and heroin, could exert such profound effects on the nervous system of animals. We now know that the surface membranes of many central neurons contain opioid receptors that normally bind to enkephalins and endorphins produced by the central nervous system. Only secondary, perhaps incidentally, they were combined with exogenous opioids. However, when opioid molecules bind to receptors, they cause such an intense pleasurable sensation that people have learned to use opioid narcotics to stimulate the receptors. However, there is a physiological problem associated with this intense high: repeated administration of exogenous opioids produces compensatory changes in neuronal metabolism, so that the removal of opioids puts the nervous system in a state

This can lead to extreme discomfort until the opioid is taken again. This metabolic dependence is called addiction. The drug naloxone, which is a competitive opioid blocker, has proven to be a useful tool for studying opioid receptors. Because naloxone interferes with the ability of opioids or opioid peptides to act on their target cells, researchers can use it to determine whether the response is mediated by opioid receptors. For example, naloxone has been found to block the analgesic effect that a placebo (an inert substance given to patients, which is suggested to reduce pain) can produce. Clearly, the very fact that a person believes that a drug or other treatment relieves pain can itself induce the release of endogenous opioid peptides, and this observation can provide the basis for the well-known "placebo effect" (i.e., almost all ) have uncovered. Likewise, naloxone rendered acupuncture ineffective in relieving pain, leading to the belief that the stimulation of acupuncture causes the release of natural opioid peptides within the central nervous system. Evidence suggests that the analgesic properties of endogenous opioids may depend on the ability of these neuropeptides to prevent the release of neurotransmitters from specific nerve endings. For example, pain perception may be reduced when neuropeptides disrupt synaptic transmission along afferent pathways carrying noxious stimulus information. In fact, enkephalins and endorphins are found in the dorsal horn of the vertebrate spinal cord as part of a pathway that transmits sensory input within the spinal cord.

Most vertebrate and invertebrate species have both fast and slow neurotransmission. What types of information processing are best supported by fast neurotransmission? What types of slow neurotransmission are there?

Postsynaptic Mechanisms Neurotransmitter molecules act through specific protein receptors in the postsynaptic cell membrane. The properties of the postsynaptic molecule thus form a critical link in the chain of events that begins when the action potential reaches the presynaptic neuron terminal and ends when the postsynaptic neuron response is completed. In this section, we examine in detail the two main classes of receptor molecules (fast and slow) that mediate chemical synaptic transmission, and what happens after binding of neurotransmitter molecules to these receptors.

communication between neurons

199

................................................... ................................ Receptors and channels in fast, direct neurotransmission

As we have seen, chemical transmitters work by directly changing the permeability of the postsynaptic membrane to specific ions. (In general, permeability increases.) This interaction requires two main events:

1. Transmitter molecules must bind to receptor molecules in the postsynaptic membrane. 2. When the transmitter molecule binds to the receptor, the closed ion channel must temporarily open (or, more rarely, the open channel close). The receptor site can be located on the same molecular complex that forms the channel, or on a different molecule than the one that makes up the channel. When a synaptic channel opens, a tiny stream of ions flows through the open channel. Many such single-channel currents often add up to generate macrosynaptic currents, generating postsynaptic potentials in response to the release of tens or even hundreds of thousands of transmitter molecules from presynaptic terminals. Much of what we know about these events has been revealed in studies of ACh-activated channels at the vertebrate neuromuscular junction. Acetylcholine Receptor Channels The number of protein molecules in postsynaptic channels is very small compared to other proteins in the membrane; therefore, the isolation, identification and characterization of these important proteins has been difficult. In earlier studies, physiologists used different pharmacological agents to differentiate receptor types, creating a pharmacological taxonomy of receptor types. As a result, various ion channels are named after substances that can alter the channel's activity. For example, there are two types of acetylcholine receptors. Nicotine is an alkaloid produced by some plants that mimics the action of ACh on channels at the neuromuscular junction in vertebrates, hence these ACh receptors (AChRs) are called nicotinic AChRs. Muscarine, a toxin isolated from certain fungi, activates another AChR found in target cells of parasympathetic neurons of the vertebrate autonomic nervous system. These AChRs are called muscarinic AChRs. Our understanding of nicotinic AChRs received a major boost when it was discovered that specialized organs in certain elasmobranchs and bony fishes contained extremely high densities of these receptors. The receptors are located on the side of the Elearoplax organ, which is made up of many flattened cells derived from embryonic muscle tissue that produce the very powerful electrical discharges that these species use to stun prey and send navigational signals. The unusually high density of nicotinic AChR in electroplax tissue makes nicotinic AChR the first ligand-gated channel to be chemically purified and electrically studied. Recently, its molecular structure

The gates are resolved; we even have pictures of the shape of the receptor channel when it opens. A second important tool for analyzing AChR is its sensitivity to α-bungarotoxin (aBuTX; see Spotlight 6-3), a component of cobra venom that irreversibly binds nicotinic AChR with high specificity sex. α-Bungarotoxin can be isotopically labeled and used to label AChR molecules for easy chemical isolation and purification. Physiological and biochemical studies have shown that AChR and ACh-activated postsynaptic channels are identical: the receptor site to which the ACh molecule binds is an integral part of the channel protein complex. Each nicotinic AChR is composed of five homologous subunits that combine to form a channel at the center of the complex (Figure 6-35). There are two identical a subunits, and three different subunits, labeled P, y, and 6. Each subunit is a glycoprotein with a molecular weight of approximately 55 kD, for a total molecular weight of approximately 275 kD for the entire complex. This molecular weight closely matched the size of the channel structures observed by electron microscopy when they penetrated the surface membrane. Channels protrude on either side of the membrane, with funnel-shaped openings protruding outward from the cell surface. Acetylcholine binds to AChR where the receptor molecule extends into the extracellular space. This location was first inferred because ACh injected into muscle cells near the endplate produced no electrical effect. Since then, experiments have shown that there are receptor sites on both A subunits. When both sites are occupied by a ligand molecule (i.e., ACh or other agonists that activate the channel, such as carbachol or nicotine), the channel is likely to switch from the closed state to the open state. The nature of this gating process has been most extensively studied in the neuromuscular junction of frog skeletal muscle. As previously described, postsynaptic ion channels at frog neuromuscular junctions leak K+ and Na+ when activated by ACh. The increased permeability allows inward current flow with a reversal potential of approximately -10 mV. Typically, these channels and their associated AChRs are restricted to the postsynaptic membrane in the region of the endplate. The density of ACh-activated channels in the postsynaptic membrane of the frog endplate is approximately lo4 per square micron. Although this high channel density has proven useful for analyzing the aggregate activity of many ACh channels, the activity of individual channels has long been poorly understood. Erwin Neher and Bert Sakmann (1976; see Figure 5-24) were awarded the Nobel Prize in 1992 for their invention of patch-clamp recording, which enabled the analysis of individual channels. Their work on single AChR channels depended on both of them developing the patch-clamp technique (see Chapter 2 and Figure 5-24) and finding a region of muscle with sufficiently sparse distribution of AChR channels that they could isolate and record channel. she

200

physical process

.................................................. .

Neurotransmitters

6 no

3 no

2 no

Figure 6-35. The nicotinic acetylcholine receptor at the neuromuscular junction is composed of five protein subunits that link together to form transmembrane channels. (A) Channels insert through the lipid bilayer and protrude into the extracellular space and cytoplasm. The CY subunit contains the site where the acetylcholine molecule binds to activate the channel. The entrance to the channel from outside the cell is a broad funnel that narrows and carries a net negative charge to the cytoplasm, thereby forming a selective filter—a region of the pores that controls which ions can easily enter. In this image, the interior of the channel is darker than the area around it. The subunit facing you is a Y subunit. (B) Top view of the five subunits connected to form the channel. These structural features are based on electron microscopy and X-ray diffraction analysis. [Adapted from Unwin, 1993.1

2 no

This sparse distribution was created by taking advantage of changes in frog skeletal muscles after the motor nerves that control the muscles are severed. When the muscle is denervated (i.e., it loses neuronal input—in the experiments, the axon was squeezed), the ACh-responsive membrane region gradually spreads across the surface. Initially, only the membranes in the endplate region are responsive, but eventually most or all membranes contain AChR and respond to ACh. (Normal inhibition of AChRs outside these connections is thought to depend on two factors: first, the trophic action of the motor neuron innervating each muscle fiber, and second, the electrical and contractile activity that occurs in the innervated muscle fiber. When the Das axis is The synapse can reinnervate the muscle, the junctional receptors disappear, and the sensitivity to ACh is again restricted to the endplate.) Neher and Sakmann exploited the widespread but sparsely distributed junctional ACh-activating channels in denervated frog muscles to gate canals using their Newly developed patch clamp method. The muscle membrane is applied to a hyperpolarizing potential (see Spotlight 5-3) to increase the driving force of the inward current. they used a

Micropipettes with smooth polished tips, 10 µm in diameter, are filled with Ringer's solution containing a low concentration of ACh or one of its agonists. They then moved the pipette to the surface of the muscle fiber and exposed all AChRs under the pipette tip to ACh. The pipette was connected to a highly sensitive, low-noise amplifier (Figure 6-36A), allowing them to record the current flowing in the extracellular pipette. When freely applied to the surface of denervated muscle fibers, the pipette captures minute (less than 5 x 10-12 A) and transient inward currents generated by the transient opening of ACh-activated channels (Figure 6-36B). With this experiment, Neher and Sakmann recorded for the first time the flow of electrical current through a single ion channel in a biological membrane. In fact, this experiment provides the first direct evidence that ionic currents flow across the membrane through discrete, closed channels rather than in any other way, such as through carrier molecules. The single-channel currents, first recorded by Neher and Sakmann in 1976, are roughly rectangular in shape; they snap on and off, all or nothing. This observation suggests that channels can only exist in one of two states: fully closed or fully open. In addition, uniform current

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. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . …… . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

r-c-

Pipette

voltage clamp

denervated muscle cells

Figure 6-36 Patch recording technique showing ion currents through a single AChR channel. (A) Muscle membrane held at a hyperpolarizing potential (-120 mV) by a voltage-clamp circuit significantly increases the driving force for ions through the acetylcholine receptor channel (AChR) while probing the muscle surface with a filled patch pipette Ringer's solution, containing 2 x lo-' M suberoylcholine (an ACh agonist). (B) A short inward current is recorded when the pipette tip is brought close to the membrane. In this experiment, the pipette records the flow of electrical current through an ion channel of a single AChR protein complex, which opens momentarily when an agonist molecule binds to the receptor site. [Adapted from Neher and Sakmann, 1976.1

If the electrochemical driving force is held constant, the current recorded by each nicotine ACh-activated channel is approximately the same as that recorded by all other nicotine ACh-activated channels. Ohm's law states that this result necessarily implies that all individual nicotine ACh channels have similar conductivities. If two or more channels in the patch to be recorded are turned on at overlapping times, the individual single-channel streams add linearly, yielding a current that is twice (or triple, etc.) the size of a single single-channel stream. These currents do not occur unless the pipette contains ACh or an agonist, and their frequency depends on the concentration of the transmitter or agonist in the pipette. Calculated from Ohm's law, the conductance of a single open nicotinic AChR channel is approximately 2 x 10-l1 S, usually expressed as 20 picoSiemens (20 x 10-l2 S); IE. H. The resistance of the channel is 5 x lolo a). Since the pioneering patch-clamp experiments of Neher and Sakmann, many ligand-gated postsynaptic ion channels have been intensively studied using this method of recording single-channel currents. statistical analysis of

These uniform flows suggest that the channel can fluctuate between several closed states and at least one open state. Binding of an agonist molecule to the receptor site of a closed channel greatly increases the likelihood that the channel will enter the open state and allow ions to flow through the channel briefly. The channel only opens for about 1 millisecond and then closes, although ACh is still bound to the receptor site. After a short time, the agonist molecule leaves the binding site and the channel remains closed until further ACh molecules bind (Fig. 6-37). Macroscopic currents and postsynaptic potentials recorded at synapses represent the sum of many such individual channel events in the postsynaptic membrane.

Other Ligand-Gated Channels Since the purification of ACh channel proteins from electroplax organs, several types of ligand-gated channels have been isolated and characterized from neurons, including glycine, GABA, and neuronal ACh receptors, all of which mediate lead to fast postsynaptic responses. These receptors share a common pentameric protein structure, each composed of two to four different types of subunits. As with muscle ACh channels, only one subunit binds a ligand. The remarkable homology between these different channel proteins makes it possible to characterize the diversity of subunit types and their distribution in neural tissues at the molecular level. Somewhat surprisingly, for each receptor type (ACh, glycine, and GABA), many different subunits assemble together in different combinations, resulting in receptors with slightly different properties. More; each receptor type is expressed in a unique characteristic pattern in the mammalian brain, suggesting that receptor subtype expression is regulated differently in different regions of the nervous system. Recognizing the large number of possible permutations, even within receptors that respond to a single neurotransmitter, has helped us understand how subtle the mechanisms that allow the brain to achieve its highly differentiated functional states are. Furthermore, comparison of the DNA sequences of ACh, GABA, and glycine receptors revealed that they are closely related, suggesting that all ligand-gated ion channels may have a common origin. DNA sequence analysis revealed that glutamate receptors belong to a separate family that bears little resemblance to nicotinic receptors. This family of receptors is of current interest because glutamate is the most common excitatory neurotransmitter in the mammalian central nervous system and because glutamate receptors are involved in changes in synaptic strength, which may It is the basis of learning and memory. Three types of fast-acting glutamate receptors have been identified, named for their sensitivity to specific agonists. Typical agonists for the three receptor classes are kainic acid, quisquitinic acid (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) and NMDA (N-methylaspartic acid acid). These receptor types are discussed in more detail later

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Figure 6-37 There are three functional states of nicotinic acetylcholine receptor channels. When acetylcholine (ACh) or an agonist molecule binds to the protein complex, ion channels through the receptor open. After about 1 millisecond, the ion channel closes, but the ACh molecule remains bound. When the ACh molecules remain bound, the channel can "blink" between closed and open states. The ACh molecules then separate and the channel closes and remains closed until two other ACh molecules bind.

, close ##b4

Joonen Canal

4----

Open

in the Mechanisms of Synapse Modification section of this chapter (see Long-Term Potentiation). Receptors in slow, indirect neurotransmission

A large class of receptors responds to a slow family of neurotransmitters. Interestingly, these receptors share many similarities with receptors that respond to light, odors, hormones and other extracellular messengers. Most of these receptors work by activating members of a group of proteins called G proteins, which attach to cell membranes and bind guanosine triphosphate (GTP). G proteins are composed of three subunits called a, P, and y. The G protein transmembrane signaling pathway was discovered and described by Alfred Gilman and Martin Rodbell, who studied its role in nonsteroidal hormone signaling (see Chapter 9 for a more comprehensive treatment of G proteins); they He was awarded the 1994 Nobel Prize for this work. When GTP binds to a G protein molecule, the protein is activated and catalyzes the hydrolysis of the bound GTP to GDP, thereby terminating its activation (Figure 6-38). This cycle of GTP binding and hydrolysis is facilitated when a membrane receptor molecule binds to its ligand, as the receptor-ligand complex catalyzes the release of GDP from the G protein, making the binding site available for new GTP molecules more quickly. Three separate proteins contribute to G protein-mediated synaptic transmission. Neurotransmitter receptor molecules span cell membranes, bind neurotransmitters on the extracellular side and catalyze G protein activation on the cytoplasmic side. Activated G proteins can regulate the activity of effector proteins, which can be ion channels or enzymes that control the concentration of second messengers in the cell, or both. We now know that more than 100 receptors operate through G proteins, signaling molecules that respond to a variety of external stimuli, from peptides to light and odors. G proteins themselves form a family of at least 20 different proteins. The combinatorial richness of

closure

This system provides another mechanism for generating subtle control within the nervous system. A well-studied example of indirect neurotransmission that regulates ion channels is found in atrial cells, the system used by Otto Loewi to first demonstrate more than 75 years ago that neurons can transmit information via chemical signals. Acetylcholine acts on muscarinic receptors in the heart to keep K+-selective channels open, prolonging hyperpolarization. Determining that this role of ACh depends on the G protein required several different types of experiments. Some experimental results are described here. Acetylcholine has been found to act on atrial cells only when GTP is present in the cells, and muscarinic activation of Kt channels is known to be blocked by pertussis toxin.

....-

Neurotransmitters

Extracellular

Joonen Canal

G protein complex

\Second Messenger, Andere Proteine

Ton

Other cellular functions Figure 6-38 Intracellular second messengers alter channel conductance at slow chemical synapses. G proteins are involved in signal transduction at many slow chemical synapses. In this type of synapse, the receptor protein spans the plasma membrane. The neurotransmitter molecule binds to the extracellular domain of the receptor and activates the G protein located on the cytoplasmic side of the membrane. Activated G proteins regulate the activity of other intracellular proteins that alter conductance directly or indirectly through ion channels in the membrane. Activated G proteins can also alter other cellular functions by altering metabolic pathways or the structure of the cytoskeleton.

Second

5 p.m. G, *Control

r 5 0 PMGa*

,A C h

atrial cell sheet

Extracellular

. muscarinic receptors

Figure 6-39 Muscarinic acetylcholine receptors in cardiomyocytes indirectly lead to opening of potassium channels in the membrane. (A) Experimental setup for measuring the effects of slow synaptic activation on guinea pig atrial cells. A nonhydrolyzable GTP analog, GTPyS, binds to G protein α-subunits to activate them, as well as activated α-subunits (activation status indicated). (indicated by an asterisk) was applied to the intracellular surface of a patch isolated from atrial cells. The net effect mimics the outcome of receptor-mediated activation of endogenous G proteins. (B) Typical recordings from experiments shown in part A. As the concentration of activated A subunits increased, K+ channels opened more frequently, resulting in more frequent current steps in single-channel recordings. (C) Schematic representation of muscarinic synaptic events in intact cells. When ACh binds to a muscarinic receptor, the G protein in the membrane is activated, and a subunit of the G protein binds to the K+ channel, opening it. [Data adapted from Covina et al., 1987.1

Thereby inactivating many G proteins. In a direct test of the hypothesis that ACh acts on these cells through the G protein, Codina and colleagues (1987) applied the G protein α-subunit activated by GTPyS, a nonhydrolyzable GTP analog, to cells derived from cardiomyocytes (Fig. 6 -39A). The results mimic the stable activation of G proteins in membranes. As the number of activated a-subunits in the bath solution increased, so did the number of open channels, which was reflected in an increase in the number of single-channel currents (Fig. 6-39B). Similar experiments have identified a variety of K+, Na+, and Ca2+ channels whose activity is also regulated by receptor-activated G protein a subunit. Neuromodulation

The postsynaptic response to fast synaptic transmitters is immediate, transient, and localized to specific sites on the postsynaptic cell. In contrast, slow synaptic transmission is not only slow and long-lasting, but also spatially widespread. In some cases, slow or indirect synaptic transmission can interact with fast synaptic transmission and modulate its effects. This interaction may affect only one postsynaptic neuron or more postsynaptic neurons.

Synaptic neurons, a phenomenon known as neuromodulation. Neuromodulation (or more specifically, modulation of synaptic transmission) is defined as a transient change in the effectiveness of a presynaptic neuron to control events in a postsynaptic neuron (i.e., synaptic availability). Neuromodulatory changes in synaptic effectiveness last from seconds to minutes. The time course distinguishes neuromodulation, described later in this chapter, from synaptic plasticity, whose effects are longer-lasting and even permanent. One of the best understood examples of neuromodulation and its role in normal synaptic excitation is found in frog sympathetic ganglion cells. The system is complex because these cells receive three different classes of synapses

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Inputs are mediated by two different neurotransmitters acting on three different types of receptors. Three distinct excitatory postsynaptic responses are produced: fast EPSP, slow EPSP, and late slow EPSP. A typical experimental setup is shown in Figure 6-40A. Both fast and slow excitatory postsynaptic potentials are generated by ACh

Second

quick eps

Slow Espp

4 m to L

late, slow, epsp

4 meters. large

1 minute

nerve stimulation

GnRH added 3m v L , 1 minute

Figure 6-40. Postsynaptic potentials with very different time courses can be recorded in bullfrog sympathetic ganglion cells. (A) The ganglia of the sympathetic chain are located on either side of the spinal cord (see Chapter 1), allowing the recording of responses from large B cells (a type of neuron in the ganglia) at the same time as the nerves innervating the ganglia. Anterior at the top of the figure. (B) Three different types of synaptic responses can be recorded in B cells: (I) fast excitatory postsynaptic potentials (latencies of 30-50 ms) when ACh activates nicotinic receptors in the postsynaptic membrane ; (2) slow EPSP (latency 30-60 ms) when ACh binds to muscarinic receptors in the postsynaptic membrane; (3) late, slow EPSP (latency > 100 ms), produced by cold-blooded spines Caused by a decapeptide messenger found in the animal brain, closely related to the release factor GnRH in the hypothalamus. When GnRH binds to postsynaptic receptors, it triggers B cell depolarization that lasts for several minutes. (Note the calibration bar below the trace.) (C) When exogenous GnRH is applied to B cells, the effect is identical in onset, magnitude, and duration to the late, slow EPSP in part B. [Adapted from Jan and Jan, 1982.1

Presynaptic nerve endings. The postsynaptic cell has both nicotinic receptors (fast responding) and muscarinic receptors (slow responding) in its membrane. In contrast, delayed, slow, excitatory postsynaptic potentials are generated by a neuropeptide very similar to mammalian gonadotropin-releasing hormone (GnRH—see Chapter 9), a hormone that Also released from presynaptic neurons, but not directly to postsynaptic neurons. The three postsynaptic potentials depolarize the postsynaptic cell to different extents at different times after stimulation and through distinct but not entirely independent mechanisms. When ACh binds to a nicotinic receptor, ion channels in the receptor complex open and Na+ and K+ can pass through, resulting in a rapid response (Figure 6-40B). This type of excitatory postsynaptic potential can be evoked by a single stimulus lasting only tens of milliseconds. Slow-moving excitatory postsynaptic potentials are generated when ACh binds to muscarinic receptors and can only be triggered after trains of APs reach the presynaptic site and release ACh. Muscarinic receptors act through G proteins, causing a type of K+ channel called an M channel to close (Figure 6-41A). When these K+ channels are closed, the steady-state Na+ influx is no longer balanced by K+ efflux and the cell is depolarized. Depolarization is small (only about 10 mV; see Figure 6-40B) because it depends on a small steady-state Na+ current. It does not generate APs in postsynaptic cells by itself, but it can dramatically alter cellular responses to fast synaptic signals, especially when interacting with delayed, slow excitatory postsynaptic potentials. Late, slow EPSP is caused by the release of another neurotransmitter, GnRH-like peptide, which closes the same M channels affected by muscarinic receptors through transmembrane receptors. Addition of exogenous GnRH to postsynaptic neurons produces the same type of response (see Figure 6-40C). The time course of the response to GnRH is slower than that of the muscarinic response; it begins 100 msec after stimulation and can last up to 40 min (see Figure 6-40B). The similarities and differences between these two slower responses are important for understanding how neuromodulation works in animals. To investigate the role of slow excitatory postsynaptic potentials in these sympathetic ganglion cells, the efficacy of injecting current into presynaptic cells before and during slow EPSP was assessed (Fig. 6-41B). Before the slow EPSP, a presynaptic stimulus elicited a single postsynaptic AP; during the slow EPSP, the same stimulus elicited a burst of APs. Apparently, slow EPSP alters the signaling in this synapse. Normally, KC currents through M channels are activated by membrane depolarization and tend to repolarize the cell by redirecting depolarizing currents entering through synaptic channels, thereby reducing the effectiveness of any excitatory postsynaptic potentials. When the M channel is held closed by ACh

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Acetylcholine

K+-Canal

third, fourth or fifth

seventh or eighth

Gonadotropins

Second

Gonadotropins

C cells

B cell

Acetylcholine

50 mV 9th and 10th current

Kaiten Ganga

Figure 6-41 Muscarinic acetylcholine receptors and GnRH receptors both depolarize postsynaptic cells by closing M-type potassium channels. (A) When acetylcholine (ACh) binds to a muscarinic receptor or when a GnRH-like neuropeptide binds to its receptor, the M-type channel closes, reducing transmembrane K+ currents and depolarizing the neuron. (B) Effects of fast excitatory postsynaptic potentials (epsps) on postsynaptic B cells before, during, and after slow epsps. During slow EPSP, the reduction of Kt currents through M channels increases B cell excitability and generates a series of action potentials in response

Quick epsp. (C) Cholinergic neurons of the seventh and eighth spinal nerves innervate C cells of the ninth and tenth sympathetic ganglia, while neurons of the third, fourth, and fifth nerves innervate only B cells in these ganglia . Only C cells received a terminal immune response to GnRH, but stimulation of the seventh and eighth spinal nerves resulted in a late, slow EPSP in both B and C cells, suggesting that GnRH was released from its release site on the C cell surface and diffused and The receptor activates B cells. [Part B, adapted from James and Adams, 1987; Part C, adapted from Jan and Jan, 1982.1

For muscarinic receptors, membrane repolarization induced by K+ currents is prevented and further excitation is enhanced. Late, slower excitatory postsynaptic potentials act similarly, but with longer latency and duration, and use M channels as the final common pathway. However, there is an additional twist, as the peptide neurotransmitter diffuses to neighboring neurons, and if the appropriate receptors are present, it can also affect it (Fig. 6-41C). Only some presynaptic neurons can release GnRH, but most postsynaptic cells appear to have GnRH receptors, strongly suggesting that neuromodulation is a normal part of these neuronal circuits. Taken together, these mechanisms can produce various postsynaptic effects following presynaptic transmitter release. Transient activity of presynaptic cells usually elicits only fast excitatory postsynaptic responses. Prolonged stimulation can additionally activate slow signaling pathways, which will effectively amplify the postsynaptic cell's response to its fast excitatory postsynaptic potential. Under greater stimuli, sluggish pathways additionally increase the potency of faster excitatory postsynaptic potentials and can also enhance responses in neighboring neurons (see Figure 6-41C), increasing the Neurotransmission potency of cells in which neurotransmission occurs. Neurons are connected to neurons that release GnRH. Furthermore, given the long time constant of the sluggish response, this modulation may persist for a long time.

In recent years, studies of the orogastric ganglion in crustaceans have demonstrated the extreme power of neuromodulatory mechanisms. This ganglion contains only 30-40 identified neurons, whose connections have been described in detail and whose output patterns are well known. When certain neuromodulatory substances, such as proctolin or cholekinin, are added to the saline solution bathing the orogastric ganglion, the properties of at least some of the membrane channels change dramatically, effectively rewiring the entire ganglion and creating Never-before-seen circuits and outputs in ganglia lack modulators. Neuromodulators thus offer a way to redesign neural circuits, allowing a range of neurons to interact in vastly different ways, even though their physical synapses Relationships remain the same.

Synaptic integration A single neuron is rarely responsible for behavior. Even the simplest behaviors require hundreds to thousands of neurons acting in a coordinated fashion. This coordination between neurons is called neuronal integration. In this sense, "integration" means "combining into a whole". At the level of individual neurons, integration consists of responding to incoming synaptic inputs by producing AP or not producing AP, and each neuron integrates different neuronal

Excitatory and inhibitory synaptic signals acting on it. The integration process is highly dependent on the passive electrical properties of the membrane located between the synapse and the spike initiation zone. Furthermore, the density and voltage sensitivity of Na+ and K+ channels determine the threshold and firing rate in response to a given synaptic depolarization. Much of what we know about neuronal integration comes from studies of large a-motor neurons in the vertebrate spinal cord (Fig. 6-42). These neurons innervate populations of skeletal muscle fibers at the neuromuscular junction. In vertebrates, these are the only neurons that directly form synapses with skeletal muscle fibers. As such, they play an extremely important role in generating overt behavior (see Chapter 10). Thousands of inhibitory and excitatory synaptic terminals touch each neuron's dendrites and cell body. motor neurons. The net effect of all synaptic activity is to control the frequency at which APs are produced in the cell. This firing rate (usually measured in counts per second) determines the strength of the contraction of the muscle fibers innervated by the motor neuron. All combined activity in neurons is focused on the production of APs (i.e. excitation) or their inhibition (i.e. inhibition). Because APs are the only events that can transmit information over distances greater than a few millimeters, only synaptic inputs that elicit APs in a motor neurons produce behavior. Any excitatory input that fails to threshold the motoneuron alone or by summing with other inputs is lost because no AP is generated in the postsynaptic cell and the signal fades away. In A motor neurons, APs are generated in the initial segment of the axon behind the axonal hillock (see Figure 5-2). This region is more sensitive to depolarization than the soma and dendrites (perhaps the membrane here has a higher density of Na+ channels), and therefore has a lower threshold for AP generation. To generate APs in cells, synaptic currents must be able to reach the threshold of the spike initiation zone membrane.

Stimulate

gray matter

a-motor neuron

How do the thousands of independent synaptic inputs to a motor neuron affect its activity? Synaptic currents propagate electronically from synapses on dendrites and soma. How much the current drops with distance is determined by the cable properties of the neuron (Figure 6-43), but in all cases synaptic potentials decrease as they propagate from their origin to the spike initiation area (see Figure 6-43) . Passive). Electrical signal propagation earlier in this chapter and Figure 6-16). Because the attenuation is distance-dependent, synaptic currents established at the ends of elongated dendrites decay more than currents near the spike initiation region, and thus farther synapses contribute less to the spiking activity of postsynaptic neurons. Small. Thus, the position of the synapse as well as the initial magnitude of the synaptic current can affect the degree of control of certain synapses. (Interestingly, recent evidence suggests that, at least in some neurons in the mammalian brain, there may be some Na+ channels in the dendritic membrane that amplify synaptic currents and prevent them from being conducted as rapidly as through electrotonus alone Attenuation.) In many cases, the density of inhibitory synapses is highest near the axonal mound, and these synapses are most effective at preventing excitatory synaptic currents from depolarizing the spike initiation region to threshold. We have learned many of these concepts from our experiments with Rana frogs. For example, in one such experiment, several sections of the spinal cord of an anesthetized frog were exposed by opening the spine. The microelectrode is then lowered into the anterior horn of gray matter and inserted into the soma of a single α-motor neuron. A small bundle of afferent axons excised from the dorsal root was placed on a silver wire stimulating electrode and stimulated some axons, causing excitation of alpha motor neurons and others causing inhibition of motor neurons. First, intracellular recording electrodes capture randomly occurring postsynaptic potentials. these characters

Figure 6-42 Neurons connected by synapses work together to process information. In the picture, the cell bodies of spinal motor neurons are located in the ventral spinal cord and are part of a synaptic reflex arc called the flexion reflex, in which the application of a noxious stimulus to the skin causes the motor neurons to fire to control flexion muscle. This pathway involves interneurons between sensory and motor neurons. Activation of motor neurons causes the muscle fibers they innervate to contract.

. . .

. . …… ,

Man

\ I

myelin

action potential threshold

.-m C

(I)

synaptic potential

Distance Figure 6-43 Each synaptic input decreases with distance as it moves toward the spike initiation area. Excitatory postsynaptic potentials emerging from dendrites propagate electrotonically and weaken with distance (upper panel). The density of Na' channels (red dots) in the membrane determines the threshold for AP generation (lower black trace). The synaptic potential decreases as it diffuses toward the axon, and no AP is generated until the current reaches dense Na+-. Channel distribution in seckill

The firing zone of the axonal hillock (or first Ranvier node) with the lowest firing threshold. The graph shows the relative values of threshold potential and synaptic potential across the membrane between the synapse and the spike initiation zone. Dashed lines show what the amplitude of the excitatory postsynaptic potential is if the AP is blocked.

Nals are elicited by synaptic inputs from motor neurons that are not experimentally controlled. Typically, activity consists of synaptic potentials with an amplitude of approximately 1 mV, similar to miniature endplate potentials recorded from muscle endplates (see Figure 6-24). Stimulation of single presynaptic neurons has been shown to cause these motor neurons to release as few as one to many transmission quanta in response to presynaptic APs. In this respect, excitatory synapses terminating on motor neurons differ quantitatively from those at the neuromuscular junction, where a single motor neuron terminal releases about 100 to 300 quanta over Responds to a single presynaptic impulse and generates an excitatory postsynaptic potential of 60 mV or higher. Transmitter delivered to a motor neuron from a single synaptic terminal only depolarizes the neuron by about 1 mV, much less than the amount required to shift the membrane potential to excitation levels. Although vertebrates are neuromuscular

Because junctions act as a single relay synapse and perform one-to-one transmissions (i.e. one postsynaptic impulse per presynaptic impulse), the motoneuron needs to activate more or less simultaneously the many excitatory synaptic inputs acting on it until the synaptic potential reaches the triggering threshold that triggers the postsynaptic AP. Thus, the decision to fire is in response to the accumulation of presynaptic input, and while each small synaptic current is ineffective by itself, single-ended activity can significantly contribute to neuronal integrative behavior. This rather democratic behavior prevents motoneurons from being activated by trivial inputs or spontaneous activity in input neurons. More importantly, it provides the ability to integrate inputs from different excitatory and inhibitory sources to determine when and how many APs a neuron generates. As the intensity of stimulating current applied to dorsal root presynaptic axons increases, more and more

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More excitatory axons become active; that is, they are attracted to increased stimuli. When these neurons fire simultaneously, the total amount of transmitter delivered to the motor neuron increases, generating more individual synaptic currents that add up to generate a larger excitatory postsynaptic potential. When inputs from multiple individual synapses are added simultaneously to alter Vm in a postsynaptic neuron, this process is called spatial summation. When all synaptic inputs are excited, summed synaptic inputs lead to more depolarization (Figure 6-44). When an inhibitory transmitter is released at the same time as an excitatory transmitter, it also produces a synaptic current that adds to the excitatory current (Figure 6-45). Opening inhibitory synaptic channels can short-circuit the depolarizing currents carried by Na+ ions through excitatory channels; that is, if a depolarizing positive charge is transported into the cell by Na+ ions, when K+ ions migrate out or Cl- ions When migrating through inhibitory synaptic channels, part of the charge is immediately removed from the cell. Activation of inhibitory synapses. Causes depolarization of the spike initiation region and reduces the likelihood of AP generation. If a second postsynaptic potential is fired a short time after the first, it can complement or "piggyback" the first, even though both synaptic events are elicited by the same presynaptic neuron. This effect is called

synaptic current

time

Figure 6-44 Synaptic inputs from multiple presynaptic neurons produce a spatial summation on a motor neuron. Two excitatory synaptic currents from two independent neurons a and b arise at spatially separated synapses. The lower right trace shows synaptic potentials recorded in the spike onset region when each input acted alone and when both inputs were activated simultaneously, resulting in a spatial sum. Spatial summation of currents from many synapses is required in order to generate synaptic potentials above the motor neuron threshold. If too few excitatory inputs are activated simultaneously, V in the spike onset area will not reach threshold and no AP will be generated.

synaptic current

inhibitory synaptic current

-\-/I,

[

- - - - - - - A and B

Figure 6-45 Sum of excitatory and inhibitory synaptic currents. Stimulation of separate presynaptic pathways results in excitatory (a) and inhibitory (b) synaptic currents. The bottom right trace shows synaptic potentials recorded from the spike onset area when a or b were stimulated individually and then together, demonstrating the summative effect. Dotted arrows indicate partial reduction of excitatory synaptic currents through patent inhibitory channels.

Time summation (Figure 6-46). The shorter the interval between two consecutive synaptic potentials, the higher the second response is to the first, and the larger the postsynaptic potential can become. Further summation can be achieved when additional stimuli arrive in rapid succession, with a third synaptic potential lying on top of the second, and so on. Under natural conditions, the sum of space and time often occurs simultaneously. For example, when different excitatory synapses on a motor neuron are active at slightly different times, the effects add up spatially and temporally. The spatial and temporal summation of synaptic potentials depends on the passive electrical properties of neurons. Spatial superposition occurs because synaptic currents occurring at the same time but at different synapses each propagate away from the synapse electrotonically (see Figure 6-43), so their effects on V can be added to the spike initiation region . Temporal summation, on the other hand, does not require summation of synaptic currents and can be done even if the individual currents do not overlap (see Figure 6-46C), because membrane electrical time constants are long relative to the time course of synaptic currents. The first synaptic current brings a positive charge into the cell, thereby partially releasing the negative resting potential of the cell membrane. The positive charge brought into the neuron by the synaptic current then slowly escapes (via the resistor K+).

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Saint ~ Sturm

S y n a p t ~ ccurrent

Second

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But by chance, V, changes. Every now and then, the excitatory inputs add up to trigger APs in the neurons, which in turn cause APs and twitches in every muscle fiber that the neurons innervate. The result of this activity is a persistent background of low tension in the skeletal muscle, as first a motor neuron and then another fire, causing the muscle fibers it innervates to contract. (See Chapter 10 for more information on muscle fibers and their control.) The membranes in the zone of initiation of motor neuron diffusion often do not fully adapt to sustained depolarization. When the synaptic input is both strong and sustained, it causes the motor neuron to fire a sustained series of APs. The frequency of pulses in the sequence depends on the degree of depolarization in the spike onset region (Fig. 6-47), which in turn depends on its

Stimulate

Figure 6-46 In temporal summation, presynaptic signals arrive at the synapse in rapid succession. (A) Setup for recording postsynaptic events. (B) A single signal elicits a synaptic current (shaded signal) and a slower-decaying synaptic potential. (C) Synaptic potential summation does not require synaptic current summation because the time constant of synaptic potential is longer than the time course of synaptic current. Arrows indicate the timing of presynaptic impulse arrival at the synapse.

channel and membrane capacitance) and V, which gradually return to their quiescent state after the cessation of synaptic current. Thus, the synaptic potential lasts a few milliseconds longer than the synaptic current, and if a second synaptic current flows before the decay of the first synaptic potential, it will cause a second depolarization, which amplifies the decay phase of the first , even if the two synaptic currents do not overlap. Thus, the charge storage capacity of the membrane allows the voltage effect of the synaptic current to accumulate over time. The longer the membrane time constant, the slower the decay of the postsynaptic potential and the more efficient the temporal summation of asynchronous synaptic inputs. The membrane time constant (T) of vertebrate motoneurons is approximately 10 ms, and that of other neurons ranges from 1 ms to 100 ms. Microelectrode recordings revealed that, under normal conditions, motor neurons are almost never electrically silent, but always exhibit synaptic noise (irregular fluctuations in membrane potential) caused by persistent activity of presynaptic neurons. The result is stable

time

Depolarization Figure 6-47 The initial frequency of impulses generated in a motor neuron is roughly proportional to the magnitude of the membrane depolarization. (A) Two electrodes, one for delivering depolarizing current and one for recording membrane potential, were inserted into spinal cord A motor neurons. (B) Three idealized traces showing that increasing depolarization (from top to bottom) results in increased throwing rates. (C) Initial firing frequency plotted against the degree of depolarization. As depolarization increases, the frequency of the AP increases up to a certain maximum.

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Sums the magnitude of synaptic inputs. Thus, the number and frequency of APs produced in motor neurons provides information about the neuronal input. In fact, most of the transmission of information in the nervous system depends on this frequency code. In summary, APs are generated in neurons when an initial low-threshold segment (usually the axonal hillock) depolarizes to or beyond threshold. With increasing depolarization, the frequency of APs in neurons increases to the maximal firing frequency. The degree of depolarization of the spike initiation zone depends on the relative timing of excitatory and inhibitory synaptic currents and the source of these currents.

Synaptic Plasticity A nervous system is less useful to animals if it cannot be modified through experience. Neuronal plasticity, the change in neuronal function due to experience, is critical to the survival of any organism. Learning and the development of motor skills and habits are common examples of neuroplasticity in our lives. This plasticity underlies human intelligence and the ability of all higher animals to respond adaptively to stimuli that allow them to transcend the fixed reflexes genetically programmed into their developing nervous systems. Almost all animals exhibit some degree of behavioral plasticity, and the mechanisms behind synaptic plasticity are currently the subject of many experiments. Synaptic plasticity is also a consequence of developmental events throughout life. Synaptic connections formed in embryos were later refined to adult patterns, and even later changes in synaptic strength were recognized as an important mechanism of learning and memory at mature synapses. Interestingly, both the developmental design of mature synapses and their changes in learning and memory appear to depend on retrograde signals sent from postsynaptic neurons to presynaptic neurons. In mature adult organisms, neuronal plasticity requires changes in synaptic efficiency. Altering synaptic availability is not the only way to alter neuronal function, but it is the one with the most experimental evidence to date. D. 0. Hebb proposed in 1949 that the potency of an excitatory synapse increases when the activity of the excitatory synapse is consistent and positively correlated with that of the postsynaptic neuron. A challenge since then has been to identify the mechanisms that might underlie this change. Two broad classes of mechanisms that may exert this effect are (1) changes in presynaptic terminals and (2) changes in postsynaptic neurons. An example of a presynaptic mechanism is a change in the amount of transmitter released from a presynaptic terminal in response to a presynaptic AP. An example of a postsynaptic mechanism is a change in the postsynaptic apparatus that alters the magnitude of the resulting depolarization

When a certain amount of transmitter is released from a presynaptic terminal. The mechanism of postsynaptic plasticity is poorly understood, although it has been demonstrated in a variety of tissues. We will consider presynaptic mechanisms of neuronal plasticity. There are two broad classes of presynaptic mechanisms that alter synaptic effectiveness. Within a class, activity in the terminal itself causes usage-dependent changes in the launcher's release. These mechanisms are therefore termed homologous synaptic modulation. In another class, changes in presynaptic function are caused by the action of modulatory substances released from another nearby nerve terminal. Therefore, these mechanisms are called heterosynaptic modulation. Typically, heterosynaptic modulation lasts longer than synaptic modulation. Synaptic Modulation: Relief

Use-dependent changes in synaptic efficacy can be observed in partially curarized endplate regions of frog skeletal muscle fibers when two stimuli are applied to motor axons in rapid succession. If the second synaptic potential occurs before the decay of the first, they will add, but the magnitude of the second response will be greater than can be explained by the sum alone. The second postsynaptic potential can still reach a higher amplitude than the first if it occurs shortly after the first postsynaptic potential has completely decayed and time summation is excluded. This effect, called synaptic facilitation, lasts 100 to 200 milliseconds at the neuromuscular junction in frogs (Fig. 6-48). Stimulate

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Figure 6-48 Synaptic facilitation occurs at the frog neuromuscular junction. In this experiment, curare in bath salts blocks some ACh receptors and reduces the amplitude of excitatory postsynaptic potentials below the triggering threshold. Two stimuli are delivered to the nerve in rapid succession. The second synaptic potential adds to the first descending phase, producing a larger postsynaptic potential. But beyond that, the amplitude of the second response (represented by the line labeled 2) is larger than can be explained by summation alone.

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After Katz and Miledi (1968). They used carefully positioned micropipettes to deliver pulses of Ca2+ ions to the external solution near the motor endplate of frog muscles immersed in Ca2+-free Ringer's solution (Fig. 6-49A). They found that when the delivery of a pulse of extracellular Ca2+ ions coincided with the arrival of the first AP, the facilitation of the postsynaptic potential evoked by the second stimulus was greatest (Fig. 6-49B). Giving the first Ca2+ pulse after the first AP reached the end did not significantly increase remission (see Figure 6-49B). Therefore, if synaptic facilitation is to occur, Ca2+ must be available for access to presynaptic terminals when APs enter the terminals. If Ca2+ ions from the external fluid are allowed to enter the port, the Ca2+ ions from the second AP will add to the remaining Ca2+ ions from the first AP, resulting in the release of more transmitter. Synaptic Modulation: Post-Tonic Potentiation

Figure 6-49 Synaptic facilitation depends on the presence of calcium ions in the extracellular fluid. (A) Motoneurons innervating muscle fibers were stimulated and the resulting postsynaptic potentials were recorded. The bath solution does not contain calcium, but a small pulse of CaCl2 is delivered directly to the endplate area via a CaCl2-containing pipette. In this experiment, the relative timing between motoneuron stimulation and CaCl2 release was changed. (0) Recordings of postsynaptic potentials in muscle fibers. The horizontal black bar shows the timing of the Ca2+ pulse. Thin vertical lines indicate stimulation of presynaptic neurons. Lane 1 shows the amplitude of the postsynaptic potential in response to a single AP in a motor neuron. In the other three lanes, the temporal relationship between the first AP and the CaCl pulse was varied. In all cases, Ca2+ ions were available at the second AP. Facilitation occurs only when Ca2+ ions are present at the endplate and both APs reach the endplate. [Adapted from Katz and Miledi, 1968.1

The best evidence suggests that synaptic facilitation depends on the amount of free Ca2+ at the presynaptic terminal. When the first AP opens a voltage-gated Ca2+ channel, the concentration of free intracellular Ca2+ ions increases terminally, and this increase in Ca2+ ion concentration lasts for a short period of time. When the second pulse reaches the end, the Ca2+ concentration is still slightly elevated, and the Ca2+ ions entering the second pulse add the remaining Ca2+ ions, resulting in a higher Ca2+ concentration at the end. Because transmitter release is a Doyle function of intracellular Ca2+ concentration near the presynaptic release site, this small increase in terminal Ca2+ concentration leads to a large increase in transmitter release after the second pulse. Experimental evidence for this hypothesis was found.

When frog motor axons are tonically stimulated (i.e., stimulated at high frequency over a relatively long period of time), synaptic transmission at the neuromuscular junction is initially inhibited after stimulation. However, responses to test pulses applied at later time points after stimulation have been found to be greater than normal. This increase in response magnitude lasts for several minutes, during which time the response is amplified. This post-tetanic potentiation is another example of a usage-dependent change in presynaptic efficacy that occurs in one form or another across many types of synapses. Figure 6-50 shows the results of such an experiment. initial,

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50 s-' Figure 6-50 Tetanic stimulation of frog motor nerves leads to inhibition and potentiation of excitatory postsynaptic potentials in muscle fibers. Curare has been used to reduce the magnitude of synaptic potentials, block APS production and reveal the magnitude of synaptic potentials. Stimulation of the motor nerve at 50 stimulations per second for approximately one minute while the nerve and muscle were bathed in normal frog Ringer's solution with a Ca2+ concentration of approximately 2 mM (above panel) initially resulted in subsequent depression of excitatory postsynaptic neurons When the extracellular Ca2+ concentration was lowered again to 0.225 mM, only one efficacy was observed after high-frequency stimulation. [Adapted from Rosenthal. 1969 i

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Excitatory postsynaptic potentials (EPSPS) are elicited at frog neuromuscular junctions by stimulating the motor nerve at a low controlled frequency (one stimulation every 30 s). The stimulus rate was then increased to 50 per second for 20 s, followed by a series of test stimuli at the initial rate of one every 30 s. In Ringer's solution containing normal concentrations of Ca2+ (see Figure 6-50, upper panel), posttetanic inhibition of EPSPS is induced immediately after tetanic stimulation. However, the amplitude of the EPSS increased within one minute; in other words, posttetanic potentiation occurred. After approximately 10 min, the amplitude of the EPSS returned to control levels. In Ringer's solutions containing lower than normal concentrations of Ca2+ (see Figure 6-50 below), depression did not occur and posttetanic potentiation declined more rapidly. These outcomes are believed to depend on events within the terminal. Upon RF stimulation at normal concentrations of extracellular Ca2+ (1.8 mM), available synaptic vesicles are released faster than they are replaced, so the amount of transmitter available for release is depleted immediately after high residual frequencies and Reduce stimulation for a while. In the late postcatatonic phase, the quantum of transmitters available for release recovers and the depression subsides. During tetanic stimulation, Ca2+ ions entering the terminal accumulate, charge available Ca2+ binding sites that normally buffer intracellular Ca2+ concentrations, and remain at the terminal until activated by active transport across the cell membrane and pumped out. Posttetanic potentiation and its slow decay are thought to reflect this increase and subsequent decrease in terminal Ca2+ concentration. In low Ca2+ Ringer's solution, fewer Ca2+ ions are accessible to the terminals, allowing fewer synaptic vesicles to bind to the membrane and release transmitters. As a result, there was less depletion of available synaptic vesicles and no tonic depression. Post-tetanic potentiation is also evident, as although repetitive stimulation brings Ca2+ ions to the terminals, the potentiation declines more rapidly, either because the terminal Ca2+ rises less, or because the presynaptic terminal is able to absorb additional The Ca2+ pumps the ion Ca2+ out more quickly because less is accumulated.

But not at the presynaptic terminals, which should act heterosynaptically, since transmission through the synapse is altered by an extra third neuron releasing the modulator. One class of heterosynaptic actions discussed earlier in this chapter is presynaptic inhibition; another, in which the amount of transmitter released is increased by the presence of a modulator, is called heterosynaptic facilitation. In heterosynaptic modulation, the modulator is thought to alter the amount of Ca2+ ions entering the terminal after the presynaptic AP. Synaptic regulators typically do not open (or close) ion channels directly. Instead, they change the way ion channels respond to different stimuli. In doing so, they increase or decrease the ionic current conducted through presynaptic AP-activated channels. This action of a modulator is usually mediated by one or more intracellular messengers that act on ion channels. Instead, fast neurotransmitters bind to membrane receptors and open (or close) the channel. The most widely studied example of synaptic heterosynaptic regulation is found in Aplysia californica, a snail-like snail mollusc widely used in the study of neuronal plasticity. Eric Kandel and his collaborators found that during behavioral sensitization, excitatory transmission between certain identified neurons in the Aplysia central nervous system is enhanced. They found that this amplification occurs through heterosynaptic facilitation of transmitter release triggered by serotonin release near the synapse (Fig. 6-51). In this context, serotonin is thought to increase levels of the intracellular messenger 3',5'-cyclic adenosine monophosphate (CAMP), which has been shown to affect the opening of a specific type of K+ channel (S channel). In particular, when CAMP is elevated in presynaptic neurons, S channels are more likely to close at any given Vm. K+ efflux through S channels contributes to post-AP repolarization, so closure of S channels prolongs presynaptic APs and allows more Ca2+ ions to enter terminals through voltage-gated Ca2+ channels. Increasing the influx of Ca2+ ions releases more transmitters and increases the magnitude and duration of postsynaptic potentials.

Long-term potentiation Heterosynaptic modulation of neurotransmitter release at certain synapses is affected by certain neuromodulators. These modulators include serotonin in mollusks and vertebrates, octopamine in insects, and norepinephrine and GABA in vertebrates. All of these drugs are also neurotransmitters (see Table 6-2). In addition, endogenous opioids have been shown to act as modulators of vertebrate neurons. Release of such drugs into the circulation or from nerve endings near synapses is thought to alter transmitter release from presynaptic terminals. When they were released nearby,

In recent years, intense interest has focused on long-term changes in synaptic effectiveness found in the mammalian hippocampus, the location of some memories. High-frequency stimulation of hippocampal inputs leads to increased amplitudes of excitatory postsynaptic potentials recorded in hippocampal postsynaptic neurons. In intact animals, the magnitude of the increase can persist for hours -- even days or weeks -- after the booster stimulus. This prolonged facilitation of synaptic transmission, known as long-term potentiation, has been shown to occur in many synaptic signaling pathways. In different locations, long-term synergies may require different requirements

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sucrose > glucose), and the sweet taste receptors of the human tongue. Like insects, many vertebrates have taste receptors on their bodies. For example, bottom-dwelling robins have modified pectoral (front) fins with taste receptors at the tips of the fin rays, which are used to probe muddy bottoms for food. In terrestrial vertebrates, taste receptors are located on the tongue and epiglottis, the back of the mouth, and the upper pharynx and esophagus. In vertebrates, taste receptor cells are located in taste buds, and taste buds and olfactory organs share certain organizational features (Fig. 7-20). Taste receptors are surrounded by supporting and basal cells, which are precursor cells for the development of new taste receptors. Basal cells are derived from epithelial cells and periodically generate new sensory receptor cells; taste receptor cells only live for about 10 days. This remarkable turnover of primary sensory cells also occurs in a specialized section of vertebrate olfactory organs and photoreceptor cells, called the outer segment. All these regularly self-renewing cells or cell parts interact directly with physical stimuli from outside the organism: taste and smell molecules in taste and smell cells and photons in the outer parts of photoreceptors. The turnover of all sensory cells presents problems for maintaining sensory specificity in an organism, since specificity is lost if the new cells are not precisely integrated into the existing network. How to preserve the integrity of taste and smell remains an unsolved but actively explored mystery. Although our subjective experience suggests a very wide range of possible tastes, these sensations can be grouped into four distinct qualities: sweet, salty, sour, and bitter. From an evolutionary point of view, these categories may be related to some basic properties of food. Sweet foods can be high in calories and are therefore useful; salt is essential for maintaining water balance (see Chapter 14); sour tastes can signal excess danger; and many bitter substances are poisonous. The finding that vertebrates respond to only four basic taste categories suggests that all perceived tastes must depend on various combinations of these basic traits. Furthermore, a separate, identifiable sensory pathway has been hypothesized to be associated with each of the four tastes.

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Figure 7-20 Chemosensory organs typically consist of receptor cells surrounded by a support structure. (A) In a vertebrate taste bud, receptor cells are surrounded by basal cells that give rise to new receptor cells and supporting cells. Transduction occurs on the apical membrane. Recipient cells do not themselves send axons to the CNS, although they can produce APs. Instead, they act as synapses in the central nervous system. These are afferent neurons that carry olfactory information in vertebrates (B) and insects (C). The receptors themselves send primary afferent axons to the central nervous system. Similar structures in vertebrates and insects are similarly drawn in Parts B and C. All three types of receptors extend delicate processes into the mucus layer that covers the epithelium. In insects, these fine extensions are true dendrites. [Part A, adapted from Murray and Murray, 1970; Part C based on Steinbrecht, 1969.1

How do molecules interact with membranes to produce different flavors? In recent years, patch-clamp recordings have been used to identify the mechanisms responsible for each taste modality (Fig. 7-21). Each individual taste receptor cell responds to a specific stimulus, and each class of taste stimulus activates specific cellular pathways in the receptor in response to it. salty

Figure 7-21 Each style is implemented by a different mechanism. (A) In the transduction of salty and some sour tastes, Na+ (or H-) ions pass through ion channels in the apical membrane and directly depolarize the recipient cell. (B) In the transduction of other sour and some buttery flavors, Ki channels are blocked by protons (sour) or certain bitter compounds, and leakage of remaining cations into the cell depolarizes the receptor (Ala), while some Other sweet compounds bind to the receptors. (C) L-Alan netoren (R) and activation of G protein (G). Activated G proteins activate adenylyl cyclase (AC), and the resulting increase in cAMP closes Kt channels in the basolateral membrane and depolarizes the cell. (D) L-Arginine (Arg) binds and opens a ligand-gated non-selective cation channel. (E) Some bitter compounds bind to receptors and activate G proteins thought to be coupled to phospholipase C (PLC), leading to an increase in intracellular inositol triphosphate (InsP), which is then released from intracellular CaZ' to release memory. The end result is increased transmitter release from recipient cells, but the mechanism is not fully understood. PIP, phosphoinositide 4,5-bisphosphate. [Adapted from Avenet et al., 1993.1

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Bacteria such as NaCl are bound in water, and Na+ ions enter the receptors through Na+ channels in the membrane, depolarizing the membrane potential. These can be blocked by Na+ channels. When they are complete, the voltage-gated Na+ channels are injected with a substance that can drug most APs. Acidic salts act through excess H+ ions, through channels (as seen in hamsters), or by blocking K+

Channels (observed in the newt Nectuuus). In both cases, the membrane is depolarized. Sweet compounds and the amino acid alanine (Ala) bind to the receptor, which engages in an intracellular cascade, closing K+ channels in the basolateral membrane and depolarizing the receptor. Other sweet substances, including the amino acids arginine (Arg) and MSG, activate nonspecific cation-selective channels in taste cells. Some bitter compounds such as Ca2+ and quinine close K+ channels in the apical membrane, depolarizing the cell. The transduction of other bitter compounds is less clear but appears to rely on intracellular second messenger systems (InsP or cAMP pathways) to excite cells. The sweet and bitter taste pathways acting through second messengers are thought to be mediated by G proteins, and recent reports point to candidate molecules. In all cases, the initial event in the recipient cell eventually leads to an increase in the intracellular Ca'+ concentration, which increases the release of neurotransmitters to secondary cells in the signaling pathway. Taste receptors produce APs but do not have axons and therefore cannot transmit information to the central nervous system. Instead, they attach to and regulate the activity of neurons whose axons line the facial, glossopharyngeal, and vagus nerves (the seventh, ninth, and tenth cranial nerves). The presence of four taste types and the specificity of the membrane transduction mechanism for each taste suggest that each receptor subtype may be associated with a distinct set of axons. In this arrangement, for example, information about "sweetness" would be carried by a specific subset of axons. This pattern is known as tagline encoding, but records show that flavor information is not nearly as neatly organized. Recordings from individual neurons show that receptors generally respond best to one type of stimulus (Figure 7-22), but many receptors also respond poorly to other classes of stimuli. Thus, the data suggest that the individual fibers that innervate the taste buds receive information from receptors belonging to different subtypes.

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Figure 7-22. Each afferent taste neuron is most efficiently stimulated by one stimulus, but also responds to others. Responses to four different taste stimuli were recorded from individual taste-affinity axons in two different hamster nerves. Each neuron responded maximally to one of the four taste stimuli; different neurons responded maximally to different stimuli. However, all axons responded at least weakly to all four stimuli, suggesting that each taste afferent is not limited to carrying information about one taste type. Abbreviations: S, sucrose (sweet), N, NaCl (saline), H, HCl (acid), Q, qulnlne HCl (batter) Number of axons per group indicated [Adapted from Hanamor et al., 1988]

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Sensory information about taste must be based on parallel analysis of many taste axons rather than simply encoding tagged lines. olfactory receptor mechanism

In vertebrates, olfactory receptors are located in the nasal cavity and are arranged to generate air or water flow

Flow through them during breathing (Figure 7-23). Animals that are particularly dependent on olfactory signals have complex cavities lined by layers of receptors. These cavities are called turbinates, and the mechanism that allows airflow through them remains unknown. Each receptor neuron has a long, thin dendrite that terminates in a small bump on the surface (Figure 7-24A). Figure 7-23 In the olfactory organs of vertebrates, air (or water) carrying an odorous substance moves past the olfactory receptors. The human olfactory epithelium covers part of the surface of the airways in the nose. Arrows indicate the path air takes through the nose when inhaled. The dashed portion of each line shows the air flow in the turbinate (shaded red) where the olfactory receptors are located. Dashed lines also indicate air vortices generated on the olfactory epithelium lining the dorsal hollow of the nasal cavity.

Figure 7-24. Receptors in the vertebrate olfactory epithelium depolarize in response to odorants. (A) Organization of mammalian olfactory epithelium. (B) Response of cultured salamander olfactory receptor neurons to a focal impulse of an odor. (Left) When a stimulating chemical pulse is directed at the receiving membrane of a cilium, it generates a large current (upper recording). When a solution with a high K+ concentration is focused at the same point, the response is low (lower trace). (Right) When the stimulating chemical pulse is directed at the soma rather than the cilia, there is little response (highest recorded). However, when the high K+ concentration solution acts directly on the somatic cells, it elicits a strong response (lower data set). [Part A adapted from Shepherd, 1994; Part B, adapted from Firestein et al., 1990.1

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Several thin cilia (about 0.1 µm in diameter and about 200 µm in length) grow from the mass and are covered with a protein solution called mucus. Molecules released into the nasal cavity are absorbed by the mucus layer and released to the cilia. Two clues suggest that cilia are the site of olfactory transduction. First, only ciliated neurons respond to odors, which means that cilia must be the place

divert. A second line of evidence comes from experiments in which olfactory neurons are grown in culture and exposed to odorants while recording receptor currents from intracellular electrodes in the soma (Fig. 7-24B). When a solution of odorant molecules was expelled onto the cilia, the cells responded strongly; on the other hand, when the same solution was sprayed onto the cell body, there was little response. Instead, from

KC1 (which depolarizes the receptor membrane) responds minimally to cilia, whereas ejection of KC1 into the soma elicits a strong response. These data suggest that only cilia are capable of responding to odors, resulting in significant changes in V,,,. The olfactory transduction cascade involves adenylate cyclase linked to G proteins. (See the discussion of transduction earlier in this chapter.) A very large family of proteins was recently discovered to be expressed only in the olfactory epithelium. The structure of each protein includes seven transmembrane domains, and other features also suggest that these molecules are homologous to proteins that mediate other transduction processes. The large size of this protein family suggests that there may be many different receptor subtypes for different odors

In contrast, there are few receptor types that encode taste. The vertebrate olfactory code is detected electrically in the frog olfactory epithelium (Fig. 7-25A). In these experiments, the activity of individual receptor axons was recorded from one electrode, while the total potential (electroolfactory graph or EOG) of a large number of olfactory receptors in epithelial cells was simultaneously recorded from another electrode (Fig. 7-25B). Pulses from individual receptors are then electronically superimposed on the current map. Using this technique, the activity of a single receptor can be compared to the overall response of many receptors when a single odorant or combination of odorants is presented. Figure 7-25 enables simultaneous study of olfactory reception in frog olfactory epithelium and organ level. (A) Various odorants can be applied to the nasal epithelium while recording the total electro-olfactory graph (EOG) and spikes of individual recipient cells. These two types of records can then be electronically aggregated into one composite record (right). (B) Details of tissue and electrodes. Electrode 1 records the entire EOG potential as it moves away from the axon, while electrode 2 records the activity of a single axon closest to it. [Adapted from Gesteland, 1966.1

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It turns out that the stimulus code in the vertebrate nose is much more complex than that in the housefly contact chemoreceptors. Different receptors respond differently to the same scent. Specific odors increased pulse frequency in some olfactory axons (Fig. 7-26A). Some odorants that smell similar to humans had similar effects on the olfactory cells of some frogs, suggesting that frogs also smell the same. However, the same odorants had different effects on other cells (see Figure 7-26A, cell a vs. cell b), suggesting that they smell differently to the frog. In the olfactory bulb, further down the chain of olfactory neurons, neurons can respond to odors by decreasing or increasing their activity (Fig. 7-26B). In fact, establishing a one-to-one relationship has proven impossible

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Between odorant categories and olfactory cell types in frogs. Instead, each olfactory receptor cell appeared to express a chimera of olfactory receptor molecules with different specificities. Therefore, the response properties of a particular olfactory receptor must depend on the ratio of its multiple receptor molecules. This situation implies that the ability of mammals to discriminate between various odors must depend on the ability of higher olfactory centers in the brain to decode combined signals from a large number of olfactory receptors.

Mechanical Reception All animals can sense physical contact on body surfaces. This stimulus is recognized by mechanoreceptors, the simplest of which consist of morphologically undifferentiated nerve endings in the connective tissue of the skin. More complex mechanoreceptors have additional structures that transmit mechanical energy to the sensing membrane. These appendages often also filter mechanical energy in some way, as previously described in mammalian Pacinian bodies, where sensitive ends are covered by capsules (see Figures 7-14). Other mechanoreceptors include the various types of muscle stretch receptors found in arthropods and vertebrates, with mechanosensitive sensory endings attached to specialized muscle fibers (see Figures 7-13), and the muscular stretch receptors emanating from the arthropod exoskeleton. Hair-like sensilla (Fig. 7). -27). The most complex accessory structures associated with mechanoreceptor cells are found in the vertebrate middle and inner ears and the vestibular system, both of which are discussed later in this chapter. The stimulus that activates the mechanoreceptor membrane is stretching or deformation of the surface membrane. In fact, strain-sensitive channels are present in all types of organisms, from the simplest to the most complex. Patch clamp data revealed that these channels respond to stress changes in the membrane plane and can be activated or deactivated by stretching. Strain-sensitive channels are difficult to easily classify in terms of selectivity because they exhibit a wide range of conductivities and fidelities. Potential sensors of mechanical stress are the cytoskeleton, enzymes or ion channels themselves. Mechanosensitive channels are the only major mechanosensors that do not rely on enzymatic activity, but instead directly exploit the free energy stored in electrochemical transmembrane gradients. Mechanoreceptors can be very sensitive, responding to mechanical displacements as small as 0.1 nm. Understanding how such small changes lead to changes in ion permeability across membranes is an ongoing challenge.

Figure 7-26 Olfactory receptors respond to various odorants in complex ways. (A) Recording of two frog olfactory receptors. Both menthone and menthol slightly inhibited ongoing activity in cell A, suggesting that cell A was unable to distinguish between the two substances. In contrast, cell b responded differently to the two substances, producing more AP in response to menthol than in the resting state, but less in response to menthone than in the resting state. Therefore, cell b may distinguish between the two substances, while cell a cannot. Note that the electroolfactory graph (EOG) is summed with individual recordings for each cell. (B) Recordings from secondary olfactory cells of the tiger salamander. Odors can reduce or increase the ongoing activity in these cells. [Part A after Gesteland, 1966; Part B after Kauer, 1987.1

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Mechanical stimuli are converted into electrical signals (Figure 7-28). You can find them in several places. Fish and amphibians have a set of external receptors, called the lateral line system, based on hair cells that detect movement in the surrounding water (Figure 7-29). The auditory organs of vertebrates and the organs that report the body's position relative to gravity (the balance organs) are also based on hair cells. The organs of balance usually include the semicircular canals and the vestibular apparatus. Hair cells are named for the many cilia that protrude from the tip of each cell. These cilia can be divided into two types: there is usually one kinetocilium and 20-300 immobile stereocilia per hair cell. The microtubules inside the kinocilium are arranged in a "9 + 2" arrangement (see Figure 7-28A), which is similar to the arrangement of other motile cilia. Stereocilia contain many elongated longitudinal actin filaments and are thought to be structurally and developmentally distinct from kinetocilia. While hair cells of the lateral line and vestibular organs have both kinetocilia and several stereocilia, some hair cells in the adult mammalian ear lack kinetocilia. Furthermore, the technically remarkable feat of microsurgical removal of kinetocilia from where hair cells normally reside did not block transduction. These two observations suggest that kinocilia may not be required for mechanotransduction. The stereocilia of hair cells are arranged in order of increasing length from one side of the cell to the other (see Figure 7-28B and C). bisected by the plane of symmetry of the kinocilium

The stereocilia give the hair cells bilateral symmetry, with a tip that slopes like a hypodermic needle. In most organs, hair bundles are connected by their kinetocilia to some sort of auxiliary structure. Stimuli affecting accessory structures are transmitted to stereocilia bundles through bonds connecting accessory structures and kinetocilia to stereocilia. Furthermore, when the tip of a stereocilia bundle was touched with a fine probe, the bundle moved as a unit regardless of the direction of the stimulus. The precise process by which external pressure or force moves stereocilia bundles depends on the specific arrangement of hair cells and support structures within each sensory organ, but ultimately it is the movement of the stereocilia that generates the electrical signal. Hair cells are depolarized when the cilia bend toward the highest cilium; conversely, the cell hyperpolarizes if they bend in the opposite direction (see Figure 7-28D). (If the stereocilia bend to either side, rather than toward or away from the kinetocilia, Vm remains the same.) At rest, about 15% of the channels in the hair cell are open, producing a resting potential of about -60 mV. Hair cells do not produce AP. Instead, they form chemical synapses with afferent neurons and release neurotransmitters in a graded fashion depending on the V in the recipient neuron; the afferent neuron then carries the message to the central nervous system. The amount of transmitter delivered to afferent neurons determines their firing rate. Note that the input-output relationship of hair cells is markedly asymmetric (see Figure 7-28D); that is,

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.................................................. Figure 7-28 (Left) Membrane Das when cilia move from rest to As the location moves, the hair cell receptor potential changes. (A) Electron micrograph of a hair cell cilium cross-section. The large cilia containing the typical 9-Z microtubule structure are kinetocilia; the others are stereocilia. (B) Scanning electron micrograph showing the structure of the neuromast hair cell in the giant Danyfish. (C) Diagram of a typical hair cell showing the anatomical relationship of stereocilia and kinetocilia. Hair cells send out transmitters to afferent neurons, which carry sensory signals to the central nervous system. It also receives synapses from efferent neurons. Depending on the direction in which the cilia is bent, hair cells can increase or decrease the frequency of APs in afferent fibers. Intracellular potential changes are generated by the linear back-and-forth motion of cilia and can be recorded using microelectrodes. Extracellular recordings from afferent axons revealed that AP correlated with changes in Vm in recipient cells. (D) Input-output relationship of hair cells. Note that the depolarization elicited by movement toward the kinetocilium is greater than the hyperpolarization in response away from the kinetocilium. [Part A of Flock, 1967; Part B by Christopher Braun; Part C, adapted from Harris and Flock, 1967; Part D, adapted from Russell, 1980.1

The depolarization caused by a given movement in the direction of the highest cilium was greater than the hyperpolarization caused by a similar movement of cilia in the opposite direction. This asymmetry is important because when hair cells are exposed to symmetrical vibrations such as sound waves, changes in membrane potential can only accurately follow the alternating phases of stimuli at frequencies up to several hundred hertz (Hz), but the sound frequencies are usually much higher than this value. At higher frequencies, the response to the vibrations coalesced into a steady depolarization; hair cells depolarized even when the stimulus moved the cilium an equal distance from zero displacement in both directions. This steady depolarization in response to high-frequency stimuli results in a steady, rather than modulated, release of transmitters from hair cells that result in high-frequency firing of afferent neurons. Details of hair cell transduction are presented later (see Cochlear Hair Cell Excitation).

balance organ

air embedded

Figure 7-29 The lateral line sensory system of fish and amphibians is based on hair cells. This diagram shows the location of these receiving organs in the African clawed frog (Xenopus). The image below shows a cross-section through part of the lateral line, showing the sucker, an accessory structure that bends when the movement of the surrounding water displaces the cilia. Compare the structure of this organ to the hair cell in Figure 7-28.

The simplest organ that has evolved to detect an animal's position relative to gravity or its acceleration is the balance sac. This organ type is found in a range of animal groups from jellyfish to vertebrates. (Interestingly, insects lack these sensory organs and seem to rely entirely on other senses, such as vision or common proprioceptors, for directional information.) The static vesicle consists of a hollow cavity lined with ciliated mechanoreceptor cells in contact with the stalactite, which It may be sand grains, calcareous nodules, or some other relatively dense material (Fig. 7-30A). Statolith is either taken up from the animal's environment or secreted by the statocyst's epithelial cells. Every time a lobster molts, for example, it loses its still stone and replaces it with new grains of sand. In any case, the static stone must have a higher specific gravity than the surrounding fluid. Static stones are located in different regions of the static sac as the position of the animal changes. When the lobster tilts to the right about its longitudinal axis, the static stones settle on the receptor cells on the right side of the static sac, stimulating them and causing tonic discharges in the sensory fibers of the stimulated receptor cells (Fig. 7-30B). . Recordings from many different fibers from the balance sac show that each cell fires maximally in response to a specific lobster orientation (Fig. 7-30C). Information from these receptors is sent to the central nervous system and triggers reflex movements of the extremities. This mode of information processing was demonstrated in a clever experiment in which molting lobsters were given iron filings instead of sand. They replaced their balance stones with iron filings, which allowed the position of the iron balance stones to be manipulated by magnets. As the magnet moved through space, pulling on the iron balance stone, the lobster, whose position relative to gravity did not change, produced a series of compensatory postural responses.

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Figure 7-30 Statocysts sense the acceleration and position of a foraminifera relative to gravity (A). Structure of the balance sac. In lobsters, astatol is located in the receptor portion of a row of hair cells. (B) Effector potentials recorded from double dissected nerve fibers when examining lobsters. Each record shown here is made from a different fiber

The traces below each photo show the tilt over time and the angle at which the animal was tilted. (C) AP frequency recorded from different fibers plotted as a function of animal position. Each battery responds with a maximum discharge rate at a different location. [Adapted from Horridge, 1968.1

vertebrate ears

A fluid moves in one direction and is constrained while moving in the opposite direction. The orthogonal arrangement of the three channels enables them to capture every movement of the head in 3D space. Below the semicircular canals are three other areas with hair cells in the larger bony compartments, the so-called macules. Mineralized concretions called otoliths are associated with spots, similar to static stones associated with static capsules. Otoliths signal their position relative to the direction of gravity; in lower vertebrates, they also sense vibrations in the surrounding medium, such as sound waves. Sensory signals from the semicircular canals combine with other sensory inputs in the brainstem and cerebellum to control postural and other motor reflexes.

The vertebrate ear performs two sensory functions, each based on the activity of hair cells. Some structures in the ear, the vestibular apparatus, function like the balance sac in invertebrates, providing information about the animal's position in space relative to gravity and acceleration. The other structure, the organ of hearing, provides information about vibrational stimuli in the environment -- stimuli called sounds when they fall within specific frequency ranges.

Vertebrate vestibular apparatus In vertebrates, the vestibular apparatus is located in the membranous labyrinth that develops from the anterior end of the lateral line system. It consists of two chambers, the balloon and the utricle, surrounded by bone and filled with endolymph, a special fluid. The endolymph differs from most extracellular fluids in that it has a high K+ content (about 150 mM in humans) and a low Na+ content (about 1 mM in humans); the implications of this unusual composition are found in the "mammalian ear" section was discussed. The three semicircular canals of the inner ear arise from the utricle and lie in three mutually perpendicular planes (Fig. 7-31). Hair cells in the three orthogonal semicircular canals sense head acceleration. As the head accelerates in one plane of the canal, the inertia of the endolymph in the corresponding canal produces a relative movement of the endolymph through the gelatinous protrusion, the ampulla, which moves the ampulla. When the cupula moves relative to the cilia of its basal hair cells, the V of the hair cells changes. All the hair cells in the ear canal are lined up and therefore on the same side, so all the hair cells connected to the cup are on the

Sounds in the ears of mammals in the environment lead to the development of hearing in many tribes. Hearing enables animals to detect predators or prey and estimate their position and distance when they are still relatively far away. Sound also plays an important role in intraspecies acoustic communication, which often requires a delicate match of production and reception. Sound is a mechanical vibration transmitted through air or water in the form of high and low pressure alternating waves, accompanied by the reciprocating motion of the medium in the direction of propagation. The nature of sound, especially the different ways it travels through air and water, places particular limits on its detection. The evolution of hearing illustrates many different mechanisms that have evolved to solve various problems arising from the physical properties of sound. A well-studied example that we examine here is the mammalian ear.

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Figure 7-31 The hearing and balance organs of the human body are located in the ear. (A) The main part of the ear. (B) Semicircular canals and cochlea. The stirrups are removed to reveal the oval window. The path of the acoustic signal is indicated by the black arrow. On the far right, part of the cochlea has been removed to reveal internal structures. (Figure 7-33 shows the structure in more detail.) (C) Detailed structure of the two parts of the vestibular organ. Re-cilia

In the colloid cup, the receptors are embedded in the semicircular canals. Suction cups bend cilia as fluid moves in the tube (left). Particles called otoliths lodge on the cilia of the receptors in the sacculus (one of the spots). Changes in head position cause otoliths to shift position, thereby changing the curvature of the cilia (right). [Parts A and B adapted from Beck, 1971; part C adapted from Williams et al., 1995.1

Outer ear, ear canal and middle ear The outer ear is structured like a funnel, collecting sound waves from a large area of air and focusing the oscillating air pressure onto a specialized surface called the tympanic membrane or tympanic membrane. The outer structures of the ear -- the pinna and tragus -- help collect sound waves. The shell-like external structure as well as the mobility of shells in some species can alter the directional sensitivity of the auditory system. In some organisms, including humans, the acoustic properties of the outer ear amplify sounds in certain frequency ranges. Furthermore, the human ear emphasizes the spatial distribution of stimuli by amplifying sounds coming from certain directions but not others (Fig. 7-32).

To be detected, air vibrations must be transmitted to the fluid-filled inner ear where the receptor hair cells are located. The difficulty of communicating through the air-liquid interface can be seen by trying to talk to someone underwater. Most of the sound energy generated in the air is reflected back by the water surface, so it is difficult to generate enough energy using airborne sound to move the water at the desired frequency and displacement. This condition is called acoustic impedance mismatch. In the ear, this difference is partially offset by a series of three small bones in series, which connect at one end to the eardrum and at the other to the oval window of the cochlea. These bones, the ossicles (labeled anvil, malleus, and stirrup in Figure 7-31A),

Figure 7-32 Selective amplification of structures of the human pinna and tragus, especially for sound frequencies. This diagram shows the pressure on the eardrum that is greater than the pressure that sound exerts on the ear canal. The outer ear structures are removed. If there is no large area, the plot will be a horizontal node intersecting the ordinate with a gain of 1. Show values above 1

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Developed from the articulation point of the posterior jaw, which is now located in the middle ear. Changes in air pressure caused by sound waves in the external auditory canal cause movement of the eardrum, which transmits energy first to the ossicles and then to the inner ear structures. In the inner ear, the first structure to receive mechanical input is the oval window that forms the outermost surface of the fluid-filled chamber (cochlea) that contains receptor hair cells. At the other end of the fluid-filled chamber is another membrane, the round window. This agreement has two important consequences. First, the mechanical coupling properties between the tympanic membrane, ossicles, and oval window amplify the signal by a factor of approximately 1.3. Secondly, since the area of the tympanic membrane is about 0.6 cm2, and the oval window is smaller, about 0.032 cm2, the signal pressure between the tympanic membrane and the oval window is greatly amplified. The ratio between the surfaces of the two membranes is approximately 17:1, which means that the sound pressure on the eardrum is concentrated in a smaller area of the oval window, creating greater pressure, which is important because of the inertia of the oval window of the eardrum The cochlear fluid on the other side is larger than the alr. The increased pressure helps transmit air vibrations efficiently to the cochlear fluid. Due to these two mechanical properties, the signal reaching the eardrum is amplified by at least 22 times before reaching the cochlea. Cochlea Structure and Function This mechanically amplified sound input is converted into neuronal signals by hair cells in the inner ear. The hair cells of the mammalian ear are located in the organ of Corti in the cochlea (Figure 7-33). Movement of fluid in the cochlea causes hair cells to vibrate, displacing their stereocilia; in turn, the hair cells stimulate the sensory axons of the auditory nerve. The hair cells in the organ of Corti are similar to those of the lateral line system of lower vertebrates, except that kinocilia are absent in some hair cells in the adult cochlea. The cochlea is a tapered tube surrounded by the mastoid bone, roughly coiled like a snail's shell (see

10 12

Gain values below 1 suppress facial signals. [Adapted from Shaw, 1974.1

Figure 7-31A and B). It is internally divided into three longitudinal compartments (see Figure 7-33A). The two outer compartments (scala tympani and scala vestibular) are connected by the spirochete, an opening in the apex of the cochlea (see Figure 7-35B). The scala tympani and scala vestibuli are filled with an aqueous fluid called the perilymph, which is similar to other extracellular fluids in that it has relatively high concentrations of Na+ (about 140 mM) and low concentrations of K+ (about 7 mM). Between these compartments - bounded by the basement membrane and Reissner's membrane - is another compartment, the mesostratum, which is filled with endolymph (rich in K+ and low in Na+), the same fluid that surrounds hair cell cilia type organs. The unusual ionic composition of the endolymph contributes significantly to the auditory transmission process. The organ of Corti, which carries hair cells that convert auditory stimuli into sensory signals, is located on the mesoscale and basilar membrane, and signal transmission through cochlear hair cells depends in part on this anatomy. Among vertebrates, only mammals possess a true cochlea, although birds and crocodiles have nearly straight cochlear ducts that contain some of the same features, including the basilar membrane and the organ of Corti. Other vertebrates do not have a cochlea. Some lower vertebrates can perceive sound waves through the activity of hair cells associated with the otoliths of the utricle and bursa and one of the three spots in the vestibular organ. The hair cells of the mammalian cochlea encode the frequency (i.e., pitch) and intensity of sounds. The adult cochlea contains four rows of hair cells, one inner row and three outer rows, with approximately 4000 hair cells in each row (see Figure 7-33B). Hair cell stereocilia contact the overlying tegmental membrane. As the hair moves through the jelly-like mucus that covers the tectum, the cilia bend due to shear forces (that is, forces perpendicular to the axis of the cilia). Sound vibrations are transmitted from the ossicles to the oval window and then through the cochlear fluid and the membrane that separates the cochlear chambers

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.................................................. ............................ A

Reissner Membrane \

Spiral glue

Corti's Staff

\incoming

membrane

and efferent nerve fibers

Figure 7-33 Sound stimuli are transmitted through the hair cells in the cochlea. (A) Cross-section through the cochlear duct at approximately the location shown in Figure 7-31B, showing the two external cavities (scala vestibular and scala tympani) and the attached organ of Corti

Basement membrane in the central canal. (B) Enlargement of the organ of Corti. The cilia of hair cells are embedded in the glial layer of the tectorial membrane, while their cell bodies are anchored to the basement membrane.

(Reissner membrane and basement membrane) before their energy is dissipated through the membrane-covered round window. The compliance of the round and elliptical windows is an important adjustment because if the fluid-filled cochlea is completely surrounded by solid bone, the displacement of the elliptical window, fluid, and internal tissues will be very small. The distribution of disturbances within the cochlea depends on the frequency of vibrations entering the oval window. To visualize this,

Imagine that the displacement of the eardrum is transmitted to the oval window through the ossicles of the middle ear. The vibrations move the incompressible perilymph along the scala vestibuli, through the helical membrane, and through the scala tympani toward the round window. Excitation of cochlear hair cells Electrical recordings at different locations in the cochlea reveal potential changes in frequency, phase, etc.

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The amplitude of the sound waves that generate them. This cochlear microphonic sound is produced by the sum of receptor currents of numerous hair cells stimulated by basilar membrane movement. The actual transduction event occurs when perturbation of the basement membrane forces the tips of the stereocilia to bend laterally because the basement membrane has moved relative to the tectorial membrane (Figure 7-34). This mechanical deflection directly leads to the opening of ion channels at the tip of the stereocilia. Our understanding of these events has grown dramatically in recent years, although questions remain about the details of transduction. The perception threshold of cochlear hair cells corresponds to a shift of 0.1-1.0 nm, which corresponds to a change in membrane current of only about 1 pA through ion channels in the hair cell membrane. Experiments have shown that these channels are permeable to many small monovalent cations (such as Li+, Na+, K+, Rb+, and Cs+). When they open in the fluid, K+ ions and some Ca2+ ions enter the cells from the endolymph. (The high K+ concentration in the endolymph creates an inward driving force on K+, in contrast to the normally outward force V, -E. This inward K+ current depolarizes the hair cell as it increases towards the interior of the hair cell positively charged cells.) Based on current measurements, it is estimated that each stereocilia bundle has approximately 30-300 channels, which means that only 1 to 5 channels per stereocilia can be responsible for transduction. It is assumed that channels are opened directly by mechanical stimulation, because in experiments transduced currents increase with extremely short delay times (approximately 40 ps) when isolated stereocilia bundles are suddenly deflected experimentally. Because of this short incubation period, it is unlikely that enzymatic or biochemical steps are involved in the process. This interpretation is supported by patch clamp experiments, which show that the channel opens faster when the displacement is larger, again suggesting a direct connection

hinge point

Cap membrane

hairpin

Mechanistic influence on channel conformational state. Several factors can affect the sensitivity of hair cells. Each hair cell in the cochlea appears to be tuned to a specific sound frequency band based on its mechanical properties and the properties of the ear canal. Each cell has a resonance frequency determined by the length of the stereocilia in the hair bundle. Long hair cells are most sensitive to low frequency sounds, while short hair cells are sensitive to high frequency sounds. In addition, each cell responds maximally to specific frequencies of electrical stimulation. This electrical resonance frequency is determined by the balance of currents through voltage-gated Ca2+ channels and through Ca2+-sensitive K+ channels in the basement membrane (perilymph-exposed). The outer hair cells of the cochlea can help regulate the cochlea by altering the mechanical properties of the organ of Corti. Outer hair cells have few afferent connections but receive a large number of efferent synapses. When these cells were electrically stimulated during the experiment, they shortened when depolarized and lengthened when hyperpolarized. Thus, outer hair cells may alter the mechanical coupling between inner hair cells and the tectum, leading to changes in transduction. That mechanism actually affects auditions remains to be proven. Hair cells adapt to changes in the position of their stereocilia, a process that has been particularly well studied in the bullfrog sacculus. When the cilia of a frog hair cell are deflected by the probe and held in a new position, the cell's working range adjusts within a few milliseconds to this new tonic position, which then results in a small change in the hair cell's position away from this new set point React. Calcium ions have been shown to play a key role in this process, apparently by changing the tension in the spring that opens the transduction channel. Finally, efferent inputs to hair cells can reduce the cell's response to sound and amplify its frequency selectivity by turning on inhibitory signaling

upward deflection

membrane

Hair Cells Figure 7-34 The movement of the basement membrane relative to the tegmental membrane produces shear forces on the stereotaxy of cochlear hair cells. The tegmental membrane slides over the organ of Cort because the tegmental and basilar membranes are roughly different

The point when it is displaced by a wave moving along the cochlea. Motion in this image is greatly exaggerated [based on Davies, 1968]

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......................... K+ channels, short-circuiting the electrical resonance of the cell. Taken together, the properties of hair cells reveal their delicate coordination. However, all the adaptations that make hair cells extremely sensitive also make them highly susceptible to overstimulation, which can lead to breakage of the base of the stereocilia. Acoustic trauma can lead to permanent hearing loss, which is most severe at the sound frequencies that actually damage hair cells. While some cold-blooded vertebrates can recover from this trauma, in mammals the loss is permanent.

The receptor currents of the hair cells faithfully transmit the movements of the basilar membrane across the entire audible frequency range. These cells transmit their excitation through chemical synapses to the sensory axons of auditory neurons, whose cell bodies are located in the spiral ganglion. Neurotransmitters released by hair cells regulate the firing rate of these axons, which travel in the vestibulocochlear nerve (eighth cranial nerve) and in synapses with cochlear nucleus neurons. In fact, the inner row of hair cells receives approximately 90 percent of the contacts of spiral ganglion neurons, suggesting that the inner row of cells is primarily responsible for sound recognition. In contrast, the outer three rows of hair cells receive many efferent synapses and may be involved in modulating cochlear sensitivity by altering the mechanical relationship between the basilar and tectorial membranes. Frequency analysis through the cochlea Pioneering work on exposed cochlea by Georg von Btktsy has greatly advanced our understanding of how the auditory system encodes information about the frequency of stimuli. His research shows:

1. In response to a pure sine wave tone, the basilar membrane perturbation has the same frequency as the tone. 2. Low frequency noise travels across the entire length of the basilar membrane in the form of traveling waves. 3. The location of the maximum displacement of the basilar membrane by the sound is a function of the frequency of the sound. High frequencies displace only the first part of the membrane, while low frequencies displace further parts. So each point along the basilar membrane most efficiently shifts a specific frequency, at that point

Varies in an orderly fashion, with the higher the frequency, the closer the basilar membrane is to the oval window, and the lower the frequency, the farther the basilar membrane is from the oval window. For sounds up to about 1 kHz, APs in auditory sensory axons appear to follow the fundamental frequency. Above this value, the time constant of the hair cells and the electrical properties of the axons in the auditory nerve prevent a one-to-one correspondence between sound waves and electrical signals. In this higher frequency range, another mechanism must inform the central nervous system of sound frequencies. In 1867, Hermann von Helmholtz discovered that the basement membrane is composed of many transverse bands that gradually increase in length from the proximal to the apex of the basement membrane (from about 100 μm in length at the base to about 500 μm in length at the base). vertex). , which reminded him of the strings of a piano and led him to develop a theory of resonance. He proposed that different locations along the basilar membrane resonate at specific pitch frequencies, while other locations remain stationary, just as corresponding strings on a piano resonate in response to the pitch of a tuning fork. This theory was later challenged by Btktsy (1960), who found that the motion of the basilar membrane was not a standing wave, as Helmholtz suspected, but instead consisted of traveling waves moving from the narrow base of the basilar membrane to the wider apex (Fig. 7- 35). These waves have the same frequency as sound entering the ear, but they travel much slower than sound does through air. A common example of a traveling wave is the motion of the free end of a rope connected to the other end. However, unlike ropes, the mechanical properties of basement membranes vary along their length. The compliance of the membrane (the amount by which the membrane stretches in response to a given force) increases from its narrow end to its wide end, causing the amplitude of traveling waves to vary along the length of the membrane (see Figure 7-35). The location of the cochlea where basilar membrane displacement is greatest (Maximum stimulation of the hair cells at this location) depends on the frequency of the traveling wave and therefore also on the frequency of the stimulating sound. When the stimulus is high frequency, the traveling wave produces the greatest displacement near the base of the cochlea. As the sound frequency decreased, the region of maximum displacement moved apically along the basilar membrane. The amount of membrane displacement at any point along the basement membrane determines the intensity of stimulation of the hair cells and thus the firing rate of sensory fibers from different parts of the basement membrane. Even at maximum amplitude, all movements were very small: the loudest sounds produced basilar membrane displacements of only around 1pm. Hair cell cilia move much less, and the stimulus detection threshold is at the limit of thermal noise.

20 base end

Oval

Stirrup distance (rnm)

top still

basement membrane

top notch

Distance from the oval window Figure 7-35 Sound produces traveling waves along the basilar membrane. (A) The wave moves in the direction indicated by the arrow. Lines a and b represent the shape of the membrane at two different times. The light dashed line shows the envelope produced by the motion, which in this case has the greatest amplitude near the tip. (The amplitude of the waves is greatly exaggerated in this figure). (B) The cochlea is straightened. The places that respond most strongly to sounds of different frequencies are given below. [Part A adapted from Von Bekesy, 1960; Part B, adapted from Moffett et al., 1993.1

Worm ear

The ears of many creatures function differently than mammalian ears, and it is instructive to look at at least one of them to understand possible differences. Crickets find their mates through vocal communication: males sing a song specific to their species, and females are attracted to their species' song. The cricket's ears are located on the first sternum and are connected to an airway called the trachea (Figure 7-36). Each ear has an eardrum that functions like a mammalian eardrum, and the changes in air pressure that produce sound are transmitted through the windpipe to the eardrum. tym. The panum is exposed to changes in air pressure from outside and inside the animal through the trachea. When the right side of the cricket makes a sound, the eardrum on the right side vibrates directly. In addition, it is transported through the tracheal system to the left eardrum, causing the left eardrum to vibrate as well.

Figure 7-36 The cricket's ears are located on the anterior sternum. The eardrum receives sound stimuli through the trachea and vibrates in response to sounds from the outside or transmitted through the trachea in animals. Nerve cells connected to the eardrum transmit sound stimuli.

Differences in the arrival times of stimuli to the left and right eardrums can be used to localize sounds, a principle that also applies to vertebrates (see Chapter 11). In some species, the hair cells are connected to the eardrum, suggesting that excitation in insect ears may resemble that in mammalian ears. Insect ears share some features with mammalian ears: the ear canal directs sound waves to moving surfaces that vibrate in response to the sound waves. When the eardrum vibrates, it directly or indirectly excites receptors and sends signals to the central nervous system. However, the tracheal system allows sound to travel through the animal's body, moving the eardrum from inside or outside the animal's body.

Electricity reception The hair cells in the skin of some teleosts and cartilaginous fishes have lost their cilia and have been modified to detect electrical currents in water. The source of these currents is either the fish themselves or currents from the active tissues of other nearby animals. Weakly electric fish (like mormyrids) have specialized electrical organs that generate the fields that these receptors sense; you can use these fields to communicate with each other and navigate murky waters. Virtually any electrically active tissue can generate an electric field, and some sharks are particularly adept at locating prey by sensing the electrical currents emanating from the animal's active muscles. The electroreceptors of fish are distributed on the head and body of the lateral line system (Fig. 7-3712). In weakly electric fish (as opposed to strongly electric fish such as electric eels), electrical impulses from changes in muscle or nerve tissue at one end of the body re-enter the fish through epithelial pores in the lateral line system. At the bottom of each well, the current encounters an electroreceptor cell (Figure 7-37B), which activates the synapse

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Stimulation Voltage (mV) Figure 7-37 Electroreceptor cells are specialized hair cells found along the lateral line of many fish species. (A) The location of the electrical organs and nerve trunks of the lateral line and the distribution of electroreceptor pores in the weakly electric fish Gnathonernus peters;. (B) At the bottom of each electroreceptor well is an electroreceptor cell whose apical membrane has a lower electrical resistance compared to the basement membrane. (C) Recipient cells release transmitter molecules

(a) Current entering the cell (b) depolarizes, increasing the rate of release, thereby increasing the abundance of APs in the cell's sensory pathway. The current (c) leaving the cell reduces the release rate. When V changes by only a few microvolts, the amount of transmitter released from the recipient cell changes. [Adapted from Bennett, 1968]

Contacts the axon of the eighth cranial nerve innervating the lateral line system. The outward-facing cell membrane has a lower electrical resistance than the basement membrane, so most of the potential drop caused by the current flowing through the cell occurs across the basement membrane, depolarizing it. Depolarization of the basement membrane activates Ca2+ channels in the membrane, and the resulting influx of Ca2+ to the cell base increases the release of synaptic transmitters by recipient cells. This transmitter increases the frequency of APs in sensory fibers innervating receptors. Instead, current flowing from the fish hyperpolarized the basement membrane of the recipient cells, reducing transmitter release below the spontaneous rate. This increases or decreases the frequency of firing in the sensory fibers, depending on the direction of current flow through the electroreceptor cells (see Figure 7-37B and C). The sensitivity of these receptors and their sensory fibers, like the hair cells in the vertebrate ear, is truly remarkable. Changes in sensory nerve firing as shown in Figure 7-37C

Response to changes in V of recipient cells of only a few microvolts. A pulse train of current flows through the water from the back of the fish to the front of the fish (Figure 7-38). Anything with a different conductivity than water will distort the current flow. Lateral line electroreceptors feel the current distribution flowing back into the fish body through the lateral line holes in the head and front of the body, and can detect the field changes generated by objects in the water. This sensory information is then processed in the fish's greatly enlarged cerebellum, allowing it to recognize and localize objects in close proximity. Electrical signals are produced by other fish species for entirely different tasks. Unlike weakly electric fish, which use electric fields for navigation and signal transmission, some eels, torpedoes, and other fish produce powerful electrical discharges to stun enemies and prey. These hyperelectric fish produce a series of continuous synchronized, relatively high frequencies

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................................................... ................... Figure 7-38 Electroreception allows electric fish to recognize and locate objects in the environment. Objects with a higher conductivity than water deflect the current along the flow axis. Objects with lower conductivity than water (inset) will deflect the current from the flow axis. [From H.W. Lissman, "The Electrical Position of Fish". Copyright 0 1963, Scientific American, Inc. All rights reserved. ]

sideline system

Their electrical organs depolarize, and the way these fish generate and utilize electrical discharges is similar to how muscles are controlled to produce movement (see Chapter 10).

Temperature Sensation Temperature is an important environmental variable, and many organisms obtain sensory information about temperature through the action of specialized nerve endings or temperature receptors in the skin. Higher-order neurons receive input from thermoreceptors and contribute to the mechanisms that regulate body temperature (see Chapter 16). In addition, some neurons in the vertebrate hypothalamus are able to detect changes in body temperature. Thermoreceptors can be very sensitive. An example is the infrared (radiant heat) detectors in the pits on the face of rattlesnakes (Fig. 7-3912). Receptor membranes consist of branched ends of sensory nerve fibers with no apparent structural specialization.

The ends appear to detect changes in tissue temperature, rather than radiant energy itself. The mechanism by which temperature changes can alter receptor production is unclear. If the temperature in the pit increases by only 0.002 °C, sensory axons from the pit organ of the rattlesnake temporarily increase their firing rate, and this change in receptor firing rate can alter behavior. For example, a rattlesnake can detect radiant heat from a mouse 40 cm away if the mouse's body temperature is at least 10 °C above the ambient temperature. In addition, thermoreceptors are located deep in the facial pits, an arrangement that enables the snake to sense the direction of a source of radiant heat (Fig. 7-39B). Both the integument and upper surface of the mammalian tongue contain two types of thermoreceptors: those that increase their discharge when the skin is heated ("thermoreceptors") and those that increase their discharge when the skin is heated ("cold" receptors ). These receptors are also very sensitive. Humans can perceive changes in skin temperature of only 0.01°C. two categories

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Time (s) Figure 7-40 The frequency of APs in mammalian thermoreceptors varies with body surface temperature. (A) Steady-state firing rates of caloric receptors suspended over the surface of the mammalian tongue. (B) Time course of cold receptor responses when the tongue is cooled first and then warmed, shown in black. [According to Zotterman, 1959.1

At temperatures around 30–3 °C, this pattern changes for both types of receptors and the frequency of APs decreases (see Figure 7-40A). Thermoreceptor responses consisted of large transient changes in firing rate followed by a longer sustained steady-state phase. The transition phase is an accurate response to any temperature change (Figure 7-40B), although the steady state phase behaves as shown in Figure 7-40A.

Vision Figure 7-39 The facial pits of rattlesnakes contain extremely sensitive temperature receptors. (A) Facial pit structure of the rattlesnake Crotalus viridis. (B) The location of the facial pit sensitizes the direction of thermoreception in the pit organ. [Adapted from Bullock and Diecke, 1956.1

The types of thermoreceptors differ from each other in that they respond differently to changes in temperature that approach normal human body temperature (about 37°C). Both hot and cold receptors increase their emissivity as the temperature gradually deviates from 30 to 35 °C (Figure 7-40A): as the temperature gets warmer, the hot receptors emit faster; The discharge rate will increase. However, if the temperature and

Since the formation of the Earth more than 5 billion years ago, sunlight has been an extremely powerful selective force in the evolution of organisms, and most organisms have been able to respond to light in some way. Light-sensing involves converting photons into electrical signals that the nervous system can interpret, and the light-sensing organ—commonly known as the eye—has evolved in many shapes and sizes, and in many different designs. Interestingly, although the physical structure of the eye varies widely between species, visual transmission relies on a remarkably conserved set of protein molecules that provide a light pathway, direct light to the photoreceptor surface and capture the photoreceptor Photons within. Preservation of this vision molecule suggests that an appropriate biochemical approach to the problem has been developed

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Despite problems with capturing light energy, these sequences were preserved despite being packaged into organs with distinct structural features. For example, opsins are protein visual pigment molecules. Each molecule contains seven transmembrane domains. Opsin is coupled to photopigment molecules, which change in structure due to the absorption of photons, thereby changing the properties of opsin (see Figure 7-3). Opsins are ubiquitous in the animal kingdom, even in extremely simple light-sensing structures that lack the features that make up an eye. In many organisms, the structure of the eye has evolved to collect and focus incoming light before it reaches the site of transmission. The eye refracts light through the highly concentrated soluble proteins that form the lens, and these refracting structures also have an interesting evolutionary history. Let's start by looking at how the eye collects and focuses light.

The physics of light severely limits the structure of the eye that produces usable images. Most possible designs were "discovered" during evolution, leading to unrelated animals with similar structures. One of the most famous examples of convergent evolution is the eye similarity of phylogenetically unrelated squid and fish. These eyes are similar in many details because the laws of optics dictate convergent solutions to underwater vision problems. On the contrary, the eyes of humans and fish are similar because they have the same things in common.

common evolutionary lineage, although they differ somewhat because the two species live in different optical media. Eye development occurs in two stages. Nearly all major groups of animals have evolved simple eyespots, consisting of receptors located in cups that shield the opening of pigment cells (Fig. 7-41A). Some biologists estimate that such photon detectors have evolved independently 40 to 65 times. Eyespots provide information about the distribution of light and dark in the environment, but not enough information to detect predators or prey. To recognize patterns or control movement, animals need an eye with an optical system that narrows the angle of acceptance of individual receptors and creates a kind of image. This stage of optical evolution is less common, occurring in only 6 of the 33 metazoan phyla (Cnidaria, Molluscs, Annelids, Claweda, Arthropoda, and Chordates). Since this phylum accounts for approximately 96% of all extant species, it is tempting to speculate that the presence of eyes confers a significant selective advantage. So far, ten optically distinct imaging eye designs have been discovered. They include almost all known possibilities in physical optics, with the exception of Fresnel and zoom lenses. In addition, there are variants, such as array optics, that have not been used by physicists who study optics. Simple eyepoints are typically less than 100 µm in diameter and contain 1 to 100 receptors. Even simple eyepoints enable visually controlled behavior. In protozoa and flatworms, the direction of light sources is detected using shielding pigments that cast shadows on photoreceptors. some flagellates such as

Second

Optical Mechanisms: Evolution and Function

shallow pit

vertebrate eyes

I am B

Simple evolutionary relationships between eye types [adapted from Land and Fernald, 1992.1].

Man

Losjog

Figure 7-41 The structure of the eye includes many different optical elements (A). The smallest eyes consist of flat open areas filled with photoreceptor cells. (B) In a slightly more complex eye, the opening of the eye is small relative to the size of the eye, and the eye functions like a pinhole camera (C). An alternative improvement to achieve image formatting is to add a refractive element between the aperture and the photoreceptor layer (D). Three lenses arranged in a row improve the optical properties of the eye The Pontella copepod (E) vertebrate eye is an evolution of ommatidia with the addition of a small aperture and a Posa lens

multiple lenses in series

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.................................... has a base of light-sensitive organelles near the flagella, surrounded on one side by Tinted eye spot occlusion. This shielded organelle provides a rough but effective orientation cue. When a flagellate swims, it rotates around its long axis once a second. When it enters a beam of light shining from one side and perpendicular to its trajectory, the eye point is shaded every time a shielding pigment passes between the light source and the light-sensitive part at the base of the flagella. Each time this happened, the whip moved just enough to rotate the whip slightly to the side with the protective paint. The net effect is to turn the flagellate towards the light source. The simplest eyes are created by reducing the aperture opening to create pinhole eyes (Figure 7-41B) or by adding refractive structures (Figure 7-41C) to enhance the eye point. The evolutionarily ancient cephalopod mollusk Nautilus had a perforated eye, which was quite advanced except for lacking a lens. It is nearly 1 cm in diameter and has a variable opening that expands from 0.4 to 2.8 mm. In addition, the extraocular muscles stabilize the eyes by compensating for the rocking motion that occurs when animals swim. Most aquatic animals have a single-chambered eye with a spherical lens (see Figure 7-41C). This type of lens provides the high refractive power needed to focus images underwater, but suffers from spherical aberration. Lenses found in fish and cephalopods prevent this

connect

Eye

vertebrate eyes

Difficult because the material of the lens is not uniform. Instead, it is dense, with a high refractive index in the center, and a gradient of decreasing density and decreasing refractive index towards the periphery. This pattern was first noticed by Matthiessen in 1877, who showed that the consequence of the density gradient was a short focal length, about 2.5 times the radius (known as the Matthiessen ratio). This pronounced density gradient has evolved eight times in aquatic animals, showing that it is a very good and perhaps the simplest solution. The eyes of other aquatic creatures have multiple lenses. For example, the eye of the copepod Pontella (Fig. 7-4 ID) contains three lenses in series that work together to correct for spherical aberration. The vertebrate eye (Fig. 7-41E) combines a relatively small aperture with a refractive lens. These two features combine to produce a very high-quality image, focused on the light-sensitive layer of the retina at the back of the eye. compound eyes

The compound eyes of arthropods are imaging eyes composed of many units, each unit has the characteristics of the eyes shown in Figure 7-41C. Each optical unit (called an ommatidium) is aimed at a different part of the field of view (Fig. 7-42A), and each unit scans an angular cone occupying approximately 2-3 degrees of the field of view. In the vertebrate eye, by contrast, each receptor may cover only 0.02 degrees of the field of view. because of the receptive field

Figure 7-42 Compound eyes produce mosaic images. (A) In a compound eye, each ommatidium scans a different part of the visual field through a separate lens. The image on the right shows a mosaic image of a butterfly as seen by a dragonfly at a distance of 10 cm. (B) In a simple eye, each receptor cell scans part of the field of view through a lens common to all receptor cells. For comparison, the image on the right shows the same butterfly as seen by a simple vertebrate eye. Arrows indicate that the optical system of the vertebrate eye inverts the image on the retina, whereas the optical system of the compound eye does not. [Adapted from Kirschfeld, 1971 and Mazokhin-Porshnyakov, 1969.1

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Each unit in the compound eye is relatively large. The vision of compound eyes is not as good as that of vertebrate eyes. Although the mosaic image produced by this eye is coarser than that produced by vertebrate eyes (Fig. 7-42B), it is still easily recognizable.

Meter readings are accessible and their activity can be monitored using simple electronic logging techniques. Limulus compound eye visual receptor cells are located at the base of each ommatidium (Fig. 7-43B and C). Each ommatidium lies beneath a hexagonal portion of the outer transparent layer (corneal lens). The primary photoreceptors are 12 retinal cells that surround the dendrites of another neuron, the eccentric cell. Each retinal cell has a striated nodule, and the cell's surface membrane is thrown into dense rnaovilli masses, which are tiny tubular protrusions of the surface membrane (see Figure 7-43D). Microvilli significantly increase the surface area of the cell membrane in the rhablast. Light enters through the lens and is absorbed by the photopigment rhodopsin molecules located in the rhodopsin receptor membrane. Temporary, random depolarization of the membrane po-

Each ommatidium of the horseshoe crab's compound eye contains multiple photoreceptors. The best studied invertebrate photoreceptors are those in the lateral and ventral eyes of the horseshoe crab Limulus polyphemus (Figure 7-43). The two lateral eyes of Limulus are typical compound eyes, similar to the compound eyes in Figure 7-42A, while the unpaired ventral eyes are simpler in structure, more similar to the eye points in Figure 7-41A. Most early electrical recordings of monovision units were made with this side eye, since this eye was an experimental eye

Light

Eccentric cells

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Light

one

Figure 7-43 Early studies of the compound eyes of Limulus polyphemus provided insights into visual transmission. (A) The side eye of Limulus horseshoe crab is located on the carapace. (B) Cross-section of a lateral eye composed of ommatidia. (C) Structure of singleommatidium (outlined in red in part B). Light enters through the lens and is intercepted by the visual pigments in the rhastoids of retinal cells. These cells are arranged like pieces of an orange around the dendrites of the eccentric cells. When light falls on the striated nodes, the eccentric cells depolarize and generate APs. (D) Electron micrograph of a cross-section through an elastomeric microvilli. [Part C of How Cells Receive Stimuli by W.H. Miller, F. Ratliff, and H.K. Hartline. Copyright 01961 Scientific American, Inc. all rights reserved. Part D was provided by A. Lasansky. ]

Light

outside of

1 pm

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................................................. When eyes are exposed Because it's very weak even lighting. The frequency of these "quantum bursts" in the image increases with increasing light intensity, causing more photons to hit the receptor. Transient depolarization is the electrical signal produced by the absorption of a single photon by a single photopigment molecule. A single photon captured by a single visual pigment molecule in Limulus produces a receptor current from A. This transduction event amplifies the energy of the photon absorbed between lo5 and lo6. How does capturing a single photon lead to the rapid release of so much energy? In this case, amplification occurs through a series of chemical reactions within the cell, including G protein activation (see From transduction to neuronal properties earlier in this chapter). The net effect is to open ion channels, allowing cations to enter the cell. In Limulus, receptor currents are carried by light-activated channels of Na+ and KC. This current induces a depolarizing receptor potential through a mechanism similar to the depolarizing postsynaptic potential generated when acetylcholine activates motor channels on muscle endplates (see Chapter 6). When the light is turned off, these channels close again and the membrane repolarizes. The sensitivity of individual photoreceptors decreases with exposure to light, and this adaptation is thought to be mediated by Ca2+ ions entering the cell when light opens ion channels, which then reduce current flow through light-activated channels. Although retinal cells have axons, they do not appear to produce AP. Instead, acceptor currents appear at

Retinal cells spread through low-resistance gap junctions in the dendrites of eccentric cells, from where the depolarization spreads to the axons of eccentric cells, where it generates APs. AP is delivered to the central nervous system in the optic nerve. Although the horseshoe crab eye is structurally simple compared with that of vertebrates, the horseshoe crab's visual system is capable of generating electrical activity that matches some of the more complex features of human visual perception (Focus 7-1). Perceiving planes of polarized light The arrangement of cells in the ommatidium confers special abilities on some arthropods. For example, some insects and crustaceans are able to behaviorally orient themselves toward the sun even when the sun itself is blocked. This ability depends on the polarization of sunlight, which varies in different parts of the sky. It has been found that many arthropods can detect the plane of the electric vector of polarized light entering the eye, and some arthropods use this information for orientation and navigation. Measurements of birefringence (the ability of a substance to absorb light of different planes of polarization) in crayfish retinal cells showed that absorption of polarized light was greatest when the plane of the electro-optic vector was parallel to the long axis of the crayfish, microvilli of the striated muscle. Each ommatidium is composed of seven cells, and the striated nodules of the seven retinal cells are interlaced to form striates. In striated muscles, the microvilli of some receptors form a 90-degree angle to the microvilli of a second set of receptors (Fig. 7-44). when photosensitive pigment molecules

rhabdomocyte retinal cell microvilli

Figure 7 4 4 The structure of the ommatidium allows some arthropods to perceive the plane of polarized light. (A) Interlocking striated muscles of individual retinal cells produce two sets of mutually perpendicular microvilli. (B) Electron micrograph of the cross-striped part

They consist of two sets of microvilli. The upper microvilli were cut parallel to their long axis and the lower microvilli were cut perpendicular to their long axis. [Part A after Horridge, 1968; Part B by Waterman et al., 1969.1

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judgment of the human subject

be asked for

COM-

Comparing the Strength of Different Lengths

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Photopigment molecules separated by photon conversion

Parameters of Photoreceptor Activity and Stimulation Al-

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Although these receptors share some features with human pho-

Illustrates differences in flash intensity and duration

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Follow the flash frequency up to almost 10 Hz (part B in the opposite picture). Above this frequency, the receptor potential can no longer follow the flash. Instead, pulses in V coalesce into a steady degree of depolarization (see Figure 7). -55 also) Actron

Potentials in sensory fibers no longer exist

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For example the nervous system.

Corresponding to the flashing frequency of sending messages

1. Frequency of APs Recorded from Axons of Individual ommatldla vs.

logarithm of intensity

The logarithmic relationship of the stimulus light (right part A of the attached figure) is also typical

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continuously,

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(A) When the flash is shorter than 1 s, the product of intensity and duration determines the amount of AP produced by Limulus photoreceptors. Responses to brief, bright flashes were indistinguishable from responses to weaker but longer-lasting stimuli. (B) Flashing light above a certain frequency cannot be distinguished from constant illumination. The on-off pattern of stimuli is shown below the recorded responses to stimuli from Limulus photoreceptors. At 10 Hz, the photoreceptor follows the flicker; at 12 Hz, the photoreceptor becomes less accurate at detecting flicker; at 16 Hz, the response in the photoreceptor is continuous. [Part A is based on Hartline, 1934; Part B of How Cells Receive Stimuli by W.H. Miller, F. Ratliff, and H.K. Hartline. Copyright O 1961 by Scientific American, Inc. all rights reserved. ]

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................................................... ................................... Polarized light planes from arthropods. In fact, in electrical recordings from individual crayfish retinal cells, the response to a given light intensity varies with the polarization plane of the stimulating light, consistent with this hypothesis (Fig. 7-45).

R e t ~ n u l a cell r

cell a

400

500

600

700

500

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Figure 7-45. The response of Krebs photoreceptors to polarized light varies with the plane of polarization. Two cells a and b are exposed to a series of flashes of polarized light of the same energy but of different wavelengths. The color (expressed as wavelength in nanometers) of each flash is shown along the bottom axis. Cell a responds maximally to light with a wavelength of approximately 600 nm; cell b responds maximally to light at 450 nm. When the plane of polarization (red arrow) is perpendicular to the microvilli, the response of both cells is low (left). When the plane of polarization (red arrow) is rotated so that it is parallel to the t6e microvilli (right), the response of both cells is enhanced. [Adapted from Waterman and Fernandez, 1970.1

Systematically arranged in the microvilli and preferentially absorbs each beam of light, its electric vector is parallel to the microvilli, and the anatomical structure within the striation can provide a physical basis for detection

The Vertebrate Eye The vertebrate eye (see Figure 7-41E) exhibits certain camera-like structural features. In a camera, the image is focused on the film by moving the lens forward or backward along the optical axis. For example, in order to focus on an object close to the camera, the lens must be placed relatively far from the film. To focus on distant objects, the lens moves forward. In the vertebrate eye, incident light is focused in two stages. In the early stages, incoming light rays are diffracted as they pass through the cornea, the clear outer surface of the eye (Figure 7-46). They bend further. Or reflect as they pass through a second structure (the lens) and eventually form an inverted image on the back inner surface of the eye (the retina). In fact, most of what happens in the eye (85% of the total) takes place at the air-cornea interface, with the rest depending on the action of the lens. Like a camera, some bony fish focus the image on the retina by moving the lens of the eye relative to the retina. (This principle of changing the distance between the lens and the photosensitive surface is also adopted by some invertebrates. For example, in the eyes of jumping spiders, the position of the lens is fixed, and the focus depends on the movement of the retina.) On the contrary, in the higher vertebra In an animal's eye, neither the lens nor the retina move. but pictures

straight muscle

Figure 7-46 In the mammalian eye, incident light is refracted by the cornea and lens and focused on the light-sensitive retina. In this figure, the refraction of light has been simplified; there is no refraction at the interface with the air cornea. The image focused on the retina is upside down

through the lens. The lens is held in place by zonular fibers. When the ciliary muscle fibers contract, the tension on the zonular fibers decreases and the elastic properties of the lens cause it to round, resulting in a shortening of the focal length.

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Focusing is done by changing the curvature and thickness of the lens. Changing the curvature of the lens surface changes the distance at which the image transmitted through the lens is focused, known as the focal length of the lens. The shape of the lens is changed by changing the stress applied to the periphery of the lens. The lens is held in place by zonular fibers arranged radially (see Figure 7-46). The fibers of the zonules exert outward tension on the periphery of the lens. The radially located ciliary muscles regulate the tension exerted on the lens. When the ciliary muscle relaxes, the lens is flattened by the elastic tension of the zonule fibers, which pulls the lens periphery outward. In this state, objects far from the eye are in focus on the retina, while objects closer to the eye are out of focus. Contraction of the ciliary muscle brings objects near the eye into focus on the retina, which relieves some of the tension on the lens and makes the lens more round. This process is called conditioning to close the object. As humans age, their ability to accommodate decreases as the lens becomes less elastic, leading to a type of "hypermetropia" known as presbyopia. Accommodation is probably not the mechanical mechanism that changes the focal length of the lens, but the neural mechanism by which images are "selected" from all the complexity of the visual environment to focus correctly on the retina as a result of nerve impulses to the ciliary muscle . A related neural mechanism produces binocular convergence, in which the left and right eyes are positioned by the ocular muscles so that images received by both eyes fall on similar parts of both retinas, regardless of the distance between the object and the two eyes. When an object approaches When moving, the eyes must turn toward the middle of the nose; when an object moves away, the eyes turn outward from the centerline. Response to changes in light intensity In cameras, the intensity of light incident on the film is controlled by adjusting the aperture of the mechanical shutter, which allows light in when the shutter is closed.

gamma rays

Ter opens. The vertebrate eye has an opaque iris with an opening called the pupil, similar to the mechanical aperture of a camera. When the circular smooth muscle fibers in the iris contract, the diameter of the pupil decreases and the amount of incident light that can enter the eye decreases. The pupil dilates as the radial muscle fibers contract. The contraction of these muscles -- and the diameter of the pupil -- is controlled by central nervous reflexes sent by the retina. This pupillary reflex can be demonstrated in a dimly lit room by suddenly shining a flashlight on a person's eyes. Changes in pupil diameter are temporary. After a few minutes of responding to sudden changes in light levels, the pupils gradually return to average size. Furthermore, the area of the pupil can only change by a factor of about five, so it cannot cope with the six or more orders of magnitude changes in light intensity that the eye typically experiences. Therefore, although the pupil can make rapid adjustments to modest changes in light intensity, other mechanisms must be available. The eye adapts to extreme light conditions through changes in the state of visual pigments and neuronal adaptation processes (see Adaptation Mechanisms earlier in this chapter). Another benefit of pupil constriction: improved image quality on the retina. The edges of the lens are less optically perfect than the center; therefore, when the pupil constricts, it prevents light from passing through the periphery of the lens, reducing optical aberrations. Depth of field (the distance at which an object is in focus when the lens is in a fixed shape) increases as the diameter of the pupil decreases, just like in a camera when the aperture decreases. Vertebrate Visual Receptor Cells The stimulus for all visual receptor cells is electromagnetic radiation in a specific energy range called visible light (Figure 7-47). The energy of electromagnetic radiation changes inversely proportional to its wavelength, and we see this energy change as a color change. Purple light, the highest light energy possessed by human beings

X-ray film

ultraviolet light

Infrared

short radio waves

wavelength log

Figure 7-47 The spectrum of electromagnetic radiation covers a wide range of energies and can be detected by different sensory modalities. Most visual receptors detect energy in a variable range, but some can also detect energy in ultraviolet light. Some snake organs can sense infrared chakra latlons [adapted from Lehnger, 1993]

400

500

600 wavelength (nm)

700

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................................................... ................................Eye response, wavelength about 400 nm. The low-energy red light has wavelengths at the end of the visible spectrum between 650 and 700 nm. Bright light provides more energy per unit of time than low light. Photoreceptors, which capture light energy and convert it into neural signals, are located in the retina of the vertebrate eye. In mammals, birds, and other vertebrates, the retina contains multiple cell types that are interconnected in networks. The visual receptor cells themselves fall into two categories: rods and cones, named for the shape of the cells when viewed under a microscope (Figure 7-48). All neurons in the retina, as well as epithelial cells, contribute to the light response of the vertebrate eye, but rods and cones have different physiological properties. Cones, for example, work best in bright light and are protective

Video offers high definition, and the wand works best in low light. Different animals use these different abilities to provide specific visual abilities. For example, animals that live in flat, open environments, such as cheetahs and rabbits, often have horizontal visual stripes, regions of the retina that contain an abnormally high density of cone receptors. Such a region corresponds to the horizon in the visual world and is intended to provide maximum resolution for that part of the scene. The visual striae also contain large numbers of ganglion cells -- cells that transmit visual information to the brain. In contrast, tree species (and humans) typically exhibit radially symmetric photoreceptor density gradients. An important feature of this type of retina is the fovea, or central area. It is a small portion (approximately 1 mm2) in the center of the retina of many mammals and provides very detailed information

Pigment epithelium

eyelash

Figure 7-48 Photoreceptors in vertebrates are classified as rod-shaped or cone-shaped according to their morphology and physiological characteristics. The outer parts of the rods and cones that capture light are facing away from the

from the light source. The light-absorbing pigments are contained in the membranous layer, and the outer layer ends in close proximity to the pigmented epithelium.

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Information about the visual world, a characteristic known as high visual acuity. In humans and certain other mammals, the fovea contains only cones, while the rest of the retina contains a mixture of rods and cones, with rods significantly outnumbering cones. In mammals, cones mediate color vision, while rods, which are more light sensitive, mediate only achromatic vision. However, this distinction between rods and cones does not apply to all vertebrates. In fact, due to morphology, the retinas of some species contain only rods but may still be capable of color vision. Rods and cones are more similar in structure and function than various invertebrate photoreceptors. Each vertebrate recipient cell contains a segment with an internal structure resembling a cilium. This underdeveloped cilium connects the outer segment containing the photoreceptor membrane to the inner segment containing the nucleus, mitochondria, synaptic contacts, etc. (see Figure 7-48). The receptor membrane of vertebrate photoreceptors consists of flattened sheets that arise from the surface membrane near the origin of the outer segment. inside

In mammals and some other vertebrates, the lumen of each lamella is open to the outside of the cell. In the rod, the flakes are completely detached from the surface membrane of the outer part, forming flat pouches or discs that are stacked like pita bread on the outside of the rod. The disk stack is completely contained in the surface film of the photoreceptor. Photopigment molecules are embedded in it. disk membrane. Since photopigments are present in the disc membrane of the rod outer segment but not in the surface membrane, the main photochemical transduction steps must occur in the disc membrane rather than the surface membrane. The eyes of many invertebrates lack the ciliary structure that connects the inner and outer parts of vertebrate rods and cones (Figure 7-49). In the eyes of these invertebrates, photopigments are present in microvilli formed by cell membranes, and these pigment-containing microvilli form the rhizomes. Since many invertebrate species have simple eyes in which the photoreceptors are of the rhizoid type, it might be tempting to conclude that rhabdomid photoreceptors are only present in simple eyes. arthropod

Ciliated Vertebrate

.Echinodermata

horizontal line

Figure 7-49 Vertebrate photoreceptors contain a typical 9 2-cilium structure connecting the inner and outer parts, but many invertebrate photoreceptors lack this ciliated structure and instead contain many Rnicrovilli. This figure illustrates the phylogenetic distribution of ciliary eyes and rhomboid eyes. However, there are exceptions. Both the scallop Pecten and the surf clam Lima have complex eyes with two layers of photoreceptors. One layer contains ciliary photoreceptors and the other contains rhastoid receptors. [Adapted from Eakin, 1965.1

However, octopus eyes are optically very complex, with bullet-shaped photoreceptors. In addition, the eyes of some bivalves, such as scallops (Pecten) and clams (Lima), have two separate layers of photoreceptors. One layer contains ciliary receptors and the other contains rhizoid receptors.

In all photoreceptors, the transfer of light energy results in a change in membrane potential; however, the transduction effect differs between vertebrate and invertebrate photoreceptors. Invertebrate photoreceptors depolarize in response to light (Figure 7-50A; see also Figure 7-45), but vertebrate rods and cones hyperpolarize in response to light stimulation (Figure 7-50B ). Membrane conductance measurements before and during light exposure revealed that the effect of light on vertebrate photoreceptors is to reduce the sodium (gNa) conductivity of the outer segment membrane. In the dark, the surface membranes of vertebrate outer rod segments are nearly equally permeable to Na+ and K+, while V, is approximately between EK and EN,. In this state, Na+ ions enter the outer segment through channels that are continuously open in the dark (Fig. 7-51A). Na+ ions that carry this inward current, hence the name dark current

Invertebrates

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raise gram,

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time

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nn lights up

light up

Figure 7-50 Most invertebrate photoreceptors depolarize in response to stimuli, whereas vertebrate photoreceptors hyperpolarize. (A) In most invertebrate photoreceptors, conversion of light energy to chemical energy results in increased surface membrane permeability to Na+ and K+, thereby depolarizing the cell. (B) Vertebrate photoreceptors respond to light with reduced surface membrane gNa leaving residual low g and shifting V towards EK. As a result, the cells hyperpolarize.

It is maximal in the dark and is prevented from accumulating in cells by stabilization of the metabolically controlled Na+, K+ ATPase. Dark current exists only in the photoreceptors of vertebrates, not in the photoreceptors of invertebrates. After the photopigment absorbs light, the conductivity of the sodium gNa in the outer part decreases, resulting in a decrease in dark current and hyperpolarization of V towards EK (see Figures 7-50B and 7-51B). When photostimulation ceases, membrane gNa returns to its high resting level and V,,, becomes more active and returns to a resting level between ENa and EK. Changes in V,,, upon light onset are transmitted electrotonically (see Passive electrical signal propagation in Chapter 6) to the interior of the photoreceptors. Internally, changes in V,, regulate the steady release of neurotransmitters at presynaptic sites in the basal portion of the inner segment. Like vertebrate auditory receptors, vertebrate photoreceptors lack axons. They connect to other neurons that carry visual signals to the central nervous system. Nerve signals are relayed by other neurons to retinal cells and ultimately affect the activity of axons projecting to the brain within the optic nerve. In vertebrate photoreceptors, the inner segment continuously secretes a transmitter while being partially depolarized by dark current. The hyperpolarization that occurs in response to light exposure reduces the amount of transmitter passed to the next neuron in the chain, altering the activity of that second-order neuron. Extracellular electrodes can record changes in membrane potential produced by a group of photoreceptors when they are illuminated, as can action potentials traveling along nerve axons. Many photoreceptors are tiny cells, making intracellular recording difficult. Consequently, this recording method, known as an electroretinogram, has proven to be very useful in vision research (Spotlight 7-2). Photoreceptors: convert photons into neuronal signals Middle) generate AP and transmit it to the central nervous system. The process of visual transduction has received a great deal of research attention, and the characterization of the visual process provides clues for physiologists to study sensory transduction in other sensory modalities. Photosensitivity studies have been performed in many different species from several phyla. Many similarities have been found between vertebrate and invertebrate photoreceptors, although it is now thought that the invertebrate photoreceptor may be more complex because it relies on two light-activated signaling pathways:

exist

Figure 7-51 Illumination reduces dark current in vertebrate rods. The g of the outer segment of the rod is higher in the dark (A) and lower in the light (B). Therefore, the dark current carried by Na+ ions entering the outer segment decreases during illumination. In the equivalent circuit (top left), the battery is Na+, K+ ATPase, and the photovaristor (R,,) represents gNa in the outer segment. [Adapted from Hag~ns, 1972.1

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rather than a single pathway found in vertebrates. There are other differences that may be relevant. For example, a single photon captured by a horseshoe crab photoreceptor produces a peak current of -1 nA, while a single photon captured by a vertebrate rod photoreceptor changes the current by -1 pA, three orders of magnitude smaller. Furthermore, invertebrate photoreceptors can respond to light intensities of seven orders of magnitude, whereas vertebrate rods only respond to within four orders of magnitude. Despite these differences in detail, all types of photoreceptors have evolved to convert photon energy into neural energy, and the study of all types of eyes contributes to our understanding of this process.

Visual pigments The spectrum of electromagnetic radiation ranges from gamma rays with wavelengths as short as 10-12 cm to radio waves with wavelengths longer than 10-12 cm (see Figure 7-47). parts of the electromagnetic spectrum

The wavelength of light between cm and lo-' cm is expressed. Humans can only see a small portion of this spectrum, ranging from about 400 nm to about 740 nm. Below this range is the ultraviolet (W) part of the spectrum, and above this is the infrared (IR) part invisible to humans and other mammals. There is nothing qualitatively special about the parts of the spectrum that make them invisible to us. Instead, what we see depends on which wavelengths are absorbed by our visual pigments. For example, with a condition called cataract, the lens becomes opaque. Treatment involves surgical removal of the lens; after surgery, patients can see light in the W range because absorption of UV light by the lens prevents people from seeing these wavelengths. The compound eyes of many insects can detect light in the ultraviolet range, which means that some flowers that contain UV-reflecting pigments are much less visible to insects than to mammalian eyes, but all animals respond to only part of the spectrum of electromagnetic radiation available for

Spot 7-2

this

Activity of photoreceptor cells and other neurons in the retina. It took us a few years to discover where every component of ERG came from, but we now believe this is a wave

electroretinogram

Receptor currents due to visual receptor cells. The b wave follows the a wave and is produced by electrical energy.

In teaching laboratories, it is sometimes useful to record the sum of the eye's electrical activity, which is technically much simpler than recording individual cells with microelectrodes. Recording electrode (can be filament or filament)

Trial activity of second-order retinal neurons receiving input from recipient cells. C waves are unique to vertebrates and appear to be generated by pigment epithelial cells attached to the outer segments of photoreceptor cells. exist

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In the developing eye of tadpoles, the ERG contains only one wave preceding synaptic contacts. also in

The ground electrode is connected to another part of the body. if

Eyes of adult frogs carry out synaptic transmission between photons

A beam of light is shone on the eye, and electrodes record a complex waveform (shown in the adjacent figure). This recording is called an electroretinogram (ERG) and records the sum

Receptors and secondary neurons are pharmacologically blocked, and the ERG consists of alpha waves only.

The vertebrate electroretinogram consists of several components, each derived from a different source. Stimulus measurements are shown below the recording [Adapted from Brown, 1974] Off

Sunlight. The visual pigments of vertebrates likely absorb only a limited portion of the electromagnetic spectrum of sunlight because vertebrate life evolved in water, which strongly filters electromagnetic radiation. The spectral region to which photopigments are sensitive in vertebrates (including terrestrial mammals such as humans) is closely related to the water-permeable spectrum. All known organic pigments attribute their ability to selectively absorb light to the presence of carbon chains or rings containing alternating single and double bonds. When a photon is captured by one of these molecules, the energy state of the molecule changes. The energy contained in a radiation quantum is equal to Planck's constant divided by the wavelength A in centimeters:

Therefore, the energy of a photon increases as the wavelength of the radiation decreases. Quanta with wavelengths below 1 nm contain so much energy that they can break chemical bonds and even atomic nuclei; quanta with wavelengths above 1000 nm lack the energy to affect molecular structure. Between these two limits, visual pigment absorption is greatest. When a certain amount of radiation is absorbed by a photochrome molecule, it increases the energy state of the molecule by increasing the orbital diameter of the electron associated with the conjugated double bond

This is the same process by which plants convert radiant energy into chemical energy through photosynthesis. Photochemistry of Visual Pigments The energy content of visible light is low enough to be absorbed by molecules without breaking them down. Pigments are essential to the process of absorbing light and converting its electromagnetic energy into chemical energy The concept originated with John W. Draper, who concluded in 1872 that light must be absorbed by molecules in the visual system R. Boll soon discovered that the characteristic reddish-purple color of frog retinas faded (bleached) when the retina was exposed to light. The photosensitive substance that produces purple, rhodopsin, was obtained in 1878 by W. Kühne, who also found that after this pigment had been photobleached, the retina could regain its reddish-purple color if the receptors were located in the eye. The cells make contact with the pigment epithelium at the back of the eye. Since then, much has been learned about the chemistry and physiology of rhodopsin. It absorbs light at a wavelength of approximately 500 nm maximally. It is present in the outer segment of rods in many vertebrates and in the photoreceptors of many invertebrates. Rhodopsin molecules pack into the receptor membrane at high density; there can be up to 5 x 1012 molecules per square centimeter, which corresponds to an intermolecular distance of approximately 5 nm.

All known visual pigments are composed of two main components: a protein (opsin) and a light-absorbing molecule. In all cases, the light-absorbing molecule was retinal or 3-dehydroretinal (Figure 7-52). Retinal, the aldehyde of vitamin A, is a carotenoid. Vitamin Al is an alcohol, also known as retinol; 3-dehydroretinal is an aldehyde of vitamin A, also known as 3-dehydroretinol. In addition to its main constituents, rhodopsin contains polysaccharide chains composed of six sugars and a variable number (up to 30 or more) of phospholipid molecules. The lipoprotein opsin, which binds phospholipids and polysaccharide chains, appears to be an integral part of the photoreceptor membrane. During the bleaching and regeneration of visual pigments, carotenoid molecules move back and forth between the photosensitive membrane and the pigment epithelium at the back of the retina. (By the way, the pigment that gives the pigmented epithelium its dark color is photochemically inactive and has nothing to do with visual pigments. Instead, it prevents light from being scattered and diffusely reflected back into the retina.) Retinal molecules assume two spatially distinct retinal conditions. In the absence of light, opsin and retinal are covalently linked via a Schiff base, and retinal exists in the 11-cis configuration (see Figure 7-3). When the 11-cis retina captures a photon, it isomerizes to the all-trans configuration (see Figure 7-52). This cis-trans isomerization is the only direct effect of light on visual pigments. The switch from 11-cis to all-trans retina triggers a series of changes in the relationship between retina and opsin, including a change in the conformation of the opsin itself.

When light hits the photopigment, an intermediate, rhodopsin II, is formed. Rhodopsin I1 activates another membrane-associated protein and binds GTP in exchange for GDP. This protein, which we now know to be a member of the G protein family, was named transducin in recognition of its key role in light transport. Activated transducin subunits diffuse across the membrane plane and encounter numerous phosphodiesterase molecules that hydrolyze cGMP to 5'-GMP. In vertebrate photoreceptors, dark current Na+ channels open only in the presence of cGMP; thus, when cGMP is hydrolyzed, these channels close (Figure 7-53). The outer rod segment membrane contains a class of channels permeable to three cations: Na+, Mg2+ and Ca2+. As cGMP levels decrease, conductance through these channels decreases. Most importantly, the internal I,= decreases and the remaining K+ current flows through other channels leading to cell hyperpolarization. When light stimulation ends, cGMP is regenerated by the action of another enzyme, guanylate cyclase. When cGMP levels rise, dark current channels open and the receptor current returns to its full value in the dark. Activated transducin collides with and activates phosphodiesterase molecules at a rate of approximately 106 molecules per second. This allows the capture of a single photon to affect the conduction of a large number of ion channels. This numerical relationship yields an impressive amplification between the capture of a single photon and the event's effect on V. Following cis-trans isomerization of retinal, further changes in the molecule appear to be irregular.

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Space-filled and complete molecular structure diagrams are shown [Part B of "Molecular Isomers In Vison" by R. Hubbard and A. Kropf, Copyright 0 1967 by Scientology America, Inc. All r ~ghights resewed. ]

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This results in excitation of the visual receptor cells, but the subsequent response (Fig. 7-54) is required for active rhodopsin regeneration. Activated rhodopsin spontaneously hydrolyzes to retinal and opsin, both of which can be reused. Free retinal is reisomerized to the 11-cis form and reassembled with opsin to form rhodopsin. The lost or chemically degraded retinal tissue is replaced with vitamin A (retinol), which is stored in the pigment epithelium and actively absorbs the vitamin from the blood. Nutritional deficiencies in vitamin A reduce the amount of retinal that can be synthesized, thereby reducing the amount of rhodopsin available. As a result, the eyes become less sensitive to light, a condition commonly known as night blindness. Rod photoreceptors can respond to the absorption of a single photon, in part because rhodopsin is tightly packed in their disc membranes. In the outer segments of the rods, there are approximately 20,000 rhodopsin molecules per square micron, which corresponds to a much higher density of acetylcholine receptors than at the neuromuscular junction. Denis Baylor of Stanford University measures the response to the capture of a single photon by recording from a single rod (Figure 7-55).

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In the experiment, the rods were pulled apart and one of them was pulled into the receiving straw, where it was stimulated by a small beam of light. When the stimulating light is very weak, small current fluctuations can be recorded, each time a single rhodopsin molecule is photoisomerized by a single photon. The current properties recorded under these conditions were similar to those measured through individual acetylcholine receptor channels at the neuromuscular junction. (The change in current associated with capturing a photon is about 1 PA.) Since photoreceptors can respond to a single photon, or quantum of energy, the sensitivity of a photoreceptor is limited by the quantum nature of light; there is no smaller quantity of light than a photon. Elucidating the process of visual transduction shows the power of comparative approaches. Although photoreceptors in vertebrates and invertebrates differ significantly at the electrophysiological level, they share many similarities at the molecular level. The combination of the molecular genetics available in Drosophila and the physiological accessibility of the vertebrate retina provides an extremely powerful set of experimental methods for addressing how visual information is captured and processed

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Figure 7-53 When light is absorbed by the retina, a series of reactions lead to the closure of Na+ channels carrying dark current. (A) Activated rhodopsin increases the activity of G protein transducers. The activated G protein then activates a number of phosphodiesterase (PDE) molecules, which reduce the intracellular concentration of cyclic guanosine monophosphate (cGMP), causing the Na+ channels that transmit the dark current to close. The recipient cells then hyperpolarize. (B) Current recordings in single-rod photoreceptors isolated from toad retinas. (Left) A flash of light reduces the internal dark current from 10 pA to zero. (Right) Photoreceptors have ruptured, external saline has converted to intracellular concentrations, and when cGMP is added, a very large anaphase is produced by external saline (exposing the interior of the external part to high concentrations of cGMP). Inward current [Part B adapted from Yau and

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Figure 7-54 All-trans retinal dissociates from opsin when rhodopsin is activated. Rhodopsin is reorganized after isomerase converts the retinal tissue back to the II-cis configuration. Retinol (vitamin A) is stored in the pigment epithelium and can be delivered to photoreceptors to create new rhodopsin molecules.

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Time (seconds) Figure 7-55 The rod can respond electrically to the capture of a single photon. (A) A single-rod outer segment is drawn into a smooth glass pipette electrode and illuminated with a narrow light bar while recording the ionic current of the pipette across the membrane. (B) Recorded membrane currents as a function of light exposure. In very low light (lower trace), small individual changes in the current display can

Perform single photon capture. As the light intensity increases (the intensity is shown above each lens), the response becomes greater and smoother. The duration of the illumination is indicated by the bar below each shot. Membrane currents are in PA. [Adapted from Baylor et al., 1979.1

photoreceptors. If the player's molecular identity had not been so strongly phylogenetically preserved, it might have taken much longer to work out the details of visual transduction.

The spectrum of activity of a photoreceptor depends on the absorption properties of its visual pigment. Furthermore, the results of such experiments confirmed that each photoreceptor synthesizes only one visual pigment. Light containing different wavelengths produces photochemical reactions in a given photoreceptor cell that are proportional to the amount of each wavelength absorbed; thus, a photoreceptor cell is excited by different wavelengths, depending on how efficiently its pigments absorb each wavelength. Any photons that are not absorbed have no effect on the pigment molecules; each absorbed photon transfers some of its energy to the molecule, as described in this chapter, "The Photochemistry of Visual Pigments." Thus, Young's theory of trichromaticity in relation to cone photoreceptors and their photopigments can be restated (see Spotlight 7-3): There are three types of cones in the human retina, each containing the Pigment, most sensitive to green or orange light. The electrical power of each class depends on the number of quanta that the pigment can absorb, thus aiding the transduction process. Color perception occurs when higher-order neurons integrate signals received from the three types of cone cells. Understanding the molecular basis of color vision has grown enormously since 1984, when Jeremy Nathans described the molecular structure of human opsin, thereby providing an explanation for inherited color blindness. For example, point mutations in individual pigment genes can lead to disturbed sensitivity to certain wavelengths. Indeed, the molecular basis for the different spectral sensitivities between opsins has been characterized using naturally occurring variants in visual pigments.

The ability of rods and cones to distinguish colors and perceive the visual world in more than shades of gray has to do with having multiple visual pigments, each maximally absorbing a different wavelength (Spotlight 7-3). In vertebrate species with color vision, different classes of photoreceptors have been found to contain spectrally recognizable visual pigments, and each class of photoreceptors has a unique spectrum of activity. That is, the electrical response of each photoreceptor is greatest when illuminated at a particular wavelength and decreases as the wavelength of the incident light increases or decreases. Three classes of photoreceptors are found in many species for which action spectra have been recorded. The action spectra of some species were then compared to the absorption spectra of individual photoreceptors. The absorption spectra of individual photoreceptors are measured using a technique called microspectrophotometry, in which a tiny beam of light is focused on each photoreceptor and the absorption properties of that cell are determined. Photoreceptors studied in this way can be grouped into distinct classes for each species; there is no intermediate spectrum, which means that each photoreceptor synthesizes one visual pigment (Fig. 7-56). Both action and absorption spectra of photoreceptors of many species have been determined, and the two types of spectra agree well with each other, confirming the

Know the environment

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Figure 7-56 Each type of cone cell in carp retina has a characteristic activity spectrum. (A) Absorption spectra of a single cone cell in a goldfish retina showing the presence of three distinct visual pigments, each with a characteristic absorption peak. These measurements were performed using microspectrophotometry, which measures the absorption spectrum of individual photoreceptors. In humans, the cone class corresponding to goldfish red-absorbing cones absorbs maximally at 560 nm, a wavelength in the yellow part of the spectrum. (B) Electrical responses of three individual cones in the carp retina to flashes of different wavelengths, as shown above. The wavelength that elicits the greatest response is different for each of the three cones. (C) When the amplitude of activity in each cell shown in panel B is plotted as a function of wavelength, three types of cones are found, each with an action spectrum close to the one absorption spectrum in panel A . [Part A adapted from Marks, 1965; parts B and C adapted from Tomita et al., 1967.1

11-cis-retinal (or 11-cis-3-dehydroretinal) appears to be the light-absorbing molecule in all visual pigments, and this prosthetic group binds to different opsin molecules to produce visual pigments with different absorption maxima. Differences in the amino acid composition of opsins—rather than changes in the light-absorbing prosthetic group—cause rhodopsins to have different absorption maxima. Nathans and his collaborators identified three genes encoding human cone opsins. The gene encoding the protein part of the blue absorbing pigment is located on an autosome, while the two genes for the "red" absorbing protein and the green absorbing protein are closely linked on the X chromosome. The "red" and green opsins differ in only 15 of their 348 amino acids, and each shares about half of them with rhodopsin in the rods (Figure 7-57). Based on the similarity of the sequences, we can speculate that the genes of these pigments may come from a common ancestral gene that has undergone duplication and divergence. A comparison of the amino acid sequences showed that, in the cone pigments, the blue-sensitive pigments appeared first, followed by the red and green pigments. Color blindness is due to the lack of

Or a defect in one of the cone opsin genes. By combining these molecular markers with visual tests, it is now possible to define the molecular basis of this perceptual problem. For example, a high incidence of

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Figure 7-57. The two opsins in human rhodopsin absorb most in the red and green parts of the visible spectrum, differing by only 15 amino acids, most of which are thought to reside in transmembrane helices. In this diagram, these variable amino acids are in red. [Adapted from Nathans et al., 19861

Spotlight 7 - 3

light, color and

The receptors will be maximally stimulated by the monochromatic "red" and "yellow" wavelengths alone, respectively, and both will be stimulated to a lesser extent by the monochromatic or "yellow" wavelengths.

color vision

Angel light. In other words, Yang proposes that the sensation of "orange" is caused by the simultaneous excitation of "red".

In 1666, Sir Isaac Newton demonstrated that white light splits into colors when passed through a prism. Each spectral color is monochromatic; that is, it cannot be split into further colors. However, by this time it was known that painters could obtain any spectral color (such as orange) by mixing two pure pigments (such as red and yellow), each of which reflected a different wavelength than the color produced. So, between Newton's argument that there are infinite colors in light and the growing awareness of Renaissance painters that all colors can be made from three main pigments—red,

yellow and blue. This paradox arises

Thomas Young's 1802 proposal to solve this problem was that the receptors in the eye are selective for the three primary colors red, yellow and blue. Young reconciles the infinite variety of spectral colors that can be reproduced with the small number of painter's pigments required to produce all colors by proposing that each class of color receptors is more or less excited

receptors and "yellow" receptors. Young knew nothing about photoreceptor physiology, which made his discovery truly remarkable. Extensive psychophysical studies by James Maxwell and Hermann von Helmholtz in the 19th century supported Young's theory of trichromaticity, and William Rushton Rushton's later work provided additional support. However, direct evidence for the existence of three types of color-receiving photoreceptors is lacking. then in

In 1965, WB Marks and E. MacNichol measured the color absorption of individual cone cells in the retina of a goldfish (see Figure 7-56A). They found three types of cones, each absorbing the most at specific wavelengths. Subsequent measurements of the absorption spectra of cone cells in the retinas of humans, monkeys and other fish reproduced these results. Thus, the retinas of species that appear to be able to perceive and respond to color contain photoreceptor cells with different absorption spectra, and in many of these species there are three different classes of receptors.

Degrees of light passing through each wavelength: "red" and "yellow"

Red-green color blindness is caused by recombination of these tightly linked red and green opsin genes. Some representatives of all classes of vertebrates exhibit color vision. In general, the retina containing cones is associated with color vision, but examples of different color categories in rods have been found. For example, in addition to cones, frogs have two types of rods -- red (containing rhodopsin that absorbs cyan) and green (containing a pigment that absorbs blue). If color vision is primarily mediated by cones, what is the role of rods? Rods are more sensitive to light than cones (the recordings in Figure 7-55 were made by rods), and their connections to the next neuron in the series converge more than cone connections (see Vertebrate visual processing). retina, Chapter 1I), leading to a greater sum of weak stimuli. Therefore, the wand is most effective in low light. Because cones produce color vision, when only our rods are stimulated by dim light, we see black and gray instead of colour. In the human retina, most images are preferentially focused on the fovea, which contains only dense cone cells. Rods exist only outside the fovea. The different distribution of rods and cones in the retina means that we are most sensitive to dim light when images outside the fovea are focused on the part of the retina where there are more rods. For example, a dim star will appear brighter if you adjust your sight so that its image is outside the fovea. If you make M you look, do it

If the image falls on the fovea, the stars will fade or even disappear. This increased sensitivity comes at a price: the wider connection between the rods reduces the sharpness of rod vision. Our visual acuity is greatest when the image is focused on the rods outside the fovea; our vision is greatest when we focus the image on the cones of the fovea. As visual pigments were explored throughout the phylogeny, some interesting patterns emerged. For example, all the visual pigments that have a retinal pseudonym are called rhodopsins. All human visual pigments—rod pigments and the three cone pigments—are rhodopsins. Visual pigments with 3-dehydroretinal as a prosthetic group are called porphyrins, and the distribution of rhodopsins and porphyrins between species shows an interesting correlation with the environment. All visual pigments of terrestrial vertebrates are rhodopsins. In addition, rhodopsin is found in invertebrates, including horseshoe crabs, insects, and crustaceans. In contrast, porphyrins have been found in the retinas of freshwater fish, saltfish (see Chapter 14) and some amphibians. This distribution suggests that there are certain properties of porphyrins that make them particularly suitable for freshwater conditions. In fact, anadromous fishes that migrate from freshwater to saltwater (and vice versa) during their life cycle switch their visual pigments between porphyrinubin and rhodopsin during the migration. In fresh water they synthesize porphyrins and in salt water rhodopsins. The absorption maximum of porphyrorubin varies with time.

wavelength, the red end of the visible spectrum, while rhodopsin absorbs most at shorter wavelengths. Perhaps the freshwater environment makes sensitivity to the red end of the spectrum important. Tracing the transfer of information from photon absorption to neural signal generation leaves unanswered the question of how all this information about incoming radiation forms a coherent picture of the world. The information gathered is sent to higher nerve centers where it can be integrated and used to shape behavior—a topic discussed in Chapter 11.

Limitations of Sensory Reception To be most effective, sensory receptors should be highly sensitive to environmental stimuli and encode information with perfect accuracy. In fact, due to the physical properties of stimuli and receptors, no receptor fulfills these requirements; all receptors represent a compromise in the way they receive and encode sensory information. Some physical principles that apply to many sensory modality receptors inevitably limit the accuracy with which cells receive and transmit sensory information. In some cases, the accuracy of sensory perception is limited by the relative strength of the signal and background noise. This signal-to-noise ratio limits the performance of all systems that receive and transmit information, whether present or not. In other cases, the performance and sensitivity of sensory systems are limited by the form of energy to which the receptors are tuned. For example, light is essentially quantized into photons. No receptor can receive less than one light quantum, since light does not exist in fractional quanta. A major source of background noise is a consequence of the third law of thermodynamics. This means that at all temperatures above 0°K the molecules have kinetic energy and are in motion. The heat energy is given by

where k is Boltzmann's constant (1.3805 x 10-16 erg K-1) and T is the absolute temperature. This equation gives the energy associated with molecular motion (i.e. Brownian motion) at the body temperature of the animal. It places a lower limit on the sensitivity of the receptor to detect the signal, since thermal energy provides a constant noise level at which stimulation occurs. In order to detect an external signal, the receiver must be able to distinguish the signal from this thermal noise floor. How easy is it for the recipient cells to accomplish this task? An example is photoreceptors. At a body temperature of 2°C, the thermal energy is approximately 0.58 kcal-mol-' or 4 x 10-14 ergs. We need to compare this energy with that of a typical sensory stimulus. The stimulus for vertebrate photoreceptors is visible light

A portion of the electromagnetic spectrum (see Figure 7-47). The energy of a single photon is given by the Einstein relation:

where h is Planck's constant and v, c and A are the frequency, speed and wavelength of light. Using the values for blue light photons (A = 500 nm), the energy can be calculated to be about 57 kcal-mol-l, almost 100 times the thermal energy. Within the visible range, detection is absolutely not limited by the thermal energy inside the detector. Instead, it was found to be limited by the quantum nature of light itself. When considered, the energy is given by the Einstein relation for a single phonon, a quantum unit of sound energy similar to a photon. Animals hear sounds in a very wide range of frequencies, from 10 to 15 Hertz. Phonons are 7 times more energetic than ergs at these frequencies. In the middle of this 7-fold range, the energy of a single phonon is 10 orders of magnitude (101°) below the detection limit determined by thermal energy. This result suggests that the detection of acoustic stimuli is fundamentally limited by thermal noise and that special mechanisms must exist that enable auditory sensory perception. In fact, there is some benefit in tweaking the detectors to limit their range, a common feature in hearing aid hair cells. A number of mechanisms have been developed to counteract the thermal noise limitation, but direct measurements have also shown that sensory cells in hearing aids faithfully reproduce thermal noise at their input. As discussed earlier in this chapter, most chemical stimuli (odours, tastes, chemotaxis) bind to specific receptors rather than altering ionic currents directly through membrane channels. In this case, the relationship between binding energy and thermal energy determines the detection limit. The binding energy measured in chemical sensor systems is typically around 1 kcal mol-I. This energy is far greater than the thermal energy that chemoreceptors can theoretically calculate for a single molecule. However, the physics of receptor binding has an important limitation. The higher the binding energy, the longer the molecule will bind to its receptor. For a binding constant of lop6M, the association time is seconds; at a binding constant of about three times lo-'' M (giving very high specificity), association persists for more than 5 minutes. Because the performance of a receptor system depends at least in part on comparisons among many receptors, long binding times require comparisons over long periods of time, a mechanism that evolution seems to have avoided. In contrast, moderately high binding constants between chemical stimuli and their receptor molecules reduce the binding energy but also the time required for chemical signal transmission and interpretation.

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The signal-to-noise ratio properties of stimuli that activate electroreceptors and thermoreceptors can be predicted. Electroreception is relatively common in aquatic organisms and is used for navigation, communication and hunting. Electric field energy transmitted through water at the frequencies used is about 10 orders of magnitude lower than thermal energy. Therefore, the electrical reception process, like acoustic reception, must be dominated by thermal noise in the detector. Thermal sensing is based on the detection of photons in the infrared region of the electromagnetic spectrum (lower frequency and longer wavelength than the visible band), and is by definition limited by the temperature difference between the object of measurement and the organ being measured. Some animals, notably beetles, have been found to be at or close to the theoretical limit; others apparently tuned so that their detectors were cooler than the rest of their bodies, reducing thermal background noise. As scientists explore the limits of recognition reached by animal senses, it becomes clear that many modes operate at or near theoretical limits dictated by the laws of physics. Many types of receptors have evolved similar molecular mechanisms to accomplish this astonishing task, and the mechanisms available for each sensory modality depend at least in part on whether the sensory input is thermally confined or quantum-confined1 species.

Summary Recipient cells are highly sensitive to certain types of stimulating energy and relatively insensitive to other types of stimulation. They convert stimuli into electrical signals, usually (but not always) depolarizing. The lower limit of sensation usually depends on how much energy is transmitted in the signal compared to the energy in the thermal noise in the organism. The transduction process is most sensitive to weak stimuli, producing receptor signals that contain orders of magnitude more energy than the stimulus itself. This sensitivity decreases with increasing stimulus intensity. In most recipient cells, the primary site of reception and transduction is the receptor molecule located in the cell membrane or in the intracellular membrane. Activation of receptor molecules alters the conductivity of certain ion channels in the membrane. Typically, a change in conductivity allows receptor current to flow, creating a receptor potential. In many sensory modalities, the recipient cells themselves do not produce AP. Instead, the receptor potential modulates the amount of neurotransmitter released by the receptor cell on the secondary neuron, which in turn initiates or regulates the amount of AP in the secondary neuron. Stimulus intensity is usually encoded in pulse frequency, which in many sensory fibers is roughly proportional to the logarithm of the intensity, up to a maximum frequency. The logarithmic relationship between stimulus and response magnitudes enables reception over long periods of time

Dynamic range combined with high sensitivity to weak stimuli. Parallel inputs from receptors covering different parts of the intensity range increase the range of perceivable stimulus intensities. Loss of sensitivity to persistent stimuli over time, known as sensory adaptation, is a common property of receptor cells; some receptors adapt quickly, others slowly. The mechanisms responsible for sensory adaptation are different. Some occur in receptor cells, and some in networks of neurons that carry sensory information. In at least one case (Limulus photoreceptors), adaptation is caused in part by an increase in intracellular Ca2+ that prevents light-dependent activation of Na+-K+ selective ion channels. Some receptor cells exist alone, but others are organized in sensory tissues and organs, such as the nasal epithelium in vertebrates or the retina of the eye. Anatomical tissue affects the function of sensory organs. For example, the quality of images produced by the vertebrate visual system depends on the presence of a lens and a large number of photoreceptor cells in the retina. Several sensory systems share common features. In particular, many receptor molecules contain seven transmembrane domains, a feature that is also present in certain neurotransmitter and hormone receptors. Many sensory systems also share common elements in the cascade of biochemical events that follow signal recognition and amplify the signal. Mechanoreception is the result of deformation or stretching of the receptor membrane, leading directly to changes in ionic conductivity. Deflection of hair cell stereocilia provides directional information by modulating up or down the frequency of spontaneously occurring axonal impulses in the eighth cranial nerve This function is the basis for reception by several sensory organs - the lateral line system in fish and amphibians, hearing in vertebrates, and the balance organs in vertebrates and invertebrates. The mammalian cochlea analyzes sound frequencies to determine their effectiveness in replacing different parts of the basement membrane that support hair cells. Mechanical waves propagate along the basilar membrane and are produced by sound-driven movements of the tympanic membrane and auditory ossicles. They stimulate hair cells, which in turn modulate their synaptic activity on auditory nerve fibers. Certain sound frequencies stimulate every site on the basilar membrane more than others, which is the basis for frequency discrimination in mammals. Electroreceptors in fish are altered hair cells that have lost their cilia. Exogenous current flow through electroreceptor cells produces a change in transmembrane potential that regulates the release of transmitters at the base of the receptor cells, thereby determining AP rates in sensory fibers. Vision receptors use pigment molecules in specialized membranes that change conformation after absorbing photons. conformational change

The photosensitive molecules initiate a reaction cascade that results in a change in the electrical conductivity of the recipient cell membrane. All visual pigments are composed of protein molecules (opsins) and a carotenoid chromophore, retinal (in rhodopsin) or 3-dehydroretinal (in porphyrin ). The amino acid sequence of opsins determines the absorption spectrum of each visual pigment. Cis-trans isomerization of carotenoids triggers all visual responses. Photon absorption is coupled to the opening (in invertebrates) or closing (in vertebrates) of intracellular second messenger ion channels. In vertebrate rods, photons captured by rhodopsin molecules lead to activation of associated G protein molecules located in the receptor membrane. Each G protein then activates many phosphodiesterase molecules, each of which hydrolyzes many molecules of the internal messenger cGMP. In the dark, cGMP continuously activates Na+ channels that carry dark current. Light-dependent hydrolysis of cGMP reduces dark current, and residual K+ current hyperpolarizes photoreceptors, reducing the steady release of neurotransmitters in the inner segment. A decrease in the rate of transmitter release leads to changes in the activity of the next higher neurons. The fovea of some vertebrates has three types of cone cells, each containing the visual pigment most sensitive to a different part of the light spectrum. Integrating the activity of all these cones results in color vision. Rods, which contain only one type of photopigment in humans, are abundant in the retinal periphery outside the fovea, are more sensitive than cones, and exhibit greater synaptic convergence. Therefore, they have lower vision but higher sensitivity.

Review Questions 1.

3. 4.

Pressure, heat, electricity and light can stimulate visual receptor cells, provided these other stimuli are high enough. How does this fact reconcile with the concept of receptor specificity? Choose a sensory modality and outline the steps from receiving energy in the recipient cell to triggering an action potential (AP) that is delivered to the central nervous system. Why does receptor potential need to be converted to AP to be effective? All sensory information enters the central nervous system in the form of APs with similar properties. How do we differentiate between different stimuli? What is the difference between sensory transduction and sensory reinforcement? Choose a sensory modality and describe how the two processes are related in that modality. Discuss the relationship between the strength of the stimulus and the strength of the signal sent by the recipient cells to the central nervous system. How is the stimulus generated?

Voltage encoding? How do sensory systems respond to stimuli that vary in intensity by many orders of magnitude? 7. Discuss three mechanisms that contribute to sensory adaptation. 8. Discuss an example in which efferent activity can modulate the sensitivity of recipient cells. 9. How is the movement of the basilar membrane translated into auditory nerve impulses? 10. Discuss the function of the inner and outer hair cells of the cochlea. How does spontaneous discharge increase the sensitivity of certain receptor systems - such as lateral electroreceptors? How do the electroreceptors of weakly electric fish sense the presence of objects? What are the main differences in the electrical responses of vertebrate and invertebrate photoreceptors to light? Compare the mechanisms that allow the auditory system to discriminate between incident sound frequencies and the visual system to discriminate between incident light frequencies. Describe the currently understood steps in light energy transmission in vertebrate visual receptors. How does our current understanding of the physiology of color vision support Young's theory of trichromaticity? Compare and contrast the morphological and functional characteristics of vertebrate rods and cones. What makes some arthropods respond to the directionality of polarized light? Man cannot do that. Why? Compare the way mammalian and teleost lenses focus images.

Recommended reading: Corey, D.P. and S.D. Roper. 1992. Sensory transmission. 45th Annual Meeting of the Society of General Physiologists. New York: The Rockefeller University Press. A series of articles discussing the latest data from research on transduction of many different sensory modalities. Dowling, J. 1987. retina. Cambridge, MA: Belknap Press, Harvard University Press. A very readable compendium of information on the vertebrate retina, written by an important contributor to our understanding of this sensory organ. Finger, T.E., and W.L. Silver. 1987. The neurobiology of taste and smell. New York: Willie. A series of articles dealing with the various chemical senses of animals. Hudspeth, A.J. 1989. How the ear works. Nature 341:397-404. A beautiful text on auditory transmission in hair cells, by a man who has been instrumental in the study of this discipline.

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Kandel, E.R., J.H. Schwartz, and T.M. Jessell. 1991. Principles of Neuroscience. 3D output. New York: Elsevier. A comprehensive and authoritative compendium of information on nervous system function, from the biophysics of membrane channels to the physiological basis of memory and learning. There are several chapters on sensory mechanisms, with a focus on vertebrates.

Land, M. and R. Fernald. 1992. Eye development. install. Neuroscience Rev., 15:1-29. Considerations of the physical and optical properties of the organ of vision throughout animal phylogeny. Shepherd, G.M. 1994. Neurobiology. 3D output. New York: Oxford University Press. A concise text that considers the different ways of feeling in vertebrates and invertebrates.

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Hierarchical organization. The retina contains the first three layers, with the remainder in the cerebrum, lateral geniculate process, and cerebral cortex. Information converges and diverges between layers, and flows bidirectionally between layers. [Part A is from "Retinal Processing of Visual Images" by C.R. Michael. Copyright O 1969, Scientific American, Inc. All rights reserved. Part B is based on Noback and Demarest, 1972.1

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Figure 11-33. The function of the vertebrate retina is based on five main types of neurons. Photoreceptors receive light stimuli and convert them into neuronal signals. Bipolar cells carry signals from photoreceptors to ganglion cells, which send their axons into the central nervous system

through the optic nervous system. Horizontal cells and amacrine cells located in the outer and inner plexiform layers, respectively, transmit signals laterally. [From "Vision Cells" by R.W. Young. Copyright O 1970 by Scientific American, Inc. all rights reserved. ]

(Figure 11-34). Vertebrate photoreceptors hyperpolarize in response to light (see Chapter 7). They release synaptic transmitters continuously in the dark, and their release decreases when they hyperpolarize in response to light. Likewise, horizontal cells only produce gradient hyperpolarization in response to light (see Figure 11-34). Bipolar cells can produce graded potential changes of either polarity. Ganglion cells respond with the same polarity as the bipolar cells they innervate. When a synapse to its bipolar cell depolarizes, it depolarizes and fires AP, and when its bipolar input hyperpolarizes, it becomes hyperpolarized and stops firing spontaneously. Amacrine cells respond transiently to switching light on and off in response to input from bipolar cells. Bipolar cells usually connect more than one receptor to each ganglion cell, and they can also connect each receptor cell to multiple ganglion cells. Thus, there is already convergence and divergence between the primary and tertiary cells of the visual system, but the degree depends on the location on the retina. In mammals, the fovea, or central area (the area), converges and diverges very little.

In the center of the retina where visual images are in sharp focus). This lack of convergence and divergence results in very high vision based on the one-to-one connections between cone photoreceptors, bipolar cells and ganglion cells. (Cones are the majority of the photoreceptors in the fovea.) Outside the fovea, each ganglion cell receives input from many receptor cells, mainly rods, making these ganglion cells sensitive to dim light More sensitive, but reduces vision. Structurally, output from the retina is carried to the optic nerve by the axons of ganglion cells, but how is the output organized? Understanding the information output by ganglion cells depends on the concept of receptive fields, an idea first proposed by Sherrington and applied to visual processing by Hartline in the 1940s. A cell's receptive field is the area of the retina where light stimuli affect cell activity. The receptive field of a ganglion cell is roughly cell-centric and its size depends on the number of photoreceptors and bipolar cells that converge on the way to each ganglion cell. Ganglion cells located in the center of the fovea

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Figure 11-34 Each type of retinal neuron has a different electrical response to light. The activity of each cell type was recorded in response to a spot of light focused directly on the receptor in the area (left) and in response to a halo around the photoreceptor (right). The duration of stimulation is shown in the lower curves for each data set. In this example, ganglion cells are activated by light shining on the center of their receptive field. Note that bipolar cells and ganglion cells respond with opposite polarity to dots and rings. This effect is thought to be due to lateral inhibition similar to Limulus. Note that non-bipolar cells and central ganglion cells are shown without synaptic connections. See Figure 11-36 for a detailed illustration of how ganglion cell responses relate to signaling in bipolar cells. [Adapted from Werblin and Dowling, 1969.1

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The receptive field extends only to one or a few photoreceptors. In the highly convergent retinal periphery, ganglion cells have receptive fields up to 2 mm in diameter. Each ganglion cell is spontaneously active in the dark, with changes in activity levels when a spot of light falls into its receptive field. Depending on the recipient cell being irradiated, the frequency of APs in ganglion cells increases—a response—when small spots of light enter the cell's receptive field. Alternatively, the AP can lower the frequency in response to the on/off response. The receptive field of a ganglion cell is usually divided into a center and a periphery, and the response of the cell depends on whether the center, the periphery, or both are stimulated (Figure 11-35). In central ganglion cells, the frequency of APs increases when the center of their receptive field is illuminated (see Figure 11-35A). When the aura illuminates the entire receptive field, with the center of the aura above the center of the field, there is less activity in the cell. A

The weak turn-off response is caused by light spots that only illuminate part of the area. The ring around the center of the receptive field is called the inhibitory environment of the receptive field. An off-center cell exhibited the opposite behavior: It stopped or reduced its activity when the center of its receptive field was illuminated, and increased its activity when the surrounding area was illuminated. The midfield organization of the receptive field depends on lateral inhibition, similar to the compound eye of the horseshoe crab. Lateral interactions in the vertebrate retina occur primarily through the activity of horizontal cells in the outer plexiform layer (see Figure 11-33). Horizontal cells have extensive lateral processes and are connected to adjacent horizontal cells by electrical connections. Furthermore, they form chemical synapses on bipolar cells and receive synaptic input from recipient cells. Light that falls near the receptive field of a ganglion cell acts through the horizontal connections of the cells. Because Hori——

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Figure 11-35 Retinal ganglion cells respond centrally or eccentrically to light stimulation. (A) Four images of typical ganglion cells in the central retina. Each recording shows ganglion cell activity over 2.5 s intervals. Stimuli are shown in the middle of the figure. In the dark, the APs in the cell are slower and more or less random. The bottom three traces show the response to a small spot, a large spot covering the center of the receptive field plus surround, and a ring covering only the surround response (B) of the off-center receiver element [adapted from The same set of pulses, Hubel, 19951

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Ribbon cells form extensive syncytial networks that communicate with each other through low-resistance gap junctions. Input of any receptor into a horizontal cell generates a hyperpolarized signal that propagates electrotonically in all directions away from the receptor. Each bipolar cell receives input from surrounding recipient cells via horizontal cells, and these inputs decay with distance as they propagate in electrotonic fashion with a graded, hyperpolarizing potential decay in the horizontal cells . The indirect input that the bipolar cell receives from external receptors through a horizontal cell network is opposed to the direct input it receives from photoreceptors and forms the basis of the organization around the center of the retinal receptive field. Local direct pathways from photoreceptors through bipolar cells to ganglion cells generate central responses. Indirect pathways from photoreceptors through horizontal cells to bipolar cells and thence to ganglion cells mediate responses to the environment. These two methods show how certain features of stimuli can be extracted by relatively simple neural networks. The characteristic responses of central and central ganglion cells arise from their association with two types of bipolar cells: bipolar cells and extracentral bipolar cells. These two types of bipolar cells respond oppositely to synaptic* input from receptor and horizontal cells (Figure 11-36). Non-bipolar cells hyperpolarize upon exposure to the receptor, while bipolar cells depolarize. In both types of bipolar cells, exposure to ambient light produces a horizontal cell-mediated response that has an opposite electrical signal to that produced by central illumination. Each bipolar cell causes a change in potential in its ganglion cells or cells with the same sign as the change in potential occurring in the bipolar cell. Thus, ganglion cells innervated by unipolar cells have receptive fields in the center, whereas ganglion cells innervated by non-bipolar cells have receptive fields off-centre. Centrally located ganglion cells are excited by light in their center

The receptive field because it receives direct synaptic input from bipolar cells. It is inhibited by light near its receptive field because horizontal cells receiving signals from surrounding photoreceptors inhibit bipolar cells on the direct path from photoreceptors to ganglion cells. The on and off response of bipolar cells depends on the cell's response to neurotransmitters released by photoreceptor cells and various neurotransmitters released by horizontal cells. Bipolar cells are continuously hyperpolarized in the dark by the constant release of transmitters from partially depolarized recipient cells. If light stimulation causes photoreceptors to hyperpolarize, they release messenger substances and bipolar cells depolarize. This depolarization leads to the release of excitatory transmitters from bipolar cells, which depolarizes the ganglion cells, thereby increasing the frequency of APs in the ganglion cells. In contrast, non-bipolar have distinct classes of postsynaptic channels with different ion selectivity and are stably depolarized by neurotransmitters released by photoreceptors in the dark. When light falls on photoreceptors and they hyperpolarize, reduced neurotransmitter release causes bipolar cells to hyperpolarize. This hyperpolarization is accompanied by a decrease in transmitter release from non-bipolar cells, leading to hyperpolarization of postsynaptic ganglion cells. In summary, the organization of the receptive field in the vertebrate retina depends on three fundamental features:

1. Two types of ganglion cells receive input from two corresponding types of bipolar cells. These connections result in central and extracentral ganglion cell responses. 2. Receptors near the receptive field act through a horizontal cellular network of electrical connections to both bipolar cells.

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Figure 11-36. Connections within the retina yield central and eccentric ganglion cell response signatures. Two types of bipolar cells, EON and BoFF, respond in opposite ways to direct input from receptors R, and indirect input from lateral transport of horizontal cells H. Bipolar cells are depolarized during activation of overlying receptor cells and weakly hyperpolarized by lateral input from horizontal cells. Non-bipolar behaves the opposite. (A) Bipo-response

Larval cells and ganglion cells in dots of light. (B) Bipolar and ganglion cell responses to halos. For simplicity, amacrine cells are omitted from the figure. The direct pathway G from photoreceptors to ganglion cells is colored. Indirect lateral paths through horizontal cells are shown in gray. Plus and minus signs indicate synaptic transmission that maintains (+) or reverses (-) the polarity of the signal.

Polarization of presynaptic cells. The postsynaptic response depends on the generation of ionic currents in the postsynaptic cell as a result of presynaptic neuron-mediated release of transmitters.

Direct input to bipolar cells via covering receptors and indirect input to cells via horizontal cellular networks are opposed to each other, resulting in contrasting center-surround organization in central and eccentric ganglion cells. Ganglion cells can be seen.

The organization of the retina reveals several general principles that apply to the rest of the central nervous system. First, neurons without APs can send electrical tension signals to each other when in close proximity. In fact, non-spiking neurons can convey more information more accurately than all-or-nothing signals. The electrotonic signal decreases with distance, limiting the range of effects such as lateral inhibition. Second, receiving stimulation does not necessarily equate to depolarization. In some neurons (such as photoreceptors and some horizontal cells), hyperpolarization is a normal response to stimulation; it regulates synaptic transmission by reducing the steady release of transmitters. Third, the postsynaptic response of neurons cannot be predicted from signs of possible changes in presynaptic neurons. In response to hyperpolarization, cells can depolarize or hyperpolarize.

Information processing in the visual cortex What happens to a retinal image after it has been translated into a series of receptive field responses within the retina? Physically, information travels through axons to visual areas in the brain. The details of this pathway vary from species to species. In mammals and birds, retinal ganglion cell axons point toward the ipsilateral or contralateral optic chiasm of the brain, where some axons cross the midline (see Fig. 11-32B); In animals, all visual fibers point opposite to the optic chiasm (see Figure 11-32A). The degree of crossing at the optic chiasm depends, in part, on the degree of overlap between the visual fields of the two eyes. In animals whose field of view in one eye is completely different from that of the other, all axons of retinal ganglion cells cross the midline. mammalian ganglion cell axon

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................................................... ................................... Synapses with quaternary cells in the lateral geniculate nucleus of the thalamus. Lateral geniculate neurons send axons to form synapses with fifth-order cortical neurons in the occipital cortex (see Figure 11-11), an area called area 17, also known as the primary visual cortex because it is the signal The first area of the cortex in the pathway receives visual information. The pattern of synaptic relationships in the lateral geniculate body represents a further step in the processing of visual input based on the source and nature of the information conveyed by retinal ganglion cells. Each lateral geniculate body, or body, is composed of six layers of cells, stacked like a folded-leg sandwich (Fig. 11-37). The top four layers contain neurons with small cell bodies, called parvoneurons, and the bottom two layers contain neurons with large cell bodies, called magnocellular neurons. The input to these neurons is strictly organized. Each lateral stifle receives information from only one half of the visual field (i.e., one of the two fields of view shown in Figure 11-32B), and cells in each layer receive input from only one retina. Each neuron in the geniculate body receives information from only one eye. Neurons in a given layer receive information from the same eye, and layers alternate from one eye to the other, changing the pattern of alternation between layers four and five (see Figure 11-37). In all slices, the topographic map of the corresponding retinal surface was accurately preserved and consistent between slices. If we move the electrodes along the path shown by the dotted lines in Figure 11-37, we encounter cells that respond to light stimulation in exactly the same way

A point in the field of view, but as the electrodes move from one layer to the next, the origin of the eye changes from left to right. Are there functional differences between the layers that receive information from each eye? Yes, the cells in each layer respond to specific properties of the stimulus, and the responses vary from layer to layer. In monkeys, for example, cells in the four dorsal layers responded to the color stimulus, while the two cells in the deepest layer did not. In contrast, the deepest two layers responded to movement, while the outer four layers did not. This spatial classification of ganglion cell output illustrates another principle of brain organization: that information about a single stimulus is divided into parallel pathways. This mode, called parallel processing, is a major topic of research in advanced brain functions. The receptive fields of geniculate neurons were not significantly different from those of retinal ganglion cells. That is, they have an eccentric or central type of concentric arrangement around the center of the organization. The puzzle of how the visual world is organized in the next area of visual projection in District 17 was analyzed extensively and insightfully in the 1960s by David Hubel and Torsten Wiesel, who received a Nobel Prize in 1981 for the importance of their work. bell award. In their experiments, they recorded the activity of individual neurons in the brain of an anesthetized cat while projecting a simple visual stimulus (such as a dot, circle, bar, or edge) onto a screen where the The cat's entire field of view can be captured (Figure 11-38A). The cells of the mammalian lateral geniculate nucleus are organized into layers, with each layer receiving information from only one eye. Histological section of the left clavicle of a macaque; the incision extends parallel to the surface. The cells of the outer four layers have small cell bodies called pa-ocellular. The deeper cells are macrocytes. In the left knee tubercle, all cells receive information about the right visual field. Furthermore, the outermost layer only receives input from the left eye, while cells in the next layer only receive input from the right eye, and so on. Recording electrodes passed from one layer to the next will reveal that cells along the path indicated by the dotted lines respond to the exact same location in visual space, but with a change in the eye receiving the information. [Adapted from Hubel, 1995.1

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The responses they recorded from cortical neurons correlated with the position, shape and motion of the projected image. In retrospect, Hubel, Wiesel, and their collaborators made two important decisions in their experiments that allowed them to discover order and regularity in the enormous complexity of the visual brain. First, they decided to use more complex stimuli than simple dots and asked which of these stimuli most effectively elicited a response in each neuron. Second, by recording how many cells each electrode penetrated, they were able to find out what neighboring cells have in common and how cells are grouped in the brain. These strategies allowed them to discover different types of commands in the connections in the visual cortex, and their findings provide a model for studying other sensory systems. The most important discovery of Hubel and Wiesel about the responses of cells in the visual cortex is that they respond to completely different stimulus properties than retinal ganglion cells. Cortical cells responded most strongly to bars projected in different directions. They named the two main types of cells they discovered simple and complex, depending on the nature of the optimal stimulus.

Figure 11-38. Neurons in cat area 17 have very different receptive fields than retinal ganglion cells or cells in the lateral geniculate body. (A) Experimental setup for studying cellular responses in the visual cortex. Electrodes are passed through the cortex while light stimuli are projected onto the screen. (B) The receptive field of a single cell in the cortex is striped. A light spot (stimulus 1) anywhere on the receptive field produces a small excitation of the simple cell. Spots of light adjacent to the bar region (stimulus2) inhibit AP in these nervously active cells. (C) Rotating a light bar (red bar) over the receptive field of a simple cell results in maximal activity in the cell when the light bar exactly coincides with the on region of the cell's receptive field (stimulus 3), and partial excitation in other regions direction (e.g. stimulus 2). [Part A of "Cellular Communication" by G.S. Stent. Copyright 0 1972 by Scientific American, Inc. all rights reserved. Part of D.H. Hubel's "Thevisual Cortex of the Bra~n" band. Copyright0 1963 by Scientific American, Inc. All rights reserved. ]

They found that each type of cell is systematically arranged in space according to their optimal stimulus. The receptive fields of simple cells are elongated, and the on region of the receptive field has a straight line boundary separating it from the off region (Fig. 11-38B), rather than the side kneeling found in retinal cells and retina. Like retinal ganglion cells and geniculate cells, the receptive fields of simple cells are located at fixed locations on the retina and thus represent specific parts of the entire visual field. There are some differences in the receptive fields of simple cells: some have a bar-shaped open area surrounded by a closed area; for others, the receptive field consists of a closed bar surrounded by open areas. For some others, it consists of a straight edge with a closed area on one side and an open area on the other. When the stimulus strip completely overlaps the cell's receptive field, it triggers the maximal activity of the simple cell (Fig. 11-38C). Rotating the bar so that it no longer matches the direction of the receptive field has no effect on or inhibits the spontaneous activity of simple cells. If you move the light bar so that it falls just outside the on area, the battery will suffer the most. Direction and Switch Boundaries

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................................................... .................... differs from one simple cell to another; thus, when a bar of light moves horizontally or vertically across the retina, It activates a simple cell as it enters one receptive field after another. What makes simple cells respond specifically to straight bars or edges with precise alignment and position? Hubel and Wiesel proposed, and recent experiments have demonstrated, that each simple cell receives excitatory connections from cells in the lateral geniculate body whose centers are arranged linearly in the retina (Fig. 11-39A). As shown in Figure 11-39B, assume input is received by simple cells that respond to boundaries rather than bars. A simple cell receives maximum input when light falls on all receptors that activate the central field of ganglion cells and knee cells. Any additional light falls on the inhibitory environment of the ganglion cells and only reduces the response of the cortical cells. Complex cells form the next level of abstraction for processing visual information. Complex cells are thought to be dominated by simple cells, which would make complex cells a sixth-order cell in the visual information processing hierarchy. Like simple cells, complex cells respond best to straight edges with specific angular orientations on the retina. However, unlike simple cells, complex cells do not have topographically defined receptive fields. Presenting appropriate stimuli to relatively large retinal regions was equally effective for activating complex cells; as with simple cells, general illumination of the entire retina was not an effective stimulus. Some complex cells respond to stripes of light in specific directions (Fig. 11-40A). Others responded "on" to the straight edge when the light was on one side, and "off" when the light was on the other. There are other complex cells that respond optimally to moving boundaries traveling in only one direction (Fig. 11-40B). For these cells, movement in the other direction elicited a weak response or no response at all. These receptive fields can be explained by a combination of synaptic inputs from simple cells. Each simple cell in turn excites the complex cell when the light-dark boundary crosses the receptive fields of the simple cells that form synapses with the complex cell. This arrangement can generate orientation sensitivity to movement of the switch boundary (see ~i~~~~1 1–4 0~) 1f. The borders move to be fired one by one sequentially, thus firing the compound cells. As each simple cell is inhibited by the dark side of the moving edge, the next cell becomes excited. On the other hand, if the boundary is changed so that simple cells are sequentially inhibited and then stimulated, the complex cells are inhibited one at a time, canceling out any excitation bias caused by the bright side of the edge. Properties of individual cortical cells suggest that they abstract features of the visual scene, such as edges

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Figure 11-39 Simple cells in the visual cortex respond from their synaptic input patterns. (A) The fixed strip-shaped receptive fields of individual cells result from the convergence of the outputs of ganglion and lateral geniculate cells, whose circular central receptive fields are linearly aligned. (B) Straight-sided switch receptive fields arise from the convergence of eccentric and central geniculate cells onto simple cells.

The first step in analysis and identification. Spatial relationships between cells in visual cortex correlate with their functional properties in an orderly fashion. In their experiments, Hubel and Wiesel found that neighboring cells responded to similar characteristics of stimuli. When they enter the visual cortex through electrodes placed perpendicular to the cortical surface and record

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Figure 11-40. Answers in complex cells can be based on their simple cell input patterns. (A) Some complex cells respond to light bars with specific angular orientations, but their location can be anywhere within the large receptive field. This pattern of responses may be caused by the convergence of many simple cells, each with similarly oriented rod-shaped receptive fields. In this example, a vertical light strip stimulates a simple cell to fire as it falls on a series of ganglion cell receptive fields that create a simple cell's strip-shaped receptive field. If you move the bar to the right, it fires another simple cell, which connects to the same complex cell and produces an activation.

Sex in Complex Cells. In contrast, horizontal light bars produced only subthreshold responses in simple cells, so no signal was sent to complex cells. (B) Some complex cells respond to edges of light moving in only one direction. This pattern of responses may arise from the aggregation of a population of simple cells, all of which are sensitive to bright and dark edges in the same direction. Complex cell firing occurs if the edge is moved to illuminate the front side of the simple cell's receptive field before illuminating the reverse side. Movement in the opposite direction will only have an inhibitory effect.

In response to the responses of cells located on that pathway, they found that cells on each pathway responded to bars with the same orientation. When they moved the electrodes sideways and re-entered, they found a column of cells that responded optimally to stimuli that had a different orientation than the optimal stimulus in an adjacent column of cells. Each of these groups of cells is called a cortical column. In contrast, cells recorded along channels parallel to the surface showed surprisingly regular changes in the direction of optimal stimulation, with each time the electrode was advanced 50 points, the preferred direction shifted by about 10 degrees. This result suggests that cells of the visual cortex are organized into columns according to the characteristics of their optimal stimuli, and that this difference varies in an orderly fashion throughout the cortex (Fig. 11-41A). Columnar organization of cells with similar response properties has been previously observed in somatosensory cortex, where adjacent columns contain cells

Response to touch or flexion of a particular joint. However, the ordered arrangement of directional columns is only the first function-based subdivision in the visual cortex. The next discovery has to do with the eye that sends out visual signals. Hubel and his colleagues determined the projection patterns of each eye on the cortical surface by injecting radioactive tracer molecules into the eyes and delivering them to the visual cortex. These experiments resulted in a second column system, where alternating columns represent one eye or the other (Figure 11-41B, but see Spotlight 11-3). A three-dimensional reconstruction of these columns, the so-called ocular dominance columns, shows their distribution on the cortical surface (Fig. 11-41C). These experiments showed that the visual cortex is divided into small functional units that analyze stimuli into their constituent parts before sending them to higher layers for further analysis. This modular organization is overlaid on a basic spatial map

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your system.

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Neurons in the visual cortex are arranged in an unusual way.

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brain. So the nasal and temporal sides of the right retina

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same direction.

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in the cerebral cortex

Figure 11-41 Neurons in the visual cortex are arranged in columns perpendicular to the cortical surface. (A) Diagram illustrating the organization of cell columns in response to stimulus targets. A column is a collection of cells where the optimal stimulation direction is the same for all cells in the column. Cells in adjacent columns have different optimal stimulus orientations, and this orientation varies systematically from column to column. (B) Eye-excited cortical neurons also switch between adjacent columns. The red pillar is activated by the left eye; the gray pillar on the right eye. (C) Simon LeVay rebuilds the eye-dominant prop in part of the 17th. [Adapted from Hubel, 1995.1

Persist through the photoreceptor layer. To understand the nature of the spatial map at the cortical level, experiments were performed to record visual fields directly onto the cortex using radioactive labeling techniques (Fig. 11-42). Injecting radioactive 2-deoxyglucose into an anesthetized monkey then projected complex, targeted stimuli onto its retina. Active neurons take up more 2-deoxyglucose than resting neurons. Cortical neurons activated by a stimulus are therefore expected to contain more radioactivity than their inactive neighbors. The radioactivity patterns observed in the visual cortex suggest that although the two-dimensional surface of the retina is perfectly delineated on the cortical surface, the cortical pattern is not an exact replica of the spatial characteristics of retinal stimuli. In contrast, the retinal area representing the center of vision (the fovea) was greatly enlarged compared to the retinal area representing peripheral vision. This pattern corresponds to differences in vision at the retinal surface and differences in convergence of primary photoreceptors with subsequent neuronal layers. This distortion of the map according to the animal's needs and habits is characteristic of all animals with well-developed visual systems. For example, animals such as rabbits that live on large open plains have an elongated horizontal specialized region called the retinal zone, which provides the greatest number of photoreceptors and the least convergence to receive stimuli along the visual horizon.

All different levels of cortical organization must combine to provide the next set of cortical cells with a complete picture of visual stimuli, and how this integration is achieved remains the subject of intense research. For example, it now appears that certain higher-order visual neurons may only become active when specific objects, such as faces, enter their receptive fields. The visual cortex has taught physiologists several important principles about the organization of sensory networks. First, the vision system is organized hierarchically. At each level, cells require more complex stimuli to excite them optimally, and this complexity arises from the fusion of cells with simpler receptive fields into cells with more complex receptive fields. Second, while convergence is evident when we follow stimuli into the system, parallel analysis of different features of stimuli also requires divergence of information. Simultaneous analysis of different features of stimuli occurring in parallel pathways appears to be an important principle of functional organization. Third, the activity of cortical neurons in areas 1–7 leads to the abstraction of certain features of visual stimuli. Fourth, the visual cortex does not receive simple one-to-one projections from the retina, either spatially or temporally. Instead, some regions of the visual field were significantly expanded in their cortical representations, while others were compressed.

Figure 11-42 Visual space is represented on the surface of the visual cortex, but in a somewhat distorted form. This targeted radial line stimulation was focused in the visual field of anesthetized rhesus monkeys 45 minutes after injecting radioactive 2-deoxyglucose into the monkeys' bloodstream. One eye remained closed. Cortices were removed, flattened, frozen and sectioned. The lower image shows a section parallel to the cortical surface. Roughly vertical marked lines represent the curve of the stimulus; horizontal marked lines represent radial lines in the right visual field. The thread was interrupted because only one eye was irritated. This dotted pattern shows the eye dominance column. [Adapted from Tootell et al., 1982.1

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Auditory Map of the Owl Brain The retinal and somatic maps described earlier are found at many levels in the brain, as sensory information is relayed through the nervous system. We recognize these maps because, even when distorted, they mimic the spatial organization of objects in the outside world. The two-dimensional arrangement of retinal surface cells creates a two-dimensional map of the environment, and spatial relationships in the environment are preserved when the image is projected into the lateral geniculate cells and cortex. In other sensory systems, the nature of the possible central map is less obvious. In the auditory system, for example, the arrangement of hair cells along the cochlea correlates with their sensitivity to specific sound frequencies (see Chapter 7). If the spatial order of these hair cells is preserved as their axons project to the cochlea, the result would be a brain map of sound frequencies, a pitch map. In fact, pitch thematic maps have been found in certain auditory regions of the brain. However, it's unclear how classifying sounds by frequency will help animals gain information about their surroundings. We know humans can locate the source of sound in space, but simply knowing the frequency of a sound doesn't help much. How do animals localize sounds in space? Information about the location of the sound source relative to the listener is encoded in the relationship between the intensity of the sound and the time it takes for the sound to reach the two ears. If there is a sound source on the left side of the animal, the sound will reach the left ear first and the right ear later. The nervous system can count the time between a sound's arrival in one ear and the other as an indication of its origin. To understand how this works, Eric Knudsen and Mark Konishi studied barn owls, a bird that relies heavily on locating noise sources in the dark. Barn owls have several characteristics that make them excellent animals for studying the neural mechanisms behind sound localization. First, owls use both vision and hearing to guide their prey when there is light, but they can catch mice in complete darkness and find their prey just by hearing the sound (Figure 11-43). Furthermore, an owl cannot move its eyes in their sockets; instead, it must move its entire head, whether toward a sound or toward a visible object, and this directional response is quite accurate. Owls can fix their heads on—

Figure 11-43 The barn owl can scratch ice in total darkness. The images are from film taken using only infrared light, which owls cannot see. The owl managed to catch the mouse in total darkness. [Contributed by M. Konishi. ]

Sound sources are detected with an accuracy of 1 to 2 degrees in azimuth (lateral distance from a point directly in front of the owl's head) and elevation (vertical distance from a point directly in front of the owl's head). To test its orientation, an owl was placed on a perch and sounded through a speaker whose position could be varied over one hemisphere of space while maintaining a fixed distance from the bird (Fig. 11-44A). The orientation of the owl's head is monitored as the owl orients itself based on the sound from the speakers. The head orientation in response to each sound is expressed in degrees elevation and azimuth (Fig. 11-44B). Careful behavioral observations reveal that owls use two types of cues in their orientation responses: the intensity of the sound to determine the altitude of the target, and the relative time at which the sound reaches the two ears to determine the target's azimuth. To study the effect of intensity signals, the right or left ear was plugged to attenuate the sound, using earplugs to either weakly or strongly reduce the sound intensity. The results of this experiment showed that an owl kept misaligning its gaze while touching one of its ears

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Figure 11-44. An owl moves its head in the direction of the sound. This behavior is easily observed. (A) Experimental setup for studying the owl's ability to localize sound sources. Target speakers can be moved to any position. Location: In the hemisphere around the front of the owl. "Hearing the Barn Owl" by E I Knudsen. Copyright 1981 by Sclent~ficArnerlcan, Inc. All rights reserved. ]

Blockage (Fig. 11-4SA). The owl's right ear is plugged back, positioning itself below the actual sound source and slightly to the left. When inserted into the left ear, it is aimed above and slightly to the right of the sound source. That is, if the sound in the right ear was louder, the owl thought it was coming from above; when the sound in the left ear was louder, it seemed to be coming from below. Small differences in azimuthal pointing angles suggest that some information about horizontal position can be obtained from intensity, but intensity does not fully account for pointing along that dimension. How do interaural intensity differences allow owls to distinguish the height of sound sources? Answer

Lying -- at least partly anatomically. The area around the owl's ear opening is made up of stiff feathers called facial folds, which provide a surface that is very effective at directing sound into the ear canal, similar to the fleshy pinnae of mammalian ears. When these feathers are removed, the owl's external auditory canal is asymmetrical (Fig. 11-4SB). The opening of the right ear faces up, while the opening of the left ear faces down. This arrangement can provide a basis for distinguishing between height and intensity features. The removal of these feathers clearly shows the importance of facial wrinkles. If it had no collar, the owl would no longer be able to detect the height of the sound source, although it would agree with its estimate

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Figure 11-45. Plugging the ears causes the owl to make mistakes in locating the source of the noise. (A) The target appears directly in front of an owl with one of its ears either hard (high attenuation, solid symbols) or soft (low attenuation, hollow symbols). Note that with the left ear plugged (circle), the owl judged the sound to be higher than its actual location. Owl blocked his right ear and returned a miss.

location direction. (B) Facial ruffles showing asymmetry of the auditory opening. The right ear canal is slightly up, and the left ear canal is slightly down. This nuance is exacerbated by the placement of the feathers in the facial folds. [Adapted from "Hearing the Barn Owl" by E. I. Knudsen. Copyright 01981 Scientific American, Inc. All rights have been modified. ]

The horizontal axis remains just as accurate without and with face frills. Therefore, the collar must reinforce the directional asymmetry of the ear and is essential to distinguish height differences between sound sources. How do owls localize sounds along the horizontal or azimuthal meridian? It is clear from behavioral experiments that differences in the timing of sound arrival to individual ears are important for this distinction. Relevant cues, however, may be differences in tone onset (or excursion) or persistent differences in overall tone duration (Fig. 11-46). Onset difference (or offset) is the difference in time when a given signal first arrives in each ear. The ear closest to the sound source receives the signal first. When a tone is sustained, there can also be an imbalance between the signals received by the two ears; just as the beginning of a sound arrives at both ears at different times, the identifiable features of the sound reach one ear first and then the other. one ear. These two types of inequality can be varied independently by implanting small speakers in the owl's ears. In response to the difference in onset, the owls failed to perform the "correct" head movement; whereas the owls, in response to a sustained difference between 10 and 80 ps, quickly pointed their heads to the correct position in azimuth corresponding to this time difference (Figure 11-46B). These experiments showed that owls can orient themselves to sounds in space with astonishing accuracy. Levels are judged on the basis of intent.

The intensity and azimuth of the sounds reaching each ear were assessed based on the difference in duration between the sounds reaching each ear. How is information about the position of sound in space represented in the nervous system? The ear does not provide the brain with a direct representation of the external world. Instead, as we have seen, the owl must calculate the difference in intensity between the sound signals perceived by its two ears to determine the height of the sound, and it must constantly evaluate the difference between the sound signals reaching its ears to determine The position of the sound in the azimuth plane. Knudsen and Konishi discovered in the late 1970s how and where these comparisons are made and how the results are represented in the brain. Knudsen and Konishi discovered a set of spatially specific neurons in the midbrain nucleus. Each of these cells responds optimally to sound signals located at a specific point in space, and each cell has a receptive field and a central external environment organization similar to that found in retinal ganglion cells (Figure 11- 47A). Sounds in the middle of a cell's receptive field (average diameter = 25 inches) excite the cell, while sounds near the receptive field dampen its response. Neurons are arranged to form a spatial map in the nucleus (Fig. 11-47B), similar to the retina-derived retinal epitope map and the derived body epitope map

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................................................... .................................................... Figure 11-46 The owl passes The difference between the sustained sounds attached to the target is used to judge the azimuth position of the ear. (A) Onset differences occur when sound reaches one ear before the other. Persistent inequality is the persistent difference in the sound waves perceived by the two ears. (B) Owls use the persistent difference between the sounds arriving at the two ears to pinpoint signals in space. The linear relationship between azimuth and the persistent difference between the signals in the two ears suggests that this type of difference is a relevant cue. [Adapted from "Hearing the Barn Owl" by E. I. Knudsen. Copyright O 1981 by Scientific American, Inc. All rights reserved.

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from the surface of the body. Cells at any point on the surface of the nucleus respond to sound at that particular point in space by firing APs. Adjacent points in the nucleus respond to spatially adjacent stimuli.

Another common feature of this map and other brain maps is that cells that receive information directly in front of the animal have smaller receptive field sizes than cells that receive information from the sides of the animal. The area directly in front of the animal covers most of the nucleus and is therefore enlarged compared to the area to the side of the animal. This representation is reminiscent of exaggerated representations of the fovea in visual cortex and large representations of hands and faces in somatosensory cortex. In barn owls, the nucleus that records these spatial fields is the dorsolateral midbrain (MLD), the avian homologue of the mammalian inferior colliculus. (The inferior colliculus, an important auditory center, lies below the superior colliculus—the mammalian homologue of the optic tectum). The MLD core transmits the map of the sound's position in space to a higher center. The difference between the signals is sensed by neurons in the nucleus located below the MLD in the midbrain. These neurons, called coincidence detectors, receive input from both ears, and their activity changes depending on whether the signals from both ears arrive simultaneously or sequentially. mechanism

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Figure 11-47. Partial auditory neurons in owl bran have spatially organized receptive fields. (A) Receptive fields of individual cells shown on the central (red) and outer (grey) hemispheres. This cell is most sensitive to sounds at an elevation angle of 0 degrees and 10 degrees to the right of center. A sound 20 degrees away from this location stimulates the cell only weakly, while a sound 40 degrees away suppresses it. (B) Spatial auditory map of the dorsal lateral midbrain nucleus in a barn owl. Data are shown for penetration of three electrodes into the core. The position and orientation of each electrode trace is shown in the figure below, which depicts the core as if it were sliced horizontally (orientation is indicated below the figure). Neurons encountered along the trajectory are numbered consecutively, and the receptive field of each neuron is shown. Neurons on a track respond to continuous positions in space; on the other hand, the Az-muthal angle of the receptive field (represented on the nucleus map) changes smoothly when the electrode is moved from one track to another. [Adapted from Knudsen and Konishi, 1978.1

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Response properties of neurons. Similar computational maps have since been found in the brains of bats, which, like owls, use auditory information to hunt for prey. The spatial representation of sound in the owl's brain is eventually transmitted to the tectum, where it encounters and matches the spatial map generated by the visual system. Adjacent layers of the tectum are then topographically interconnected, with one layer processing information through sound and the other through visual input. This arrangement suggests that behavior can be organized more efficiently if all sensory information about objects in space is first collected in one place. The next problem in understanding behavioral production is to consider where and how sensory information leads to decisions to take action. Movement Network Optic Cap

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The difference in sound intensity is calculated by the owl's brain and is still being studied. The acoustic spatial map of the barn owl is the first example of a brain map derived from de nouo.

The sensory part of the nervous system captures and analyzes information about the external world that is critical for the development of behavior adapted to the animal's current environment. This information then needs to be passed on to the neurons responsible for generating coordinated movements. Little is known about the details of the interface between the sensory and motor aspects of this process, in part because researchers work independently to understand sensory or motor systems. In some cases, however, this sensory-motor relationship has been successfully explored in very simple reflexes in vertebrates or in more complex behaviors in invertebrates. We will consider motor control systems of increasing complexity, from those eliciting simple reflex responses to networks controlling repetitive actions

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Uncovering complex networks of general principles of central nervous motor organization. Motor patterns of varying complexity exhibit varying degrees of flexibility. Fixed patterns of action are relatively inflexible; they recur and vary slightly, but many behaviors are extremely malleable. Animals can adapt them to any new situation. One of the challenges in motor control research is understanding the neural activity that allows an organism to produce behavior that changes over time as the situation changes. Levels of motor control The study of how neurons control muscle activity has largely focused on repetitive movements in animals with simple nervous systems or in more complex animals. Neural control of fixed action patterns is a major theme of this work, as the all-or-nothing nature of these behaviors suggests that a single neural decision must generate behavior. This notion of decision-making does not imply a conscious process, but rather the activation of a neural "switch" in the central nervous system is sufficient to trigger a behavioral pattern. Conceptually, this idea can be formalized as a hierarchical engine control system that uses sensory input to select specific engine performance. The lowest level of control is the motor neuron, which is connected to the muscles. The activity of motor neurons is regulated by integrated neural inputs (Figure 11-48). Initially, some physiologists thought that short feedback loops between stretch receptors in the leg muscles and the spinal motor neurons that control these muscles might be responsible for the walking motion of vertebrates. However, it is clear that repetitive motor performance (such as walking, swimming or flying) depends on activity in a central network that generates the fundamental features of motor patterns. Patterns of walking, swimming or flying can be altered based on sensory feedback and follow the terrain, currents or wind currents in the area. Finally, control is carried out by centers in the upper layers of the nervous system, whose decisions or commands are also influenced by sensory input. Note that in this control hierarchy, the same strict chain of command is not followed in all cases. Various environmental influences result in related types of engine performance, and feedback control operates at every level of the system. Simple reflexes The simplest circuits that control skeletal muscle activity are the reflex arcs. It requires only two types of neurons—stretch receptors (also known as la afferent neurons) and spinal A motor neurons—to wire up to trigger a strong or stretch reflex (Figure 11-49A) . Because the basic form of this reflex requires only synapses between afferent and efferent neurons and no interneurons, it is a monosynaptic reflex.

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Figure 11-48 The motor control system is arranged in layers. Neurons in the brain and nerve cords exert control over the entire motor side of the nervous system and determine motor performance. These decisions regulate activity within groups of neurons called central pattern generators, which activate motor neurons according to more or less preset patterns. Motor neurons provide the only link between the nervous system and the muscles that ultimately trigger behavior. Feedback occurs at all levels of the hierarchy and can affect output.

The sensory terminals of stretch receptor neurons are present in every muscle and are connected to sensory structures called muscle spindle organs. Each spindle organ contains a small bundle of specialized muscle fibers called intrafusal fibers to distinguish them from most contractile fibers called extrafusal fibers. Extrafusal fibers are the skeletal muscle fibers discussed in Chapter 10 and are innervated by motor neurons. The quality and quantity of fibers within the fusilli are low and do not contribute to the generation of muscle tone. Instead, they participate in a feedback loop that regulates the stretch sensitivity of the spindle organ. The muscle spindles run parallel to the extrafusal fibers; therefore, when something happens to stretch the muscle (such as putting weight on an isolated muscle or flexing a joint and the muscle passing through the joint is stretched), the muscle spindles are also stretched. Stretching the central region of the muscle spindle increases the frequency of APs in la afferent axons. These afferent axons form excitatory synapses directly on the motor neurons that control the muscles containing the spindle organ; thus, as the activity of the afferent axons increases, movement tends to excitatory—

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455 Behavior: initialization, modes, and control................................... ................................................................. Figure 11-49 Only two One type of neuron requires red color to generate the muscle stretch reflex (A). Uniform weight condition of light-holding muscle (B). When weight is applied to a muscle, it stretches the muscle and activates stretch receptors, which trigger synapses, or motor neurons, on the same spinal segment, causing the muscle to contract more. If the sensory axon is severed, the motor neuron will have no feedback and the weight will cause the muscle to elongate (dashed line). Ihnes) (C) Sequence of events leading to initiation of the stretch reflex.

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Neurons cause reflexive contractions in stretched muscles (Fig. 11-49B, C). Streceptors provide negative feedback because stretching a muscle triggers neural activity that causes the muscle to contract and resist the stretch. A well-known example of a stretch reflex is the knee jerk, which is evoked when the tendon running through the kneecap (also known as the patella) is tapped. Tapping the tendon causes a sudden stretch in the quadriceps muscle on the belly of the thigh, activating the muscle and stretching the knee joint. The arcuate nature of the reflex becomes evident when the dorsal root is cut into the appropriate portion of the spinal cord. Severing the dorsal root leaves all motor innervation intact but eliminates sensory input to the spinal segment. When the dorsal root is severed, the muscles innervating the spinal segments relax, although their motor drives are intact.

Note that when a muscle contracts under the influence of the stretch reflex, the muscle spindles are unloaded. If nothing else is going on, the afferents will quiet down; and if the muscle is stretched a little more, the muscle spindles won't be able to respond unless they can adjust their length. Intrafusal fibers - under the control of another group of motor neurons called Y efferents - regulate the length of stretch receptors. When a muscle shortens, driven by its motor neurons, the activity of the Y efferent nerve also causes the intrafusal fibers to shorten, thereby maintaining a constant tension on the spindle fibers. In this way, the Y effect allows the spindle fibers to remain sensitive to muscle stretch over a wide range of muscle lengths. Centrally Generated Movements Rhythmic movement and breathing usually consist of rhythmic movements generated by repetitive patterns of muscle contraction. Each phase of this neuromotor cycle is preceded and followed by characteristic activity of motor neurons. Bursts of activity are always correlated in time. Logically, these repetitive actions could depend on either instantaneous sensory input to the nervous system, or on the voluntary motor output of a pattern-generating network that occurs entirely independently of sensory input, or on a combination of these two mechanisms (Fig. 11 -50). Regulation of repetitive locomotor performance has been studied in a number of animal systems, and both mechanisms appear to be involved. These experiments are usually performed in semi-intact specimens, animals whose nervous systems have been exposed for recording but are still able to perform recognizable behaviors. Some Behaviors Can Cause Isolated Nerve Cords

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.................................. Figure 11-50 Motor output of the nervous system due to sensory input and central A combination of schema generation. Sensory input comes partly from the environment and partly from sensory receptors in the animal. The central pattern generator, represented by the recorder, plays an important role in shaping behavior as these neurons generate identical output patterns above the armpit, but they provide only part of the input to the motor neurons.

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Generates all the features of the engine's output patterns; while the concept of isolated nerve cords may seem strange, these behaviors can be studied in semi-intact specimens or isolated nerve cords, whichever is most practical. Central motor patterns are most clearly demonstrated in the nervous systems of some invertebrates—for example, in the neuromotor control of rhythmic locomotion. Grasshopper flight is controlled by muscles.

These muscles move the two pairs of wings alternately up and down, and these muscles receive the appropriate sequence of nerve impulses transmitted by multiple motor axons. (See Chapter 10 for more on insect flight.) Even when sensory input from wing muscles or joints is eliminated by cutting sensory nerves, the activity patterns of these motor neurons continue to occur in proper phase relationship (Fig. 11) .-SlA). This persistence suggests that movement patterns are primarily generated

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Figure 11-51 Both the central pattern generator and sensory feedback contribute to the generation of locust flight. (A) Experimental setup. An eviscerated grasshopper or locust is fastened so that it can flap its wings when air hits receptor hairs on its face. Electrodes are attached in place for sensing motor output and stimulating receptor nerves. (B) When sensory receptor neurons at the base of the wing are disrupted, the central pattern generator generates low-frequency patterns. Electrical stimulation of receptor axons increases the frequency of endogenous motor output. The time at which the receptor nerve was stimulated is indicated by the black line. After stimulation ceased, the rhythm returned to a low rate. (C) Cyclic organization of aircraft engine power. External sensory input, such as a puff of air on hair receptors, stimulates aircraft engine performance. Wing movement activates stretch receptors that control the flight engines. Note that this loop is similar to a positive feedback loop

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................................................... ................................... Within the central nervous system, different The time between muscle contractions. Do sensory inputs play a role in controlling locust flight, which appears to be controlled by centrally programmed motor outputs? Sensory feedback from stretch receptors at the base of each wing, stimulated by wing movement, can alter motor output and increase rhythmic frequency, intensity, and precision. When these receptors were disrupted, neuronal output to the pteroid muscle slowed to about half its normal rate, although the phase relationship between the spikes in the various motor neurons was preserved. The original rhythmic rate was restored when the nerve root containing the alar joint receptor axons was electrically stimulated (Fig. 11-SIB). Interestingly, although the frequency of motor rhythms increases when sensory input is provided, the timing of motor output does not correlate strongly with the timing of impulses in sensory nerves. Occasional stimulation of wing joint receptor axons can accelerate motor performance, although sensory input is most effective when it occurs during specific phases of the wing flapping cycle Proprioceptive feedback is therefore not required for proper coordination of motor impulses with flight muscles; however, sensory feedback amplifies the output of the central flight pattern generator when it is activated (Fig. 11-SIC). What turns an aircraft engine on and off? When a grasshopper jumps off a substrate to fly, hair receptors on its head are stimulated by passing air. This specific sensory input triggers the output of the aircraft's engines. When the insect lands, the central flight pattern generator is turned off by a signal from the mechanoreceptors in the feet (called tarsals in insects). Endogenous pattern-generating networks have been shown to exist in various invertebrate nervous systems. For example, the cyclic motor output of crayfish abdominal swimmers is present not only in isolated nerve cords, but even in a single isolated abdominal ganglion. This intrinsic rhythm is initiated and maintained by the activity of "command" interneurons whose cell bodies are located in the brain's supraesophageal ganglia. Although the burst pattern of each abdominal ganglion requires the sustained activity of one or more interneurons, there is no simple one-to-one relationship between the firing patterns of these interneurons and the swimmer's motor output patterns. Key interneurons appear to provide a general level of arousal that keeps the central pattern generator active. One of the best-studied rhythmic patterns is the escape swim of the nudibranch Tritonia (Fig. 11-52A). This sea cucumber avoids noxious stimuli by alternately contracting the dorsal and ventral flexor muscles, causing the body to bend alternately dorsally and ventrally. Central patterns arise from the connections of three neurons: cerebral neurons (C2), dorsal swimming interneurons, and

Ventral swim interneurons - form synapses with flexion neurons (Fig. 11-52B). Cerebral neurons (C2), dorsal swimmer interneurons, and ventral swimmer interneurons are connected by interconnections, many of which are a mixture of excitatory and inhibitory synapses. Mutual inhibitory synapses between neurons are found in many central pattern generators that generate rhythmic output; reciprocal inhibitory synapses in the central pattern generator of Tritonia swimming have been shown to be required for swimming production in this species. After the first stimulus, dorsal and ventral swim interneurons generate alternating bursts of neuronal activity that activate flexion neurons responsible for motor performance. Intracellular recordings from all five neuron types revealed that swimming rhythms depend on the membrane properties of individual neurons and their synaptic connections. Rhythms are thus neurogenic, arising from interactions between neurons. It was recently shown that the strength of synapses between neurons in this network can be modulated during swimming to alter the properties of the network even as it produces swimming performance. Autonomic central neuronal control also exists to varying degrees in vertebrates. The respiratory movement, driven by cells in the brainstem, persists in mammals when sensory input from the pectoral muscles is eliminated by severing the corresponding sensory nerve roots. Toads in which all sensory roots had been severed except for the cranial nerves were still able to perform simple coordinated walking movements, although these movements were difficult to detect because the loss of muscle reflex arcs resulted in muscle relaxation. When part of the sensory input was eliminated, the motor output of the shark's and lamprey's swimming muscles continued in the normal alternating pattern. However, the sequence of intersegmental motor performance that normally runs from front to back may be disrupted. After transection of the brainstem above the medulla oblongata (so-called feline spinal preparation), the walking movement of cats supported on a treadmill was studied. These studies demonstrate that walking sequences can be completed without input from the brain. Furthermore, basic gait rhythms were observed to persist even after the DR was transected, thereby eliminating sensory input. Thus, even in vertebrates, certain aspects of rhythmic movement are programmed into intrinsic connections between spinal cord and hindbrain neurons that persist even when sensory feedback and other sensory inputs are disrupted. Central Command System Stimulation of appropriate neurons in the central nervous system can induce coordinated movements of varying complexity. Electrical stimulation of this command system in the crayfish's nerve cords causes the animal to adopt a defensive posture, with the paws up and the body arched upward on the outstretched forelimbs.

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Figure 11-52. Swimming in the slug Tritonia is controlled by a central pattern generator consisting of three types of neurons. (A) When a Tritonia is threatened (for example, by a slug-eating starfish), it rises from the bottom of the water and swims, rhythmically contracting its dorsal and abdominal flexors. (B) Three interconnected neurons work together to form swimming movement patterns. Excitatory synapses are indicated by bars; filled circles indicate inhibitory synapses; the combination of these two symbols represents a multifunctional synapse.

Attributes and synaptic interactions determine swimming motility patterns. Activity records that vary as these parameters change. (C) Floating central pattern generating (CPG) neurons in the isolated brain after electrical stimulation of the pedal nerve. Abbreviations: C2, cerebral neuron; DSI, dorsal swimming interneuron; VSI, ventral swimming interneuron; DFN, dorsal flexor neuron; VFN, ventral flexor neuron. [Part A courtesy of P. Katz; Parts B and C adapted from Katz et al., 1994.1

Appropriate sensory input excites this system through specific interneurons, and this interneuron varies widely, resulting in excitation of some motor neurons and inhibition of others. The command system of arthropods typically activates many muscles in a coordinated fashion and produces interactions in specific body parts; that is, antagonists are inhibited and synergists are excited. Perhaps unsurprisingly, the command interneurons that are most effective at eliciting coordinated motor responses are often the least likely to be activated by simple sensory inputs. The discovery of such command neurons in crayfish initially led physiologists to hypothesize that most of the animal's behavior may be controlled by a small population of command neurons, each responsible for generating and shaping specific behaviors. In this case, the "choice" between behaviors would depend on which command neuron was most active. However, further research into the neural basis of behavior revealed that most command functions occur in neural networks in which all involved neurons play important roles. In order to determine experimentally whether a neuron fulfills a command function, the activity of the neuron must be demonstrated

Neurons are both necessary and sufficient to generate the motor output at hand. That is, removing a neuron from the network must prevent or greatly alter behavior (necessity), whereas simply activating that neuron must elicit behavior (sufficiency). Three observations recur when testing of necessity and sufficiency is used to determine the neural basis of many behaviors. First, many neurons are multifunctional, acting differently under different conditions. For example, some bipolar retinal cells have been found to transmit signals from rod photoreceptors in dim light and from cone photoreceptors in bright light. As ambient light levels change, their connection patterns need to change. Second, a neuron can belong to different layers of a hierarchical control system (see Figure 11-48). For example, a neuron in the Tritonia swim control network functions as a central pattern generator for swimming and a command system for escape. Third, since the network can be reconfigured according to the situation, there must be mechanisms that can change the neural connections. Anatomical connectivity can limit the range of possible outputs for many novel

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................................................... ................... rons, but functional linkages define their output at a given point in time. One of the best understood mechanisms for switching neural networks between possible functional configurations is neuromodulation (see Chapter 6). Neuromodulators can induce changes in synaptic efficiency, dynamically reconfiguring a group of neurons into a new functional unit. The recognition that "anatomy is not destiny" of the nervous system has changed the way systems are analyzed. In this section, we consider two systems that have been analyzed in sufficient detail to provide example

Follow these three principles when organizing your management system. Many invertebrates use stereotyped movements to evade potential enemies. A well-studied example is the crustacean Procambarus clarkii, which exhibits two types of escape responses depending on the location of the stimulus (Fig. 11-53A). In every behavior, at least one giant axon was part of a control circuit, a pattern typical of neurons controlling many escape responses. Large axons can transmit signals quickly, allowing animals to escape more quickly. inside

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Figure 11-53. Tactile stimulation of a giant interneuron causes a crab to change posture. (A) Stimulation of the abdomen (upper left image) induces flexion of the abdomen, moving cancer cells upward and forward. This behavior is mediated by lateral giant interneurons. Anterior stimulation of the antennae (upper right) induces different forms of abdominal flexion, propelling the animal backwards. This response is mediated by medial central neurons. In both cases, the behavior distances the animal from the stimulus. In the figure, the time after stimulation is shown in the shape of seconds and the time progression from top to bottom. (B) Diagram of an example of a circle eliciting an escape response to abdominal touch in a crab. Sensory input is transmitted to lateral Grant neurons via chemical triangular and electrical (recovery sign) synapses, resulting in fast electrotonic synapses in motor Grant neurons. Movement of the ganteuron synapse with the abdominal flexors. Giant axons of large size generate high conduction velocities, and electrotonic synapses ensure rapid communication between neurons. Electrical stimulation of the giant lateral interneurons produced flexion only in abdominal segments 1 to 3. Compare this effect to the terminal pose of a crab touching the abdomen - the flexion of the anterior abdominal region is evident. [Adapted from Wine and Krasne, 1972, 1982.1

The crab has two giant fibers: the medial giant interneuron, which controls flexion to propel the animal backwards; and the giant lateral interneuron, which plays a key role in the animal's upward and forward movement. The basic circuitry surrounding the macrolateral interneuron is shown in Figure 11-53B. The neural networks around the media giant are very different at both the input and output ends of the network, which explains why crayfish behave so differently when they are touched by their tentacles. The crab's flight response illustrates some other features of the motor control system. First, when the cancer was stimulated repeatedly, it stopped responding to the stimulus after about 10 minutes. The response should take some getting used to. Although habituation can occur at many different points in the network, it has been found that this behavior requires habituation because fewer neurotransmitters are released from sensory afferent neuron terminals when stimuli are repeated over longer periods of time. Second, the overall control of the crayfish tail involves a second parallel pathway that also elicits the tail-flick response. The fast flexor motor neuron, which is not a giant motor neuron, can more precisely control the tail beat, although it is neither as fast nor as powerful as the beat produced by a giant neuron. When tail flicks are triggered by giant motor neurons, the second pathway is also activated, although the slow pathway can also function independently. Third, when levels of serotonin, a neuromodulator, are altered in cancer, the cancer's response to specific stimuli changes dramatically. Aggressive Cancers can become submissive and vice versa. Therefore, neuromodulation must alter the connections between sensory and motor neurons. The crab's escape response is a typical fixed behavior pattern, and the neurons controlling it embody several features of the command system described earlier. Perhaps the most important feature is the presence of multiple points of control in the network, providing multiple opportunities to initiate or slightly alter the performance of behavior. This flexibility within the constraints of fixed behavioral patterns is a source of insight into behavioral organization.

Condition. The best example of dynamic network assembly is the assembly of 30 large neurons that make up the gastrogastric ganglion (STG) in crustaceans. The esophagus and stomach of lobsters and crabs are complex structures responsible for food intake, storage, chewing, grinding, and filtering (Figure 11-54). The orogastric system has four functional areas: esophagus, pericardium, gastric tube, and pylorus. Neurons of the STG control all muscle compartments responsible for food intake and peristaltic movement. They also control the bony teeth responsible for chewing and grinding. Since the majority of neurons in the STG are motor neurons innervating the muscles of the orogastric system, their intrinsic properties have been of direct interest in efforts to discover the functional architecture of any subnetwork that can be formed by this small group of neurons. The ganglia can be divided into three neuronal networks that control the muscles of the esophagus, gastrointestinal tract, and pylorus of the gastrointestinal system. The esophageal, gastric, and pyloric networks can all generate rhythmic output patterns independent of the other two (Fig. 11-55A). The output frequency of each network is a characteristic of the network. Inputs from modulating neurons drastically change the behavior of those neurons. For example, two electrically coupled neurons called PS neurons reconfigure the network. When PS neurons fire, the valve between the esophagus and stomach opens and the act of swallowing begins. Then a whole new rhythm begins, harmonizing

dilator

The recognition that synaptic neuromodulators can alter network properties opens up new ideas. A central command system once thought to accomplish a single mode of behavior must now be seen as plastic, with neurons forming different synaptic relationships as needed

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Figure 11-54 The orogastric nervous system controls the activities of the lobster esophagus, gastrointestinal tract and pylorus. The thoracogastric ganglion (STG, one of the four ganglia in the system) contains only 30 neurons, most of which are motor neurons, all of which have been identified and characterized. The output of these neurons controls the contraction of muscles, which cause food to be swallowed, chewed and transported to the rest of the digestive system. (Muscles controlling the pylorus are shown. The constrictor closes the pylorus, preventing food from escaping. The dilator opens the pylorus, allowing food to pass to the next part of the digestive system. These muscles receive input from STG neurons.) [Adapted from Hall , 1992.1

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All three parts of the STG system produce a series of peristaltic waves that propagate from the esophagus to the pylorus (Fig. 11-55B). During this behavior, all other rhythms are suppressed. When the activity of PS neurons ceases, another rhythm briefly emerges, but eventually all neurons in the swallowing network revert to their original activity patterns. The neurons that control swallowing behavior are called the swallowing network. This included neurons that were active when no PS neurons were active in the esophageal, gastric, or pyloric networks. Several neuromodulators that control the activity of STG neurons have been identified. Both the biogenic amines serotonin and the neuropeptides proctolin and cholecystokinin alter the output pattern of at least some neurons in the STG. Reconfiguration of a small subset of neurons into multiple functional networks suggests a new view of the neurons responsible for controlling motor performance. Previous work has shown that individual anatomically defined networks can generate different forms of output in response to neuromodulators, but the gastrointestinal system suggests that network composition may also be plastic. The dynamic specification of many functional networks within a defined set of neurons greatly increases the number of possible ways to control motor performance. One challenge is clearly figuring out where the control of this mechanism lies and how it is regulated.

Summary All behavior is controlled by the motor output of the nervous system. Motor neurons are organized into networks.

Figure 11-55: Modulatory input to an orogastric ganglion significantly alters neuronal output and reconfigures subnetworks within the ganglion. (A) When regulatory PS neurons are silenced. Neurons in the gastrogastric ganglion are organized into three separate subnetworks, namely, the esophageal network, the gastric network, and the pyloric network. Each of these subnetworks produces rhythmic outputs, however: These outputs are not temporally coordinated with each other. In this state, food is chewed and moved (arrows) in the gastrointestinal tract and gastric socket, and no food enters or leaves this part of the digestive tract. (B) When PS neurons are active, neurons from all three subnetworks are recruited into a new network where their activity is coordinated to produce "swallows" (indicated by red arrows). The abbreviations Es, Gast, Pyl and PS denote the activity of neurons in the esophageal, gastric and pyloric subnetworks and PS neurons. [Adapted from

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Pieces can be somewhat malleable, allowing flexibility in behavioral responses. To understand behavior at the neural level, it is necessary to understand how neurons interact to produce behavioral output. During evolution, the primitive, distributed, and anatomically diffuse "neural network" features of coelenterates condensed into nerve cords and ganglia, seen even in some jellyfish. In segmented animals, the front end, initially specialized to house many sensory organs, differentiates and contains a supraganglion or brain. The most complex nervous systems are found in vertebrates. These systems can be divided into central nervous system and peripheral nervous system. All neurons in the nervous system are afferent neurons, efferent neurons, or interneurons, and most neurons in complex neural networks are interneurons. The connections in the central network appear to be largely genetically programmed; but they are maintained and can be changed by use during development and thereafter. The integration of inputs to each neuron in a network and the generation of subsequent activity depends primarily on two main factors: (1) the organization of circuits and synapses formed by interacting neurons, and (2) the individual neuron processing or integration How to pass in information to create a custom AP. The comprehensive properties of neurons depend on the neuronal anatomy, connections, and properties of the cell membrane and ion channels. The identification of specific behaviors known as fixed action patterns facilitates the study of neurobehavioral control. these very old engines

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Patterns are usually evoked by specific stimuli or key stimuli. The goal of neuroethology is to understand the behavioral capabilities of animals at the neural level. The sensory neural network sorts and distills the information available to the animal. They amplify, amplify, filter and reconfigure raw sensory input. The mammalian visual system teaches us a lot about how sensory systems work. Electrical recordings from cells in the visual cortex reveal that individual central neurons are activated by stimuli and extract specific features of the stimulus, rather than producing point-by-point representations of peripheral inputs. Furthermore, studies of the visual system have shown that there is a hierarchical arrangement of neurons and that the specificity of the sensory features eliciting neuronal activity increases with each level until, at higher levels, only some of the visual stimulus responses These features are still the cause. Some cells can only be activated by stimuli as complex as a face. Studies of barn owls have shown that the map of the auditory space is calculated from the intensity and temporal differences between the sounds received by the two ears. This computed spatial map is anatomically consistent with other sensory maps, such as the retinotopic map of the visual system. The simplest neuronal network is the monosynaptic reflex arc, best known as the stretch reflex in vertebrates. More complex behaviors include movements based in part on core "motor programs" that determine, for example, the sequence of muscle contractions that produce coordinated movements. Feedback from proprioceptive neurons can influence the intensity and frequency of motor performance and also help fine-tune coordination in most rhythmic motor activities. Muscle performance is controlled by a rating system. An example of a minimal level of control is the monosynaptic stretch reflex arc responsible for maintaining postural tension. The next layer down are rhythmic movement patterns like walking, swimming and crawling. Finally, at the top of the hierarchy are controls for complex, fixed patterns of behavior. Attempts to understand the relationship between levels of control have been most successful in relatively simple invertebrate locomotion systems. In these model systems, it is clear that a given neuron can participate in multiple motor networks functioning at different levels. In addition, neuromodulatory substances dynamically control the configuration of networks in certain systems.

Review Questions 1. The action potential of all neurons is basically the same; how does the central nervous system recognize the shape of input from various sensory organs? 2. Describe the general organization of the vertebrate brain and spinal cord. 3. Comparing and Contrasting Sympathetic and Parasympathetic Areas of the Autonomic Nervous System

system. How do they differ anatomically? How do they differ functionally and biochemically? All sensation is said to take place in the brain. Explain what this sentence means. How does an increased firing rate of inhibitory interneurons lead to increased activity of other neurons? What is the source of persistent low-level synaptic inputs and slow tonic discharges in vertebrate spinal cord and motor neurons? Describe the organization of the vertebrate retina. Each primate eye sees roughly the same field of view, but the right hemisphere "sees" the left half of the field of vision, while the left hemisphere "sees" the right half of the field of vision. How did this happen? Why does the evening sky seem to have a lighter band outlining the mountains? What does the "receptive field" of a cortical neuron mean? How could the receptive field of a simple cell in the visual cortex be a bar or a straight edge when cells in the lateral geniculate body have circular receptive fields? What would happen to your posture if all your muscle spindles suddenly stopped working? y How do efferent fibers alter the sensitivity of muscle spindles? Some general insights into neural organization derived from studies of the retina and visual cortex are discussed. The nervous system is sometimes likened to a telephone system or a computer. Discuss some properties of the nervous system that make this analogy a good one, and other properties that make it a bad one. What evidence is there that certain complex patterns of behavior are inherited and cannot be attributed to learning alone? What supports the claim that it is the properties of neuronal circuits rather than individual neurons that are responsible for the differences in function across different nervous systems? What are trigger stimuli and fixed action patterns? Give at least one example of each. What is a Central Pattern Generator? What are the properties of central pattern generators and what role do they play in controlling behavior? Some examples of central pattern generators are described. What are command neurons? What is an order system? Describe some examples of the command system. How can one neuron play different roles in multiple central pattern generators?

Recommended Reading Camhi, J. 1984. Neuroethology. Sunderland, MA: Sinauer. (An excellent textbook summarizes many aspects of this rapidly developing discipline.)

Carew, T.J. and C.L. Sahley. 1986. Invertebrate learning and memory: From behavior to molecules. install. Pastor of Neuroscience. 9:435-487. (A review of advances in understanding this important form of plasticity in the nervous system.) Dowling, J. 1987. Retina: An accessible part of the brain. Cambridge, MA: Belknap Press. (The description of the structural and functional organization of the vertebrate retina has made an important contribution to our current understanding of this remarkable organ.) Ewert, J.-P. 1980. Neuroethology. Berlin: Springer Verlag. (Introduction to a relatively new field: the study of the neural basis of behavior.) Grillnel; S. and P. Warren. 1985. Central pattern generators of locomotion, with special reference to vertebrates. install. Pastor of Neuroscience. 8:233-261. (An overview of the properties of the central pattern generator, with emphasis on the CPG for lamprey swimming.) Gwinner, E. 1986. The internal rhythms of bird migration. Scientific American 25494-92. (A biological approach to this seemingly mysterious ability to navigate.) Hubel, D. 1995. Eyes, brain and vision. New York: American Library of Science paperback. (A highly readable survey of information processing in the visual system, written by one of the most prolific and creative researchers in the field.)

Kandel, E., J. Schwartz, and T. Jessell. 1991. Principles of Neuroscience, 3rd ed. New York: Elsevier. (Compendium of extensive information on the nervous system, with emphasis on vertebrates, especially mammals.) Knudsen, E.I. 1981. The Barn Owl's Hearing. Scientific American 245:113-125. (A very readable discussion of this bird's remarkable auditory nervous system, including descriptions of some very inventive physiological experiments.) Konishi, M. 1985. Birdsong: From behavior to neuron. Ann. Pastor of Neuroscience. 8:125-170. (A review of the neural basis of bird song production, by one of the foremost experts on the avian brain.) McFarland, D. 1993. Animal Behavior: Psychozoology, Ethology, and Evolution. New York: Willie. (A classic text on the study of animal behavior.) Nicholls, J. G., A. R. Martin, and B. G. Wallace. 1992. From neurons to brain: cellular and molecular approaches to the nervous system, third edition. Sunderland, MA: Sinauer. (A very readable text describing the properties of individual cells and circuits.) Nauta, W. J. H. and M. Feirtag. 1986. Basic neuroanatomy. New York: W.H. Freeman & Co. (A comprehensive description of mammalian neuroanatomy.)

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Physiological system integration

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So far, we have reviewed the fundamentals of animal physiology (Chapters 1-4) and then discussed the nervous, muscular, and endocrine systems and the processes by which they regulate physiological function (Chapters 5-11). Still to be discussed in Part I11 (Chapters 12 to 16) are the various regulated physiological systems involved in the daily efforts of animals to absorb and store nutrients and energy, excrete waste products, and respond to changing environments and reproduction. In the past, animal physiology textbooks treated each regulated physiological system of an animal more or less separately, with little emphasis on their functional and structural interdependence. This approach has persisted, both to facilitate discussion and because, in part, it reflects a biologist's pattern of interest in a particular animal system. Physiologists, for example, often refer to themselves as "cardiovascular physiologists"; few emphasize the more general aspects of their field, such as calling themselves

For example, "energy transfer physiologists", who study the coordinated transport of nutrients, waste products and heat between the environment and the interior of animals. Also, since the circulatory systems of all animals share similarities, it is convenient to discuss each system in one chapter. However, dividing physiological systems into units helps organize a course or a book, giving generations of students the wrong impression that animals function as a collection of loosely connected physiological systems that happen to contained within an organism. For this reason, we want to emphasize that animals function as integrated systems, responding to and constrained by their environment. These interconnected systems function in a highly coordinated manner when faced with environmental stress (temperature, pressure, etc.) or biotic stress (predation, disease, etc.). The actual structure and function of a single physiological system changes due to the constraints imposed on it because it is part of a larger physiological network. Because these systems are highly dependent on each other

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Because environmental pressures are interdependent, they can place conflicting demands on individual systems. It is important to consider these interactions in space and time. Examples abound. In some snake species, lung capacity is initially reduced after eating large prey due to space constraints in the visceral cavity. However, lung capacity is slowly restored as food is digested (the interplay of breathing and digestion in space and time). A similar situation exists in humans after a large meal or during pregnancy. As another example, muscle strength responds to exercise over time, but it's not just about gains in muscle mass. In addition, blood flow to the muscles must be increased, which can lead to changes in the heart and breathing (the interaction between the musculoskeletal and cardiopulmonary systems over time). Additionally, the skeletal framework needs to be strengthened to withstand the added stress of this exercise on the body. While we would like to emphasize the importance of a comprehensive understanding of animal physiology, we also recognize that it is impractical to require students to simultaneously learn everything about all regulated physiological systems. Therefore, we divide the regulated systems into different chapters. Each of these chapters focuses on

Examples are used throughout a given system and its functions, highlighting the interactions between physiological systems and the way they respond to environmental changes in a coordinated manner. Chapters 12 to 14 of Part I11 discuss truly multifunctional systems. The circulatory system (Chapter 12) is a means of distributing substances between tissues, especially oxygen, carbon dioxide, and various nutrients and wastes. Oxygen uptake and carbon dioxide removal are the subject of Chapter 13. The circulatory and respiratory systems of animals work together in homeostasis, for example by regulating acid-base status and, in some systems, ionic and osmotic conditions in vivo (Chapter 14). As discussed in Chapter 15, animals use a variety of mechanisms to obtain energy, from filter feeding to feeding on predators. This chapter discusses the mechanisms, controls and chemistry of food intake, digestion and absorption. The last chapter (Chapter 16) is in many ways a summary of the book's theme, dealing with animal energies. Examine the energy expenditure of locomotion, reproduction, growth, and maintaining homeostasis and place it within the overall goal of reproductive survival.

In animals with a diameter of 1 mm or less, substances are transported within the body by diffusion. In larger animals, however, sufficient mass transport rates can no longer be achieved in vivo by diffusion alone. In these animals, the circulatory system has evolved to transport respiratory gases, nutrients, waste products, hormones, antibodies, salts and other substances between different parts of the body. Blood, the medium through which these substances are transported, is a complex tissue containing many specialized cell types. It acts as a vehicle for most homeostatic processes and plays a role in nearly all physiological functions. This chapter examines blood circulation and how it is controlled to meet tissue needs. The circulatory system of mammals has received the most attention because it is the best known. Mammals are very active, primarily aerobic land animals whose circulatory systems have been adapted to their specific needs. The mammalian system is just one of several types of circulation. However, all circulatory systems are composed of the following basic components, which have similar functions in different animals:

1. The main engine that drives blood throughout the body, usually the heart. 2. An arterial system that is used both for blood distribution and as a pressure accumulator. 3. Capillaries, where the transfer of substances between blood and tissues occurs 4. The venous system, the system that acts as a blood (volume) reservoir and returns blood to the heart. Arteries, capillaries, and veins make up the peripheral circulation.

General plan of the circulatory system The movement of blood throughout the body is caused by any or all of the following mechanisms:

The force produced by the rhythmic contraction of the heart. Elastic recoil of arteries after systolic filling. Blood vessels are squeezed during physical activity. Peristaltic contraction of perivascular smooth muscle. The relative importance of each of these mechanisms in generating flux varies across animals. In vertebrates, the heart plays a major role in blood circulation; in arthropods, limb movement and dorsal contraction of the heart are equally important for generating blood flow. In the giant earthworm Megascolides australis, peristaltic constriction of the dorsal vessels is responsible for moving blood forward and filling the lateral hearts, which pump blood into abdominal vessels for distribution throughout the body (Fig. 12-1A). The worm, which can grow up to 6 meters long, is divided into segments separated by membranous structures (diaphragms). Tracer studies showed that the first 13 segments, each containing two lateral hearts, perfused rapidly, but the remaining segments lacked lateral hearts and perfused very slowly. Due to the peristaltic contraction of the dorsal vessels, the blood pressure of the dorsal vessels was significantly higher than that of the ventral vessels (Fig. 12-1B). In all animals, valves and/or diaphragms determine the direction of flow, and perivascular smooth muscle changes vessel diameter, thereby regulating the volume of blood flowing through a given pathway and controlling the distribution of blood throughout the body. open circulation

Many invertebrates have an open circulatory system in which blood pumped by the heart empties through arteries into an open fluid space, the hemocoel, located between the ectoderm and endoderm. The fluid in the blood cavity, called hemolymph or blood, does not circulate through the capillaries, but directly infiltrates the tissues. Figure 12-2 (A and B) illustrates the organization of the main ships

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Pump into ventral vessels. (B) Peak blood pressure in dorsal vessels is approximately twice that of ventral vessels due to peristaltic contraction. [Adapted from Jones et al., 1994.1

Open circuit of two groups of invertebrates. The hemocoel is usually large, accounting for 20% to 40% of the body volume. For example, in some cancers blood volume is about 30% of body volume. An open circulatory system exhibits low pressure, with arterial pressure rarely exceeding 0.6-1.3 kiloPascal (kPa) or 4.5-9.7 mmHg (1 kPa = 7.5 mmHg). Higher pressures were measured in the partially open circuits of the land snail Helix, but these were the exception. In snails, these high pressures are generated by heart contractions, while in some mussels, high pressures in the feet are generated by surrounding muscles rather than heart contractions.

Animals with an open circulatory system typically have a limited ability to alter the rate and distribution of blood flow. Consequently, in mussels and other open-circulating animals that use blood for gas transport, changes in oxygen uptake are generally slow and maximum oxygen transfer rates per unit weight are low. However, these animals have some control over the flow and distribution of hemolymph; moreover, in animals with an open circulatory system, blood is distributed in many small channels in the tissues. Without these features, even modest oxygen consumption would not be possible due to extensive diffusion.

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12-2 Most invertebrates have an open system, but cephalopods have a closed system. The major blood vessels in the open circuit of crabs (A) and mussels (B) empty into a large surrounding space, the hemocoel, accounting for approximately 30% of the body volume. The closed-circuit system in cephalopods (C) has higher blood pressure and more efficient oxygen delivery compared to the open-circuit. In all diagrams, only major blood vessels are shown. Arrows indicate blood flow.

Oxygen ion distance between hemolymph and living tissue. Insects have an open circulatory system but do not rely on it for oxygen delivery, so can achieve high speeds

In a closed circuit, blood flows from arteries to veins through capillaries in a continuous tubular circuit. All vertebrates and some invertebrates such as cephalopods (squid, cuttlefish) have this cycle (Fig. 12-2C). In general, the separation of duties is more complete in closed-loop systems than in open systems. The blood volume in closed circulatory systems of vertebrates is typically about 5-10% of body volume, much smaller than in open circulatory invertebrates. Extracellular volume in vertebrates is expressed as a percentage of body volume and is similar to the hemocoel volume in invertebrates. The closed circulatory system of vertebrates is a specialized part of their extracellular space. In a closed circulatory system, the heart is the main engine that pumps blood into the arterial system and maintains high arterial pressure. In turn, the arterial system acts as a pressure accumulator, pushing blood through the capillaries. Capillary walls are thin, allowing high rates of material transfer between blood and tissues by diffusion, transport, or filtration. Every tissue has many capillaries, so each cell is no more than two or three cells away from a capillary. The parallel capillary network enables precise control of blood distribution and tissue oxygen supply. Animals with closed circulatory systems can increase oxygen delivery to tissues very rapidly. Because of this, squid, unlike many other invertebrates, can swim rapidly and maintain high oxygen uptake; that is, their closed-loop system allows sufficient hemolymph flow and efficient distribution to muscles to support brief bursts of high-intensity activity . The blood is under a sufficiently high pressure in the closed circuit to allow ultrafiltration of the blood in the tissues, especially in the kidneys. Ultrafiltration is filtration through a semipermeable membrane (capillary wall) using pressure (blood pressure) to force fluid through the membrane, thereby separating the ultrafiltrate free of colloidal particles from plasma. Ultrafiltration occurs in the kidneys of most vertebrates, resulting in a net movement of protein-free plasma from the blood into the renal tubules. Normally, all capillary walls are permeable, and due to the high pressure, fluid slowly seeps through the walls and into the spaces between the cells. In conjunction with the high pressure closed circulatory system of vertebrates, the glymphatic system has evolved to recycle fluid from blood lost to tissues. The degree of filtration depends largely on blood pressure and the permeability of the capillary walls. Filtration through the capillary wall can be reduced by

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In the permeability of capillary walls or in blood pressure. For example, blood vessel walls in some tissues are less permeable than others. In the liver and lungs, which are highly permeable for other reasons, the pressure is lower than in other parts of the body. The systemic (body) and pulmonary (lung) circuits of mammals can maintain different pressures because the mammalian circulatory system is equipped with a fully divided heart (Figure 12-3). The right side of the heart pumps blood into the pulmonary circulation, and the left side pumps blood into the systemic circulation. However, this means that the flow in the pulmonary and systemic circulation must be equal, as blood returning from the lungs is pumped throughout the body. In other vertebrates, the heart is not completely divided and the flow to the lungs can vary independently of the blood flow in the body. The venous system collects blood from capillaries and carries it to the heart through veins. These are usually compliant low-pressure structures in which large changes in volume have little effect on venous pressure. Therefore, the venous system contains most of the blood and acts as a bulk reservoir. blood donors donate blood from

Since there is little change in pressure as venous volume decreases, volume and flow in other areas of the circulatory system do not change significantly.

Heart The heart is the ventilated muscular pump that pumps blood throughout the body. The heart consists of one or more muscular chambers connected in series, protected by valves, or in some cases, sphincters (such as in the hearts of some molluscs), which allow blood to flow in only one direction. The mammalian heart consists of four chambers, two atria and two ventricles. The contraction of the heart causes blood to be pumped into the circulatory system. Multiple ventricles allow a gradual increase in pressure as blood moves from the venous side of the circuit to the arterial side (Figure 12-4).

Cardiac and skeletal muscle fibers in vertebrates are similar in many respects, except that the T-tube system is less distributed in lower vertebrate cardiomyocytes, and cardiomyocytes are electrically coupled (see Chapter 10). Apart from differences in Ca2+ uptake and release, the contraction mechanisms of vertebrate skeletal and cardiac muscles are generally considered to be the same. Myocardium (i.e., cardiac muscle) is composed of three types of muscle fibers that differ in size and functional properties: Cardiomyocytes in the sinoatrial node (or sinoatrial node) and the atrioventricular node are generally smaller than other cells, less contractile, self-regulated, and cell-to-cell conduction very slowly. The largest cardiomyocytes, located on the inner surface of the ventricle wall, are also less contractile but specialized for conducting rapid conduction and forming a system that propagates stimuli throughout the heart. The medium-sized cardiomyocytes are highly contractile and make up most of the heart. Electrical activity of cardiac arterioles, C a ~ i l l a i e s (5-7%)

Figure 12-3 The closed circulatory system of mammals consists of a completely separate heart, allowing differential pressures in the lungs and body parts. This diagram illustrates the major components of the mammalian circulatory system, with oxygenated blood in red and deoxygenated blood in blue in the systemic and pulmonary systems. The associated lymphatic system (yellow) returns fluid from the extracellular space through the mammary sac to the blood. Percentages indicate the relative proportions of blood in different parts of the circulation. The lymphatic system and associated lymph nodes also play a key role in the immune response.

The heartbeat involves the rhythmic contraction (contraction) and relaxation (relaxation) of the entire muscle mass. The contraction of each cell is associated with an action potential (AP) in that cell. Electrical activity initiated in the pacemaker region of the heart propagates through the heart from cell to cell as cells are electrically coupled through membrane junctions (see Chapter 4). The nature and degree of coupling determine the mode of electrical excitation wave propagation in the heart and also affect the excitation rate.

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Neurogenic and Myogenic Pacemakers In the vertebrate heart, the pacemaker is located in the sinus venosus, or in a remnant called the sinoatrial node (see Figure 12-4). Pacemakers consist of small, weakly contracted, specialized muscle cells that are capable of spontaneous activity. These cells can be neurons, such as the neurogenic pacemakers in many invertebrate hearts, or muscle cells, such as the myogenic pacemakers in vertebrate and some invertebrate hearts. Hearts are often classified by the type of pacemaker and are therefore called neurogenic or myogenic. In many invertebrates, it is unclear whether pacemakers are neurogenic or myogenic. However, decapods (shrimp, lobster, crab) have neurogenic hearts. In these animals, the cardiac ganglion of the heart functions as a pacemaker. When a cardiac ganglion is removed, the heart stops beating, although the ganglion remains active and exhibits an inherent rhythm. Cardiac ganglia are composed of nine or more neurons (depending on the species), divided into parvocytic and magnocellular. A small battery acts as a pacemaker and connects to a large follower battery, all galvanically coupled. The activity of small pacemaker cells is fed to and integrated by large follower cells, which then distribute to the myocardium. Cardiac ganglia in crustaceans are innervated by excitatory and inhibitory nerves from the central nervous system (CNS); these nerves can alter the firing rate of the ganglia and thus the heart rate (beats per minute). The hearts of vertebrates, mollusks, and many other invertebrates are powered by myogenic pacemakers. these tissues

Figure 12-4 The multichambered mammalian heart allows pressure to increase as blood flows from the venous to the arterial side. This cutaway view shows the rear of the human heart with the pathways of impulses shown in color. Impulses originate from the pacemaker in the sinoatrial node and travel to the atrioventricular node, from where they are sent to the ventricles. In some invertebrates, pacemaker cells are modified neurons; in others and all vertebrates, they have been described primarily as altered muscle cells. [After E. F. Adolph, 1967. Copyright 0 1967 by Scientific American, Inc. all rights reserved. ]

Has been extensively studied in a variety of species. A myogenic heart may contain many cells capable of pacing, but since all cardiac cells are electrically coupled, the cell (or population of cells) with the fastest intrinsic activity is the one that stimulates the contraction of the entire heart and determines the heart rate. These pacemaker cells normally eclipse cells with slower pacemakers; however, if the normal pacemaker stops for any reason, the other pacemaker cells die; this creates a new, lower heart rate. Therefore, cells capable of spontaneously generating electrical activity can be classified as pacemakers and latent pacemakers. If a latent pacemaker is electrically disconnected from the pacemaker, it can beat and drive a part of the heart muscle, usually an entire chamber, at a different rate than a normal pacemaker. This ectopic pacemaker is dangerous because it desynchronizes the pumping action of the heart chambers. Pacemaker Potential An important feature of pacemaker cells is the lack of a stable resting potential. Thus, during each diastole, the cell membranes in pacemaker tissue undergo a steady depolarization, known as the pacemaker potential (Figure 12-5). An all-or-no cardiac action potential is generated when the pacemaker potential brings the membrane to threshold potential. The interval between cardiac APs of course determines the heart rate, depending on the depolarization rate of the pacemaker potential and the degree of repolarization and its threshold potential

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................................................... .................................................................................................... Figure 12-5 Pacemaker cells undergoing spontaneous membrane desquamation Polarization, called pacemaker potentiation Cardlac may be autologous cells (curve A) faster depolarization increases heart rate (curve B) and heart rate, while slower depolarization slows heart rate (curve C) and heart rate.

Latency time (seconds)

Heart Al? Faster depolarization causes the membrane to reach the firing level sooner, thereby increasing the firing rate, resulting in a faster heart rate, while slower depolarization does the opposite (see Figure 12-5). Pacemaker activity arises from time-dependent changes in membrane conductance. In the frog sinus, pacemaker depolarization begins immediately after the previous AP when the potassium conductance of the membrane is very high. Subsequently, the potassium conductance gradually decreases and the membrane shows a corresponding depolarization due to the accumulation of intracellular potassium ions and a moderately high, stable sodium conductance. Pacemaker depolarization continues until sodium conductance is activated. The Hodgkin cycle then predominates, causing a rapid regenerative uprush of the cardiac AP (see Chapter 5). Acetylcholine released from the parasympathetic endings of the vagus nerve (tenth cranial nerve) slows the heartbeat by increasing the potassium conductance rate of pacemaker cells. This increased conductance keeps the membrane potential closer to the potassium equilibrium potential for longer, slowing pacemaker depolarization and delaying the onset of the next ascending stroke (Fig. 12-6A). exist

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On the other hand, norepinephrine released from the sympathetic nerve accelerates the depolarization potential of the pacemaker, thereby increasing the heart rate (Fig. 12-6B). Although norepinephrine increases sodium and calcium conductance, this may not be the primary mechanism responsible for pacemaker rhythm acceleration. Norepinephrine may decrease the time-dependent potassium efflux during diastole, thereby increasing the rate of pacemaker depolarization. Cardiac Action Potentials Action potentials in all vertebrate cardiomyocytes last longer before contraction than in skeletal muscle. APs in skeletal muscle are intact and the membrane is in a non-refractory state before contraction begins; thus, repeated stimulation and tetanic contraction are possible (Fig. 12-7A). In contrast, in the myocardium, the action potential plateau and membrane remain refractory until the heart returns to relaxation (Fig. 12-7B). .Therefore, there cannot be a sum of myocardial contractions. Cardiac AP begins with rapid depolarization, which is caused by a sharp and rapid increase in sodium conductance. This is different from slow depolarization

B Sympathetic Nerve Stimulation ~ o t e n t i a l

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Figure 12-6 Parasympathetic and sympathetic stimulation of the vagus nerve have opposite effects on pacing potential and heart rate (A). Effects of vagus nerve stimulation on Dlastol preserve transmembrane potential, reduce depolarization rate and

Reduction in action potential (B) duration and frequency leads to increased frequency of disruption of pacemaker cell pathogenesis [Hutter and Trautweln, 19561

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Figure 12-7 Action potentials in skeletal muscle are of very short duration (A), whereas cardiac action potentials exhibit prolonged repolarization or plateau phases during which the myocardium is refractory

stimulus(0). Therefore, during contraction and contraction sum, skeletal muscle may perform repetitive movements, but cardiac muscle does not

Pacing potential characterized by stable sodium conductance and decreased potassium conductance. Cell membrane repolarization is delayed, while the membrane remains depolarized for hundreds of milliseconds in the so-called plateau phase (see Figure 12-7B). The long AP duration of the heart results in a longer contraction time, allowing the entire chamber to fully contract before part of it begins to relax—a process essential for efficient pumping. The prolonged cardiac AP plateau is due to the maintenance of high calcium conductance and a delay in the subsequent increase in potassium conductance (as opposed to the situation in skeletal muscle). Due to the high calcium conductivity in the plateau, Ca2+ ions can flow into the cell because the equilibrium potential of calcium is strongly inward. This influx is particularly important in lower vertebrates, where most of the calcium required to activate contraction passes through the surface membrane. In birds and mammals, the surface area-to-volume ratio of larger heart cells is too small for sufficient calcium penetration and full activation of contraction. Thus, most calcium is released from the extensive sarcoplasmic reticulum of the higher vertebrate heart by depolarization of the T-tubules (see Chapter 10). Rapid repolarization ends the plateau due to a decrease in calcium conductance and an increase in potassium conductance. The duration of plateaus and the rates of depolarization and repolarization vary in different cells of the same heart. The sum of these changes is recorded on the ECG (Figure 12-8). Atrial cells generally have a shorter AP duration than ventricular cells. The duration of AP in atrial or ventricular fibers also differs in different heart types. The duration of the AP is a factor related to the maximal rate of the heartbeat; in smaller mammals, the duration of the vein-

The triocular AP is shorter, so the heart rate is usually faster than in larger mammals. Due to the diversity of hearts from different invertebrate phyla, few generalizations have been made regarding the ionic mechanisms that generate cardiac APs in invertebrate hearts. The only general feature is the involvement of calcium. For example, mussel heart contains calcium AP. The electrical activity initiated at the pacemaker region by the heart's conducted impulses proceeds throughout the heart, with depolarization of one cell causing depolarization of neighboring cells due to current flow through the gap junction (see Figure 4-35). These connections between cells are located in tightly juxtaposed regions between adjacent cardiomyocytes, called intervertebral discs. The adhesion of cells to the intervertebral disc is enhanced by the presence of desmosomes. The contact area is increased by folding and interlocking the membrane (Figure 12-9). The degree of folding and interlocking increases during heart development and also varies between species. Gap junctions are areas of low resistance between cells that allow electrical current to flow from one cell to the next through the intermediate disc. Although the connections between cardiomyocytes can conduct in both directions, the transmission is usually unidirectional because pulses start in and travel only from the pacemaker region. Due to the multitude of intercellular connections, there are often multiple pathways that can excite individual cardiac fibers. When part of the heart stops functioning, excitation waves easily flow around that part, allowing the rest of the heart to keep firing. The extended nature of the cardiac AP ensures that multiple connections do not result in multiple stimulation and reverberation of myocardial activity. Activate the AP in the pacemaker area

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. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ein ECG

Kern

Figure 12-9 Electrical activity can spread throughout the heart because cardiomyocytes are tightly attached to intervertebral discs densely populated with gap junctions. Shown here is a schematic diagram of a cardiomyocyte in a mammalian heart. Folding and interlocking of membranes are characteristic of interlaminar discs. Although desmosomes are present in these regulators, which enhance cell-cell adhesion, they are not easily distinguishable.

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Time (ms) Figure 12-8 The electrocardiogram represents the sum of electrical activity in different parts of the heart (A) The main components of the electrocardiogram (ECG) reflect atrial depolarization (P), ventricular depolarization (QRS) and ventricular repolarization (T) reflection . (6) The amplitude, shape and duration of cardiac action potentials are different in different parts. Possible changes were noted in the following locations: (I) sinoatrial node, (2) atrium, (3) atrioventricular node, (4) bundle of His, (5) Purkinje fibers in the pseudotendon, (6) Terminal Purkinje fibers (7) Ventricular myofibers. The numbers indicate the firing order of the different bursts. [Part B of Hoffman and Cranefield, 1960.1]

This results in a single AP being conducted through all other cardiomyocytes and requiring another AP from the pacemaker region for the next excitation wave. In the mammalian heart, an excitation wave propagates concentrically from the sinoatrial node over both atria at approximately 0.8 ma s-'. The atria are electrically connected to the ventricles only through the atrioventricular (AV) node; in other regions, the atria and ventricles are connected by connective tissue that does not conduct excitation waves from the atria to the ventricles (see Figure 12-4). The excitation propagates to the ventricle through the thin connecting fibers, where the velocity of the excitation wave is slowed to about 0.05 m. S-'. Connecting fibers connect to nodal fibers, which in turn connect to His bundles via transition fibers; this structure

The structure divides into right and left tracts that divide into Purkinje fibers that extend into the myocardium of both ventricles. Conduction through nodular fibers is slow (about 0.1 ms-'), but conduction through histidine tracts is fast (4-5 ms-1). His and Purkinje bundles transmit excitation waves very rapidly to all regions of the ventricular myocardium, causing all ventricular myofibers to contract. For each excitation wave, the ventricular cardiomyocytes contract almost immediately, and the excitation wave propagates at a velocity of 0.5 m. s-' From the inner layer (endocardium) to the outer layer (epicardium) of the heart wall. The functional importance of the electrical organization of the myocardium lies in its ability to generate independent synchronized contractions of the atria and ventricles. Thus, slow conduction through the AV node allows atrial contraction to precede ventricular contraction and also allows time for blood to flow from the atria to the ventricles. Because of the large number of cells involved, the electrical currents flowing during synchronized heart cell activity can be detected as tiny changes in electrical potential at various points throughout the body. These underlying changes, recorded as an electrocardiogram, reflect the electrical activity of the heart and can be easily monitored and analyzed. The P wave is associated with atrial depolarization, the QRS wave is associated with ventricular depolarization, and the T wave is associated with ventricular repolarization (see Figure 12-8A). The electrical activity associated with atrial repolarization is overshadowed by the much larger QRS complex. The exact shape of the ECG varies between animal species and is influenced by the type and location of the recording electrodes and the nature of the heart contraction. An EKG has medical value because it can be used to diagnose heart defects. As previously described, release of acetylcholine (ACh) from cholinergic nerve fibers increases the spacing between APs in pacemaker cells, thereby slowing the heart rate (see

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................................................. Figure 12-6A). This reduction in heart rate is sometimes called a negative chronotropic effect. Parasympathetic cholinergic fibers in the vagus nerve innervate the sinoatrial and atrioventricular nodes of the vertebrate heart. Acetylcholine also reduces the conduction velocity from the atria to the ventricles through the AV node as the heart rate slows. High levels of acetylcholine block conduction through the AV node so that only every 2 or 3 waves of excitation are conducted to the ventricles. In these abnormal conditions, the atrial rate will be double or triple the ventricular rate. Alternatively, high levels of acetylcholine may completely block conduction through the AV node (AV block), resulting in ectopic pacemakers within the ventricles. The result is that the atria and ventricles are controlled by different pacemakers and contract at completely different rates, and the two heartbeats become uncoordinated. For fish where atrial contraction is important for ventricular filling, this would be devastating. This is not so disruptive to mammals, since atrial contraction only fills the ventricles, which are filled primarily by blood flowing directly from the venous system through the relaxed atrium. The catecholamines epinephrine and norepinephrine have three distinct positive effects on cardiac function:

Because the heart primarily uses the aerobic pathway to generate energy, it relies heavily on a constant supply of oxygen. Therefore, sustained coronary blood flow is required to maintain cardiac output. Increased heart activity depends on increased metabolism, which in turn requires increased coronary blood flow. Adenosine may be a key metabolite that maintains the relationship between coronary blood flow and cardiac activity. Adenosine formed from adenosine triphosphate (ATP) during cardiometabolic processes and other local metabolic factors lead to dilation of coronary arteries, thereby increasing coronary flow. The formation and release of adenosine increases with increased metabolism or during periods of myocardial hypoxia (oxygen depletion), resulting in higher coronary blood flow. Sympathetic nerve stimulation is a second but less important mechanism for increasing coronary blood flow. Circulating catecholamines increase myocardial contractility and cause PI adrenergic receptor-mediated coronary vasodilation.

Increases myocardial contractility or heart rate (positive chronotropic effect) Increases myocardial contractility (positive inotropic effect) Increases the conductance of pulses across the heart (positive chronotropic effect) These catecholamine effects on contraction rate are pacemaker-mediated induced, and increased contractility is a general effect on all cardiomyocytes. Norepinephrine also increases conduction velocity through the AV node. It is released by adrenergic nerve fibers innervating the sinoatrial node, atria, atrioventricular node, and ventricles, so that sympathetic adrenergic stimulation acts directly on all parts of the heart.

Cardiovascular

The cardiovascular system provides nutrients and oxygen to the heart. The coronary artery supply of the heart is extensive, and the myocardium has a higher capillary density and more mitochondria than most skeletal muscles. It is also high in myoglobin, which gives the heart its characteristic red color. Blood pumped by the heart nourishes the spongy layer of the heart in many fish and amphibians as it travels through the heart. But even in these animals, the coronary supply is necessary to transport oxygen and other substrates to the outer, denser regions of the heart wall. In general, the heart has access to a variety of nutrients, including fatty acids, glucose, and lactate; which substrate to use depends largely on availability.

mechanical properties of the heart

The mechanical aspects of heart function are related to changes in heart pressure and volume, resulting in the ejection of blood with each heartbeat. We will now examine these properties and the certainty of what the heart does. Cardiac Output, Stroke Volume, and Heart Rate Cardiac output is the volume of blood pumped from the ventricles per unit of time. In mammals, it is defined as the volume ejected from the right or left ventricle, rather than the combined volume of both ventricles. The amount of blood ejected with each heartbeat is called stroke volume. Average stroke volume can be determined by dividing cardiac output by heart rate. Stroke volume is the difference between the volume of the heart chamber before systole (end-diastolic volume) and the volume of the heart chamber at the end of systole (end-systolic volume). Changes in stroke volume may be due to changes in end-diastolic or end-systolic volumes. The end-diastolic volume is determined by four parameters: the venous filling pressure, the pressure developed during atrial systole, the compliance of the ventricular wall, and the time available to fill the ventricle

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However, there is no single Starling curve that can describe the relationship between venous filling pressure and venous system workload.

frank starling

Tricycle. The mechanical properties of the heart are influenced by many factors, including the activity level of the internal nerves.

Mechanism Otto Frank observed that the more the frog's heart was filled, the greater the stroke volume. That is, increased venous return results in increased stroke volume. Frank deduced the length-stress relationship of the frog myocardium and showed that the systolic stress increases with increasing strain until it reaches a maximum

Heart function and the composition of blood flowing through the heart muscle. For example, the relationship between ventricular load and venous filling pressure is strongly influenced by sympathetic stimulation of the innervating heart. Starling was an accomplished researcher who, together with William Bayliss, discovered the hormone secretin. he coined the word

It then decreases with further stretching. Ernest Starling, a ruler

Hormones and defines their basic properties (see Chapter 9). Starling has also contributed a lot to our understanding

Frank, an important figure in many fields of physiology in the early 20th century, came to similar conclusions as Frank. Although starlings don't

cycle. In addition to the committee's opinion

Although Frank does not consider mechanical work, the increase in ventricular mechanical work caused by an increase in end-diastolic volume (or venous filling pressure) is called Frank-

The Starling mechanism (Figure A). measured curve

As the Frank-Starling mechanism, he proposed the Starling hypothesis that the fluid exchange between blood and tissue is due to the filtration of capillary walls and the difference in colloid osmotic pressure. This hypothesis was later largely confirmed by the work of E. Landis.

The work performed by the ventricles at different venous filling pressures is called the Starling curve (Panel B).

Stellar curve (measured in mammalian hearts)

The Frank-Starling mechanism in the frog heart

/ filling pressure

The end-systolic volume is determined by two parameters: The pressure developed during ventricular systole. Pressure in the outflow channel of the heart (aortic or pulmonary artery pressure). Increased venous filling pressures lead to increased end-diastolic volumes and to increased stroke volume in isolated mammalian hearts (Focus 12-1). End-systolic volume also increased, but not as much as end-diastolic volume. Cardiac muscle therefore behaves like skeletal muscle in that stretching a flaccid muscle over a certain length results in increased tension during contraction. Elevated arterial pressure also increases end-diastolic and post-systolic volumes with little change in stroke volume. In this case, increased mechanical work is required to maintain stroke volume

Increased catecholamine stimulation. Left atrial medium pressure - +

The increase in arterial pressure is the result of increased stretching of the myocardium during diastole. As noted above, epinephrine and norepinephrine released from sympathetic nerves or circulating in the blood increase ventricular contractility; thus, these catecholamines increase the rate and extent of ventricular emptying. Cholinergic (ie, vagal) activity has a much less pronounced effect on the rate and force of ventricular output during each beat than the adrenergic sympathetic effect. This difference is due to the much wider innervation of the ventricles by adrenergic nerves than by cholinergic nerves. The effects of sympathetic stimulation and/or elevated levels of circulating catecholamines represent a series of combined effects. Stimulation of pacemaker cells results in an increase in heart rate. Increases conduction velocity across the heart to produce near-synchronous motion.

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................................... The normal beating of the ventricles. Both the rate at which ventricular cells produce ATP and the rate at which chemical energy is converted to mechanical energy increase, resulting in increased ventricular work: the rate of ventricular emptying during systole increases, so stroke volume remains constant or increases over a shorter period of time. This increased contractility is mediated through the action of catecholamines on alpha- and beta-adrenergic receptors (see Chapter 8 for details). Therefore, when adrenergic nerve stimulation increases heart rate, the same stroke volume is ejected from the heart in a shorter period of time. Thus, while the emptying and filling times available to the heart decrease with increasing heart rate, stroke volume remains fairly constant over a wide range of heart rates. For example, in many mammals, exercise is associated with large increases in heart rate with little change in stroke volume; stroke volume decreases only at peak heart rates (Fig. 12-10). This occurs because, over a wide range of heart rates, increased sympathetic activity leads to faster ventricular emptying and increased venous pressure leads to faster filling as heart rate increases. However, there is a limit to how shortened diastole can be, depending on the maximum possible rate at which the ventricles can fill and empty and the nature of the coronary circulation. When the heart muscle contracts, the coronary capillaries are blocked, so blood flow is severely restricted during systole. Diastolic blood flow increases sharply, but shortening diastole tends to shorten the period of coronary blood flow. Catecholamines also cause dilation of coronary vessels and increase coronary blood flow. As we have seen before, increases in cardiac output during mammalian exercise are generally associated with large changes in heart rate and small changes in stroke volume (see Figure 12-10). After cardiac sympathetic denervation, exercise results in similar increases in cardiac output, peak O2 consumption I II

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Figure 12-10 In humans and many other mammals, increased cellular oxygen demand during exercise is met in part by increases in heart rate rather than stroke volume, resulting in higher cardiac output. When oxygen consumption is high, heart rate levels off, stroke volume increases and then decreases again. In addition, there is increased removal of oxygen from the blood in the capillaries during exercise, which is manifested as an increase in the arteriovenous (A-V)0 difference. [Adapted from Rushmore, 1965b.l.]

But in this case, stroke volume, not heart rate, varies greatly. Increased cardiac output may be caused by increased venous return. The sympathetic nerves are not involved in increasing cardiac output per se, but in increasing heart rate and maintaining stroke volume, thereby avoiding the large pressure fluctuations associated with large stroke volumes and keeping the heart at or near its optimal stroke volume amount for effective shrinkage. hold. Thus, the sympathetic nerve plays an important role in determining the relationship between heart rate and stroke volume, but other factors also play a role in mediating the exercise-induced increase in cardiac output. Pressure and flow change during a heartbeat. Systole causes fluctuations in cardiac pressure and volume, as shown by the curves in Figure 12-11A. The following sequence of events occurs during mammalian systole (Figure 12-11B):

1. During diastole, the closed aortic valve maintains a large pressure differential between the relaxed ventricles and the systemic aorta and pulmonary arteries. The atrioventricular valves open and blood flows directly from the venous system into the ventricles. 2. As the atrium contracts, the pressure in the atrium increases and blood is expelled from the atrium into the ventricle. 3. As the ventricles start to contract, the pressure inside the ventricles increases and exceeds the pressure in the atria. At this point, the atrioventricular valves close, preventing blood from flowing back into the atria, and ventricular contraction continues. At this stage, both the atrioventricular and aortic valves are closed, so the ventricles form closed chambers with no change in volume. That is, ventricular contraction is isometric. 4. Rapid increase in ventricular pressure, eventually exceeding the pressure in the aorta and pulmonary artery. The aortic valve then opens and blood is drained into the aorta, causing a reduction in the volume of the ventricles. 5. When the ventricle begins to relax, the intraventricular pressure is lower than the aortic pressure, the aortic valve closes, and the ventricles undergo isometric relaxation. Once ventricular pressure falls below atrial pressure, the atrioventricular valve opens and the ventricles begin filling again, and so on. In the mammalian heart, the volume of blood pushed into the ventricles by atrial contraction is about 30% of the volume of blood ejected by ventricular contraction into the aorta. Thus, the filling of the cardiac chambers is largely determined by the venous filling pressure, which pushes blood from the veins directly through the atria into the cardiac chambers. Due to atrial contraction, the nearly full ventricle is only filled with blood; however, if atrial contraction is affected, peak cardiac output may be affected. Myocardial contraction can be divided into two phases. The first is an isometric contraction, during which

................................... A Pressure and volume changes during a heartbeat. left side of heart

Sequence of events in a B heartbeat

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(1) Mid-diastole

(3) Isometric ventricular contraction

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Diastole Diastole Diastole Diastole Systole Systole Figure 12-11 During a cardiac cycle, successive contractions of the atria and ventricles and opening and closing of the valves result in characteristic changes in pressure and volume. (A) Pressure and volume changes in the ventricles and aorta (left) and pulmonary artery (right)

ing a single cardiac cycle. (B) Sequence of events in mammalian heart contraction. Black indicates contracted muscles; gray, relaxed muscles. See discussion text. [Part A adapted from Vander et al., 1975.1

Muscle tone and ventricular pressure increase rapidly. The second phase is essentially isotonic; there is little change in tension because blood is rapidly ejected from the ventricles into the arterial system when the aortic valve opens, with only a slight increase in ventricular pressure. Thus, the voltage is initially generated with little change in length; the muscle then shortens with small changes in tension. In other words, with each contraction, the heart muscle changes from isometric to isotonic contraction.

The ventricles expel the same amount of blood, but the pulmonary circulation (right ventricle) creates much less pressure. Therefore, the right ventricle does much less external work than the left ventricle. As mentioned in the previous section, when intraventricular pressure exceeds arterial pressure, blood is expelled from the ventricles. When arterial pressure rises, the heart has to do more external work to raise intraventricular pressure and keep stroke volume at its original level. Of course, this means that high blood pressure puts extra strain on the heart. Not all the energy used by the heart is reflected in changes in pressure and flow; some energy is expended overcoming friction in the heart muscle, and more energy is dissipated as heat. The external work done by the heart, expressed as a fraction of the total energy expended, is called systolic efficiency. The external work done can be determined from pressure and flow measurements and converted to milliliters consumed. Again, this can be expressed as a fraction of the total O1 uptake by the heart to measure contraction efficiency. In fact, only 10-15% of the total energy expended by the heart is expressed as mechanical work. Energy is expended increasing wall tension and increasing blood pressure in the heart. According to Laplace's law, the relationship between wall stress and pressure is

Work done by the heart It's a simple principle of physics that the work done externally is the product of mass and distance traveled. In this context, work can be calculated as pressure change times flow. Flow is directly related to the change in volume with each heartbeat. Pressure is measured in grams per square centimeter, volume is measured in cubic centimeters, and pressure times volume equals grams times cubic centimeters divided by square centimeters, which equals grams times centimeters—equal to mass times distance traveled, or work. Thus, the pressure of a single contraction of a ventricle multiplied by the volume map produces a pressure-volume loop whose area is proportional to the external work done by that ventricle. Figure 12-12 shows the pressure-volume loops of the left and right ventricles of a mammalian heart. the two of them

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Volume (mL) Figure 12-12 The area of the ventricular pressure-volume loop is proportional to the external work done by the ventricle in one cardiac cycle. Shown here are the circuits of the right and left ventricles of a mammalian heart. One cycle counterclockwise equals one heartbeat. Ventricular filling occurs at low pressure; the pressure rises sharply (the sharp rise on the right side of each cycle) only when the ventricles contract. As blood flows into the arterial system, ventricular volume decreases and ventricular pressure drops rapidly as the ventricles relax. Then start filling again. Note that while volume changes are similar in both ventricles, pressure changes are much greater in the left ventricle than in the right ventricle. Therefore, the left ventricle has larger circuits and thus does more external work than the right ventricle.

The size of the pericardium depends on the stiffness of the pericardium and the size and rate of change of heart volume. Membranes can be thin and flexible (compliant). In this case, the pressure changes in the pericardial cavity with each heartbeat are negligible. Or the pericardium is very stiff (non-compliant), so the pressure inside the pericardium fluctuates with each heartbeat. The compliant pericardium that encloses the mammalian heart consists of two layers, an outer fibrous layer and an inner serous layer. The serous layer is double layered, forming the inner layer of the pericardial cavity and the outer layer (epicardium) of the heart itself. In mammals, the serous layer secretes a fluid that acts as a lubricant and facilitates cardiac activity. Pericardium maladaptive in crustaceans and mussels. In these animals, ventricular contraction reduces pressure in the pericardial space and improves flow from the venous system to the atria (Figures 12-13). Thus, the tension created in the walls of the ventricles serves both to eject blood into the arterial system and to draw blood from the venous system into the atria. The pericardium of elasmobranchs (sharks) and lungfishes is also non-conformal, whereas that of teleosts is compliant. The elasmobranch heart consists of four chambers—sinuses, atria, ventricles, and cones—all contained within a rigid pericardium (Fig. 12-14). Atria, thereby increasing venous return to the heart. When the pericardial cavity opens, cardiac output decreases; therefore, venous return increases due to decreased pericardial pressure

Hollow structures depend on the radius of curvature of the walls. If the structure is a sphere, then

where P is the transwall pressure (the pressure difference across the wall of the sphere), y is the wall stress, and R is the radius of the sphere. According to this relationship, a large heart would have to generate twice as much wall stress as a heart half the size to generate similar pressures. Therefore, larger hearts require more energy to generate pressure, and we would expect these hearts to have a greater ratio of muscle mass to total heart volume. Of course, the heart is not a perfect sphere, but has complex macroscopic and microscopic morphology; however, Laplace's law generally applies. The energy required to eject a given volume of blood from the heart depends on the efficiency of the contraction, the pressure generated, and the size and shape of the heart.

Pericardium The heart lies within the pericardial cavity, surrounded by a membrane of connective tissue called the pericardium. print size in

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Figure 12-13 In the edentulous heart, the contraction of the ventricles not only expels blood, but also reduces the pressure in the pericardial cavity and improves atrial filling. This occurs due to pericardial noncompliance. These numbers are seawater pressure expressed in centimeters relative to ambient pressure. Large black arrows indicate contraction wall motion; small arrows indicate expansion wall motion. Red arrows indicate the direction of blood flow. AAV, anterior aortic valve; PAY, posterior aortic valve; AVV, atrioventricular valve. [Adapted from Brand, 1972.1

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Figure 12-14 Because the cartilaginous gill heart is contained within a noncompliant pericardium, ventricular contraction reduces pressure in the pericardial cavity and helps fill the atria. In some elasmobranchs, fluid loss through the pericardial peritoneal tube during exercise, feeding, and coughing results in increased heart size and stroke volume. Black arrows indicate the direction of wall motion during muscle contraction or relaxation. Red arrows indicate the direction of blood flow.

Important for increasing cardiac output. In some elasmobranchs, a pericardial peritoneal tube exists between the pericardium and the peritoneal cavity. In resting fish, little or no fluid flows through this tube, but during exercise, coughing, or eating, loss of fluid from the pericardial space through the tube results in increased heart size and stroke volume. This fluid is slowly replaced by plasma ultrafiltrate. For example, the thin, flexible pericardium of mammals, although protective, has little effect on cardiac output, whereas the stiffer pericardium of sharks with potentially fluctuating pericardial fluid volumes can have a significant effect on cardiac output. The Vertebrate Heart: Comparative Functional Morphology Heart structures vary among vertebrates, and comparative analysis of the vertebrate circulatory system provides insight into the relationship between heart structure and function. many cardiovascular differences

These characteristics distinguish air-breathing vertebrates from non-air-breathing vertebrates. Air-breathing vertebrates vary in the degree to which the whole body (body) and lungs (breathing circuit) are separated. The pulmonary circulation in birds and mammals is maintained at a much lower pressure than the systemic circulation. This is possible because they have two parallel rows of chambers. The left side of the heart pumps blood into the systemic circulation, and the right side pumps blood into the pulmonary circulation (see Figure 12-3). The advantage of hypertension is that there can be rapid transit times and sudden changes in flow. This is easily accomplished when blood flows through small diameter capillaries. However, when the pressure differential across the vessel wall (ie, transmural pressure) is high, fluid will filter across the vessel wall. capillary walls; therefore, extensive lymphatic drainage of the tissue is required. In mammalian lungs, relatively low inlet pressure maintains capillary flow, reduces the need for lymphatic drainage and avoids the formation of large extracellular fluid spaces that could increase the diffusion distance between blood and air and damage the lungs gas transmission capacity. As in mammals, a split heart has the advantage of maintaining blood flow to the body and lungs with different input pressures. The disadvantage of a completely split heart is that, to avoid shifting blood volume from the systemic to the pulmonary circulation and vice versa, the cardiac output must be equal on both sides of the heart, regardless of the demands of the two circuits. In contrast, lungfish, amphibians, reptiles, bird embryos, and fetal mammals either have an undivided ventricle or some other mechanism that allows blood to be diverted from one circuit to the other. These shunts usually cause blood to move from the right side of the heart (breathing, lungs) to the left side of the heart (body-wide) when air transport to the lungs is restricted. In this case, blood returning from the body is not pumped to the lungs, but is diverted from the right side of the heart to the left, and then bypasses the lungs and returns to the general circulation. In lungfish, amphibians, and reptiles, flow to the lungs is often reduced during prolonged dives due to gas transfer through the skin and/or depletion of the body's oxygen stores. Blood flow to the lungs is also reduced during the development of the mother (mammals) or egg (birds) before the lungs allow gas exchange to fully function. Although a single undivided ventricle allows for changes in the flow ratios of the pulmonary and systemic circulation, the same pressure must be generated on both sides of the heart. Water-breathing fish The heart of water-breathing fish, including elasmobranchs and some bony fishes (bony fishes), consists of four chambers connected in series. All chambers are collapsible except for the elastic ball of the bony fish. Unidirectional blood flow through the heart is maintained by the sinus node and valves at the atrioventricular junction and ventricle outlets.

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................................................... ................................... In elasmobranchs, the outlet from the ventricle to the cone is controlled by a Pair flaps protect the valve from two to seven pairs of flaps along the length of the cone, depending on the species (see Figures 12-14). The length of the cones varies between species. In general, more valves were found in species with longer cones. Just before the ventricles contract, all valves open except the one furthest from the eccentric chamber; that is, the cones are connected to the ventricles, but a closing valve at the exit of the cones maintains the pressure differential between the cones and the abdominal aorta . During atrial contraction, both the ventricles and cones are filled with blood. Ventricular contraction in elasmobranchs does not have an isovolumic period like in mammals, because blood moves from the ventricle into the cone at the onset of contraction. The pressure in the ventricles and cones increases, eventually exceeding the pressure in the abdominal aorta. The distal valve opens and blood drains into the aorta. During the conical contraction, which begins after the ventricles begin to contract, the proximal valve closes, preventing backflow of blood as the ventricles relax. The cones away from the heart and toward the aorta constrict relatively slowly; each set of valves closes sequentially to prevent backflow of blood. As shown in Figures 12-15, in a typical underwater breathing fish, blood pumped by the heart passes first through the gill (breathing) circuit and then into the dorsal aorta, which supplies the rest of the body (systemic circuit). Unlike mammals, the respiratory and systemic circuits of fish are not connected in parallel, but in series, and the pressure of the gill circuit is higher than that of the systemic circuit. The gills of fish are involved in ion regulation as well as gas transport and many other functions

you will take it out

afferent reproductive artery

The kidneys of mammals are located in the gills. The effect of hypertension in fish gills on ion and gas transport is unknown.

Air-breathing fish Air-breathing has evolved many times among vertebrates, often in response to hypoxic conditions, high water temperatures, or both. Typically, air-breathing fish remain in the water, but surface to inhale air pockets and replenish their oxygen supply. Since gill filaments usually collapse and stick together when exposed to air, they cannot be used for airborne gas transport. Thus, air-breathing fish typically use structures other than gills to breathe air, such as parts of the gut or mouth, the swim bladder, or even the general surface of the skin. Although the gills of air-breathing fish are not used to absorb oxygen from the air, they are used to excrete carbon dioxide and regulate ions and acid-base. However, in many air-breathing fish, the gills are smaller, presumably to reduce the loss of oxygen from the blood to the water. The gills of the air-breathing bony fish arapaima found in the Amazon are so small that even in normoxic water, only one-fifth of the oxygen is absorbed through the gills. Most of the fish's oxygen uptake is through the swim bladder, which is rich in blood vessels and has many septa to increase the surface area for exchange. In fact, the gills of the arapaima are too small for the animals' oxygen needs, and without access to the air, the fish would die; that is, the arapaima is an obligate air-breather. Air-breathing fish have evolved various blood shunts to alter blood distribution

arteries - arteries

abdominal aorta

primary cycle

handkerchief

ventricle

Figure 12-15 In a "typical" underwater breathing organism like the trout, the respiratory circuit through the gills and the systemic circuit are connected in series. In a complete four-chambered heart, the pacemaker is located in the sinuses. The ventricles eject blood into the compliant bulb and the short abdominal aorta. Blood flows through the gills and into the stiff, slender back

aorta. Most teleosts have a low hematocrit secondary system that provides nutrients to the skin and gut, but not much oxygen. Black arrows indicate deoxygenated blood flow; red arrows, oxygenated blood flow. BV, body volume.

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Gills and air-breathing organs. In the tropical freshwater fish Hoplerythrinus, the celiac artery originates from the posterior gill arch, supplies the swim bladder, and is connected to the dorsal aorta by a narrow duct. When an animal breathes water, most of the cardiac output is directed to the first two gill arches and to the body. After inhalation of air, the proportion of blood flow to the posterior gill arch and thus to the swim bladder is increased, increasing the opportunity to absorb oxygen from the swim bladder.

There are far more species of breathing fish in the tropics than in the temperate zones. Why?

The air-breathing fish Channa argus uses several mechanisms to achieve some degree of separation of oxygenated and deoxygenated blood in the circulatory system. The most important mechanism is the division of the abdominal aorta into two vessels, the posterior abdominal aorta and the anterior abdominal aorta. The anterior vessels supply the anterior two branchial arches and the respiratory organs, while the posterior vessels supply the posterior arch (Fig. 12-16). The posterior arch is reduced and the fourth arch is modified so that the afferent and efferent branchial arteries communicate directly. Oxygenated blood preferentially flows to the rear arches, and deoxygenated blood preferentially flows to the front two arches. This is achieved without splitting the heart. However, the ventricles are spongy (trabecular), which may help prevent previous blood from mixing in the ventricles

Sponge heart recommended for amphibians. In addition, the absence of sinus valves and the arrangement of veins in Channa's heart may play an important role in preventing the mixing of oxygenated and deoxygenated blood, as this flow returns to the heart through the normal blood vessels. Finally, muscular ridges on the bulb wall prevent oxygenated and deoxygenated currents from mixing as they exit the heart. The situation is also similar to that of amphibians. The lungfish (dipnoi) has a more complete heart division, which has gills, lungs, and pulmonary circulation. The atrium and ventricle of the African lungfish Protol)terus have partial septa and a sphere with five helical folds (Figs. 12-17). This arrangement keeps blood at the junction of the oxygenated and oxygen-poor hearts. The prebranchial arch lacks lamellae, allowing oxygenated blood to flow directly into the tissue from the left side of the heart. Within the lamellae of the posterior branchial arch there is a basilar-arterial junction that allows blood to bypass the lamina when only the lungs are functioning (eg, during drowsiness, summer numbness). Blood from the posterior gill arch goes to the lungs or through the duct into the dorsal aorta. Ducts are richly innervated and undoubtedly participate in the control of blood flow between the pulmonary artery and the systemic circulation. The initial segment of the pulmonary artery is muscular and is called the pulmonary vasomotor segment. This vasomotor segment and the catheter likely work in tandem: when one narrows, the other widens. The ductus of the lungfish is similar to the ductus arteriosus of fetal mammals, acting as a pulmonary bypass when the lungs are incompetent.

anterior cardinal vein

you will take it out

Germ artery

Gill afferent artery ulcers Figure 12-16 Although the heart in the air-breathing layer (Channa argus) is intact, the flow of oxygenated and deoxygenated blood is partially separated. Deoxygenated blood (black arrows) flows preferentially through the first two branchial arches and the respiratory organs, while oxygenated blood (red arrows) flows into the branchial arches through the posterior branchial arches

ventricle

dorsal aorta. The fourth branchial arch is modified such that the afferent and efferent branchial arteries connect. Compare Figures 12-15 for a more typical aquatic teleost cycle. . [Adapted from lshiratzu and Itazawa, 1993.1

Tissue A

dorsal aorta

~you forgot'

~enkicle~ give it a try

Figure 12-17 The circulatory system of the African lungfish Protopterus is characterized by an almost complete separation of oxygenated blood (red arrows) and deoxygenated blood (black arrows). This separation is achieved by the septum that separates the atria from the ventricles and by long helical folds in the bulb. This fish has lungs and has

EJ oxygenated blood

Pulmonary circulation tincture. There are no lamellae in the prebranchial arch, and blood can flow directly into the systemic circulation through the dorsal aorta. Ducts and pulmonary vasomotor joints work together to direct blood to the dorsal aorta or lungs, depending on whether the fish breathes water or air. [Adapted from Randall, 1994.1

Child-

inokutan

Amphibians Amphibians have two completely separate atria, but only one ventricle. In the frog's heart, oxygen-rich and oxygen-poor blood are separated, even though the ventricles are intact. Oxygenated blood from the lungs and skin flows preferentially to the body through the systemic arch, while deoxygenated blood from the body flows to the pulmonary cutaneous arch. The helical folds in the conus of the heart arteries facilitate the separation of oxygenated and deoxygenated blood (Fig. 12-18). During systole, deoxygenated blood first leaves the ventricles and enters the pulmonary circulation. The pressure in the pulmonary cutaneous arch is elevated, similar to the pressure in the systemic arch. Blood then begins to flow into the two arches, with helical folds that partially separate systemic and pulmonary percutaneous blood flow in the conus arteriosus. The amount of blood flowing into the lungs or body is inversely proportional to the flow resistance of the two circuits. After inhalation, the pulmonary blood flow resistance is low and the blood flow is high; the resistance between breaths gradually increases, accompanied by a decrease in blood flow. These fluctuations in pulmonary blood flow are possible due to the partial division of the amphibian heart. Although deoxygenated blood goes directly to the lung cutaneous arch, but-

Figure 12-18 Although the frog's heart has only one ventricle, deoxygenated blood enters the lungs through the pulmonary artery arch, while oxygenated blood enters the tissues through the systemic arch. This ventral view of the inner workings of a frog's heart shows the location of the spiral folds that help separate the two blood streams. [Adapted from Goodrich, 1958.1

Can regulate the ratio of pulmonary blood flow to systemic blood flow. That is, when the animal is not breathing, blood flow to the lungs may be reduced, allowing most of the blood pumped by the ventricles to be directed to the body. A more even distribution of flow to the lungs and body is maintained as the animal breathes. This distribution is only possible if the ventricles are not completely divided into right and left ventricles (as is the case in mammals).

Noncrocodilian reptiles Most noncrocodile reptiles, including turtles, snakes, and some lizards, have partially separated ventricles and left and right body arches. In these animals, the ventricles are partially separated by an incomplete muscle septum called the horizontal septum, muscular crest, or muscular crest. This horizontal septum separates the pulmonary cavity from the venous and arterial cavities; the latter two are partially separated by a vertical septum (Fig. 12-19). The right atrium contracts slightly in front of the left atrium, expelling deoxygenated blood through the free edge of the horizontal diaphragm into the lung cavity. This blood is ejected into the pulmonary artery by ventricular contraction. Oxygenated blood from the left atrium fills the venous and arterial cavities; from here blood flows into the main arteries. Measurements in sea turtles support the hypothesis that oxygen-rich blood enters the systemic circulation from the left atrium, while deoxygenated blood enters the pulmonary artery from the right atrium. Pulmonary diastolic pressure is usually lower than systemic diastolic pressure; as a result, when the ventricles contract, the pulmonary valve opens first. Therefore, in each cardiac cycle, blood flow occurs earlier in the pulmonary arteries than in the body arch. In sea turtles there may be some arterial recirculation in the pulmonary circulation; ie there is a left to right shunt in the heart. The ventricles remain functionally intact throughout the cardiac cycle, and the relative flow to the pulmonary and systemic circuits is determined by the flow resistance of the various parts of the circulatory system. When the turtle breathes, there is resistance to flow through the right atrium

yes, one or one

right pulmonary artery

brachiocephalic artery

artery

'~ vertical

horizontal diaphragm

yes

diaphragm

vena cava

Pulmonary blood flow is low and blood flow is high. When not breathing, as in diving, pulmonary vascular resistance increases but systemic vascular resistance decreases, resulting in a right-to-left shunt and decreased pulmonary blood flow. As in many other animals, cardiac output is reduced during diving, accompanied by a marked slowing of the heart rate (bradycardia). Similarities in pulmonary and systemic outflow tract pressures in turtles, snakes, and some lizards suggest that their hearts have a single ventricle that is partially divided into multiple sub-chambers even during systole (Fig. 12-20A). However, in Varanus and related monitor lizards, the pressure of pulmonary effluent is much lower than that of systemic effluent during systole (Fig. 12-20B). For example, the systolic pressure in the lung cavity of a python may be only one-third of the pressure in the venous cavity. Lizard lizards achieve this pressure differential during systole through pressure tight contact between the muscular crest (horizontal diaphragm) and the heart wall (Figures 12-21). Crocodile Reptiles Unlike other reptiles, crocodile reptiles have a heart with completely separated ventricles. The left body arch arises from the right ventricle; the right body arch, from the left ventricle. Adjacent to the ventricles, the systemic arches are connected by scare holes (Fig. 12-22A). The full body bow is also connected by a short anastomosis at the tail of the heart. When the crocodile reptile breathes normally, there is little resistance to blood flow through the lungs, and the pressure developed by the right ventricle is lower than that produced by the left ventricle during all phases of the cardiac cycle. In this case, during systole, blood is pumped from the left ventricle into the right arch, and the open aortic valve closes the panic hole (Fig. 12-22B). During sysiole, there is a slight backflow of blood from the left aorta to the right aorta through the anastomosis. Because of this connection, the pressure in the body's left arch remains higher than the pressure in the right vein. Figure 12-19 In the non-crocodile (turtle) heart, the ventricle is partially divided by the horizontal septum into the venous cavity and the ventral pulmonary cavity. The common pulmonary artery originates from the pulmonary cavity, while all systemic arteries originate from the venous cavity. In this ventral view of a sea turtle heart, arrows schematically indicate the movement of oxygenated (red) and deoxygenated (black) blood, but do not represent the flow of individual blood streams through the heart. [Adapted from Shelton and Burggren, 1976.1

Cavum venosum Gemeinsame Lungenarterie Cavum venosum und Cavum pulmonale

time

I I I I I Time (100 ms)

Figure 12-20 Systemic and pulmonary outflow pressures during systole are nearly identical in tortoises, whereas there is a marked difference in lizard lizards. Display the blood pressure value measured at the same time

Specified locations during a heartbeat of (A) the turtle Chrysemys scripta and (B) the monitor lizard Varanus exanthematicus. [Part A of Shelton and Burggren, 1976; Part B, Burggren and Johansen, 1982.1

tricycle; thus, the valve at the base of the left arch remains closed throughout the cardiac cycle (Fig. 12-22C). All blood ejected from the right ventricle enters the pulmonary artery and travels to the lungs. Therefore, crocodile reptiles are functionally identical to mammals in that systemic and pulmonary blood flow are completely separated. However, crocodile reptiles have the additional ability to transport blood from the pulmonary circulation to the systemic circulation. S shunt is achieved by actively closing the valve

systole. In some experimental situations, right ventricular peak pressure equals left ventricular pressure and exceeds left systemic pressure. This opens the valve at the bottom of the left ventricle and expels blood from the right ventricle into the systemic circulation late in systole (Fig. 12-22D, E). In this case, partially deoxygenated blood returning from the body to the heart via the right atrium is recirculated in the sysS shunt circuit. The exact duration of P's normal existence in animals is unknown. The role of the hole panizzae also remains a mystery. it is only open during opening hours

Diastole

pulmonary sinus

shrink

Second

leave and

Sinus

To the right

courtyard

left atrium atrioventricular

Figure 12-21 In the veranidae lizard, there is a pressure-tight separation between the lung cavity and the venous cavity during systole. (A) During diastole, the muscular crest only partially separates the venous and pulmonary lumens. Oxygen-rich blood (red arrow) left in the venous cavity after the previous systole is flushed into the lung cavity by deoxygenated blood (black arrow). The lumen of the artery is filled with oxygen-rich blood. The arterial and venous lumens are separated by at least one atrioventricular valve. (B) During contraction, the rear of the muscle is tightly compressed

abuts against the outer core wall and forms a pressure-tight barrier. Deoxygenated blood left in the venous lumen during previous diastole mixes with oxygenated blood from the arterial lumen and rushes into the aortic arch. Deoxygenated blood mixed with oxygenated blood shoots from the lung cavity into the lung arch. If there is no connection between the vena cava and the lung cavity, different pressures will develop in the outflow tract. [Adapted from Heisler et al., 1983.1

486

integrated physiological system

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . …… . A

Diastole

Olamen von Panisa

B Spätsystole-Nr

shunt

Man

c without P+S shunt

Second

Heart

Figure 12-22 In some cases, PS shunts operate during late systole in alligators. These schematics, along with pressure and flow curves, illustrate what happens during the cardiac cycle with and without shunts. See discussion text [adapted from Jones, 1995]

systolic shunt

P+ shunt

Heart

I late diastolic systolic time (s)

Diastole, allowing flow between the aortic arches as the heart relaxes.

Mammals and Birds The hearts of mammals and birds are made up of four chambers, actually two hearts beating in one. The heart is made up of two separate tubes that have joined over time.

time

It continues to develop and forms the multichambered heart of postpartum animals. The right side pumps blood to the lungs, and the left side pumps blood throughout the body. Blood returning from the lungs enters the left atrium, enters the left ventricle, and is excreted into the systemic circulation. Blood from the body collects in the right atrium, enters the right ventricle, and is pumped to the lungs (see Figure 12-3).

The valves prevent backflow of blood from the aorta to the ventricles, atria, and veins. These valves are passive, opening and closing by pressure differences between the ventricles. The atrioventricular valves (mitral and tricuspid) are connected to the ventricular wall by fibrous cords (see Figure 12-4). These cords prevent the valve from everting into the atria when the ventricles contract and the pressure inside the ventricles is much higher than that in the atria. The walls of the ventricles, especially the left ventricle, are thick and muscular. The inner surface of the ventricular muscle, or myocardium, is lined with an endothelial membrane, the endocardium. The ventricular muscle is covered by the epicardium. Mammalian Fetus At birth, mammals switch from placental to pulmonary circulation, a process that requires several key cardiovascular readjustments. The lungs of mammalian fetuses are collapsed and exhibit high resistance to blood flow. In the fetus, the pulmonary artery is connected to the body arch by a short, large-diameter ductus arteriosus (Fig. 12-23). Cardiac function in mammalian fetuses has three important features:

Figure 12-23 In the mammalian fetal heart, most blood expelled from the right ventricle returns to the systemic circulation through the arterial mitral valve. Oxygenated blood returning from the placenta flows from the right atrium to the left atrium through the foramen ovale and then pumps into the aorta. After birth, the ductus arteriosus is usually closed, separating the systemic and pulmonary circulation. These numbers refer to the percentage of total cardiac output flowing into and out of the right and left ventricles in different parts of the body.

Most of the blood drained from the right ventricle returns to the systemic circulation through the ductus arteriosus. Blood flow through the pulmonary circulation is severely restricted. A clear right-to-left splitter (P+S) is operating. That is, blood flows from the pulmonary circulation to the systemic circulation. At birth, the lungs expand, which reduces the resistance to flow in the pulmonary circulation. Blood expelled from the right ventricle enters the pulmonary vessels, causing increased venous return to the left side of the heart. At the same time, the placental circulation disappears, and the flow resistance in the systemic circulation increases significantly. Systemic pressure is higher than pulmonary pressure; if the ductus arteriosus is not closed after birth, this pressure differential results in a left-to-right shunt (SP), flow of blood from the systemic to the pulmonary circulation. Generally, however, the ductus arteriosus becomes occluded and blood flow through the ductus arteriosus stops. If the ductus arteriosus remains patent after birth, blood flow to the lungs exceeds systemic flow because part of the left ventricular output passes through the ductus arteriosus to the pulmonary artery and lungs. In these cases, systemic flow is usually normal, but pulmonary flow may be twice systemic flow, and left ventricular cardiac output may be twice that of the right ventricle. The result is marked hypertrophy of the left ventricle. The work done by the left ventricle during exercise is also much greater than usual, with limited ability to improve exercise performance. Therefore, if the ductus arteriosus remains patent after birth, maximum bodily functions will be severely limited. In addition, the condition increases blood pressure in the lungs, leading to increased fluid loss from the walls of the capillaries in the lungs and possible lung congestion. These problems only become harmful when the left ventricle becomes enlarged. A patent ductus arteriosus can be corrected easily and simply with surgery. Fetal blood is oxygenated in the placenta and mixes with blood returning from the lower body through the inferior vena cava, a vein that drains into the right atrium (see Figure 12-23). A hole in the interatrial septum, the foramen ovale, is covered by a valve; oxygenated blood returning through the inferior vena cava is directed through the foramen ovale into the left atrium. Oxygen-rich blood is then pumped from the left atrium into the left ventricle and drains into the aorta, from where it travels to the head and upper extremities. Deoxygenated blood returning to the right atrium via the superior vena cava is directed primarily to the right ventricle, from which it enters the systemic circulation through the ductus arteriosus. At birth, left atrial pressure exceeds right atrial pressure; this closes the foramen ovale, but its position is later indicated by permanent deepening.

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The vascular network of the avian embryo forming the chorionic allantoic membrane is located just below the avian eggshell. Oxygen diffused through the eggshell is absorbed by the blood flowing through the chorion. Oxygenated blood leaving the chorion and deoxygenated blood from the head and body enters the right atrium of the bird's heart. Oxygenated blood from the chorioallantoic circulation flows from the right atrium to the left through several large and numerous small holes in the interatrial septum. Oxygenated blood is then pumped into the left ventricle and drains into the aorta, from where it travels to the head and body. After the chicks hatch, the holes in the interatrial septum close, completely separating the pulmonary and systemic circulation.

capillaries

5000 rpm

Arteriole I

I Venolen I

Hemodynamics As we've established, heart contraction produces blood flow through the blood vessels -- the arteries, capillaries and veins that make up the circulatory system. Before examining the properties of these vessels in detail, it is worth discussing the general pattern of blood flow in these vessels and the relationship between pressure and flow in the circulatory system. The laws describing the relationship between pressure and flow apply to both open-loop and closed-loop systems. In vertebrates and other closed-loop animals, blood flows in continuous circuits. Since fluid is incompressible, blood pumped by the heart must create an equal volumetric flow in every other part of the circuit. This means that the number of liters flowing through arteries, capillaries and veins is always the same per minute. Furthermore, unless there is a change in total blood volume, a decrease in volume in one part of the circuit must result in an increase in volume in another part. The flow velocity at any point does not depend on the distance of the heart, but on the total cross-sectional area of the part of the circuit, namely H. Sum of all capillary or arterial cross-sections from that point in the circulation. Just as water velocity increases where a river narrows, in a circulatory system blood velocity is highest where the total cross-sectional area is smallest (and lowest where the cross-sectional area is largest). Arteries have the smallest total cross-sectional area while capillaries have the largest cross-sectional area. In mammals, therefore, the highest velocities occur in the aorta and pulmonary arteries; then, the velocities decrease considerably as the blood flows through the capillaries, but increase again when the blood flows through the veins (Figs. 12-24 ). Slow blood flow in capillaries has an important function because of the time it spends in capillaries. There is an exchange of substances between blood and tissue. laminar and turbulent flow

Blood flow in the many smaller blood vessels in the circulatory system tightens. This continuous laminar flow is characterized by a parabolic velocity profile throughout the vessel (Fig. 12-25A). Flow is zero, maximum at the wall

Figure 12-24 The velocity of blood flow is inversely proportional to the cross-sectional area of the circuit at a given point. Arteries and veins have the highest blood flow velocity, while capillaries have the lowest blood flow velocity; the cross-sectional area is the opposite. [Adapted from Feigl, 1974.1

continuous laminar flow

B pulsating laminar flow

Figure 12-25 Flow through smaller vessels approximates continuous laminar flow, but pulsatile laminar flow is observed in large elastic arteries. From these velocity curves it can be seen that the flow velocity is higher towards the center of the vessel. (A) The presence of red blood cells flattens the blood profile compared to plasma. (6) Pulsatile flow is characterized by a flat profile and flow reversal with each heartbeat.

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489

................................... Midpoint along the ship's axis. A thin layer of blood close to the vessel wall does not move, but the next layer of liquid slides over that layer, and so on, with each subsequent layer moving at an increasing speed, with the maximum velocity at the vessel's center. The pressure differential provides the force needed to push adjacent layers past each other, and viscosity is a measure of resistance to sliding between adjacent liquid layers. An increase in viscosity requires a greater differential pressure to maintain the same flow rate, as described later. The pulsatile laminar flow characteristic of large arteries has a more complex velocity profile than the continuous laminar flow characteristic of small vessels. In the large arteries, blood speeds up and then slows down with each heartbeat; moreover, because the walls of blood vessels are elastic, they stretch and then relax with the pressure fluctuations of each heartbeat. Near the heart, the direction of flow reverses every time the aortic valve closes. The end result is that velocity across large arteries has a flatter profile than velocity across more peripheral vessels, and the direction of flow oscillates (Fig. 12-25B). In turbulent flow, the liquid moves in a direction that is not aligned with the flow axis, increasing the energy required to move the liquid through the vessel. Laminar flow is silent; turbulent flow is noisy. The turbulent flow in the blood creates vibrations that create circulatory noise. Detection of these sounds with a stethoscope can locate turbulent points. Measuring blood pressure with a sphygmomanometer depends on hearing the sound of blood flowing through a pressure cuff as systolic blood pressure rises. When the velocity of blood flow exceeds a certain threshold, noise is heard in the blood vessels and as the heart valves open and close. While turbulence in peripheral circulation is rare, it does occur in some situations. The Reynolds number (Re) is an empirically derived value that indicates whether the flow is laminar or turbulent under certain conditions. A high Reynolds number indicates that the flow is turbulent, while a low Reynolds number indicates that the flow is laminar. Re is proportional to flow rate Q (in ml/s) and blood density p, and inversely proportional to vessel diameter r (in centimeters) and blood viscosity q:

The ratio of viscosity to density (qlp) is kinematic viscosity. The greater the kinematic viscosity, the less likely turbulence will occur. Relative viscosity, as well as kinematic viscosity, increases with hematocrit (the volume of red blood cells per unit volume of blood), so the presence of red blood cells reduces the occurrence of turbulence in the blood stream. In general, turbulent flow in undivided smooth-walled vessels is sufficiently high except during periods of very high blood flow associated with

Strenuous exercise. The highest flow velocities in the mammalian circulatory system occur in the proximal portions of the aortic and pulmonary arteries, and distal to the aortic and pulmonary valves, and turbulence can occur during peak ventricular ejection or during regurgitation when these valves close. Typically, in the portion of circulation where the vessel wall is smooth and the vessel is not divided, the flow is turbulent only when Re is greater than about 1000, a value that is rarely observed. Small back vortices can form at arterial branches, like back vortices in rivers, breaking off from the main flow regime and carried downstream as small, discrete regions of turbulence. These vortices form in the circulation at Re as low as 200. The relationship between pressure and flow

Flow occurs between two places with different potential energies, which can be measured as pressure differences. Therefore, a pressure difference between two points in a flow path causes a pressure gradient and thus the direction of flow - from high pressure to low pressure. (The exception is fluids that are at rest under gravity, where pressure increases steadily with depth but no flow occurs.) When the heart contracts, the potential energy (pressure) in the heart chambers increases. The pressure generated by the heart contraction is released by the blood flow as energy is expended to overcome the resistance to flow through the vessels. Therefore, as blood flows from the arterial to the venous side of the circuit, blood pressure drops (Figure 12-26). Capillaries and arterioles! ! venule

Figure 12-26 The pressure (potential energy) generated by each contraction of the heart muscle is reduced by overcoming the resistance of the blood vessels to flow. This area of the circulatory system experiences the greatest pressure drop because resistance is highest in the arteries [adapted from Frelgl, 1974.1

The role of kinetic energy Blood movement consumes kinetic energy. After the movement, the flowing blood has inertia; that is to say, the fluid in motion has kinetic energy. In a static liquid, potential energy is measured as pressure; in a moving liquid, potential energy is measured as pressure and kinetic energy. However, as we shall see, the contribution of kinetic energy to blood velocity is usually negligible. The kinetic energy per milliliter of liquid is given by t(pv2), where p is the density of the liquid and u is the flow rate. If velocity is measured in centimeters per second and density is measured in grams per millimeter, kinetic energy is measured in dynes per square centimeter, just like pressure. The maximum blood flow velocity in mammals occurs at the base of the aorta, the peak ventricular ejection is about 50cmss-', and the blood density is about 1.055g-ml-l. Therefore, the kinetic energy of the blood in the aorta during peak ejection is calculated as f x 1.055 x 502 or 1 mm Hg. This is small compared to the peak systolic transmural pressure of about 120 mmHg. Blood flow in the ventricles is slow, but accelerates as blood is drained into the aorta. That is, blood gains kinetic energy as it leaves the ventricles. As blood is expelled from the heart, pressure is converted into kinetic energy, and this conversion is responsible for most of the small pressure drop that occurs between the ventricles and the aorta. Kinetic energy is highest in the aorta. In a capillary, the velocity is about 1 nanometer per second, so kinetic energy is negligible.

Poiseuille's law Poiseuille's law describes the relationship between pressure and continuous laminar fluid flow in a rigid pipe, which states that the flow rate Q of the fluid is proportional to the pressure difference PI - P along the length of the pipe and is proportional to the fourth power of the pipe radius r Inversely proportional to pipe length L and liquid viscosity77:

Since Q is proportional to r4, small changes in r can have a profound effect on Q. For example, doubling the diameter of the vessel results in a 16-fold increase in flow if the differential pressure across the vessel (PI - P2) remains constant. Although Poiseuille's equation is valid for steady-state flow in straight rigid pipes, it has been used for Analyzes the relationship between pressure and flow in arterioles (arterioles), capillaries, and veins, even though these are not "rigid", some limitations will be explained later. are "tubes. Blood pressure and blood flow are pulsatile, While blood is a complex fluid composed of plasma and cells. Since the vessel wall is not rigid, the fluctuations in pressure and blood flow are not synchronized; therefore, the relative

The difference between the two can no longer be accurately described by Poiseuille's law. The magnitude by which the relationship between pressure and flow deviates from that predicted by Poiseuille's law is given by the value of the dimensionless constant a:

Where p and 77 are the density and viscosity of the liquid, respectively; f is the vibration frequency; n is the order of the harmonic component, and r is the radius of the container. When a is below 0.5, the relationship between pressure and flow can be described by Poiseuille's equation. Since the value of a in the peripheral arteriole and vein is about 0.5, this equation can be used to analyze the relationship between pressure and flow in this part of the circuit. In contrast, mammalian and avian arterial systems have Cu values ranging from 1.3 to 16.7, depending on the species and the physiological state of the animal. Therefore, Poiseuille's law does not apply to this part of the circulation. Microcirculation in vivo has been poorly studied due to the difficulty of measuring blood flow and pressure in capillaries. In tissues where the relationship between pressure and flow in the microcirculation was measured, it was found to be non-linear, suggesting that the Poiseuille equation cannot accurately describe the microcirculation. There are two reasons: capillary branches have tributaries that can be opened and closed, and are so small that red blood cells deform when they squeeze through capillaries. Flow resistance Since it is often difficult or impossible to measure the radii of all vessels in a vascular bed, we express 8Lrl17rr4 as the inverse term in Poiseuille's law (Equation 12-3), since the flow resistance R is equal to the pressure difference (PI - P2) Divide by the flow rate Q at the vascular bed:

Resistance to flow in the peripheral circulation is sometimes expressed in peripheral resistance units (PRU), which correspond to the resistance of the vascular bed when a pressure differential of 1 mm Hg results in a flow of 1 m1.s-'. Blood flow through a vessel increases as the pressure difference across the vessel increases and the resistance to flow decreases, which is inversely proportional to the fourth power of the vessel radius. As the pressure in the elastic container increases, the radius increases; as a result, the flow rate also increases. Consider a blood vessel with a constant differential pressure along its length, but operating at two pressure levels:

................................... Example 1: Inlet pressure 100 mm Hg and outlet pressure 90 mm Mercury; A = 10 mm Hg Example 2: Inlet pressure 20 mm Hg and outlet pressure 10 mm Hg; A = 10 mm Hg If the vessel is malleable, the flow rate in this vessel is at the higher pressure (Example 1) will be much larger, simply because the radius increases and the resistance to flow decreases. Blood Viscosity According to Poiseuille's law, blood flow is inversely proportional to its viscosity. Plasma has a viscosity relative to water of about 1.8; the addition of red blood cells increases the relative viscosity so that mammalian and bird blood has a relative viscosity between 3 and 4 at 37°C. Thus, blood behaves as if it is three to four times thicker than water, largely due to the presence of red blood cells. This property implies that a greater pressure gradient is required to maintain blood flow through the vascular bed than to perfuse the vascular bed with plasma alone. However, blood flowing through small vessels behaved as if its relative viscosity was greatly reduced. In fact, in vessels with a diameter smaller than 0.3 mm, the relative viscosity of blood decreases with increasing diameter and approaches that of plasma. This phenomenon, called the Fahraeus-Lindqvist effect, will be explained later. As we saw earlier, the velocity profile on a continuous laminar liquid flow vessel is parabolic, as seen in plasmas (see Figure 12-25A). The maximum flow rate is twice the average flow rate and can be obtained by dividing the flow rate by the cross-sectional area of the pipe. The velocity change is greatest near the vessel walls and decreases towards the vessel center. In flowing blood, red blood cells tend to cluster in the center of the vessel, where velocity is greatest but velocity variation between adjacent layers is minimal. This accumulation makes the walls relatively acellular, so the fluid flowing from this area into the small lateral vessels contains a small fraction of red blood cells and is almost entirely plasma. Such a process is called plasma skimming. The accumulation of red blood cells in the center of the blood flow results in blood viscosity being highest in the center and decreasing towards the wall. This difference in viscosity between the center of blood flow and the wall alters the velocity profile of blood compared to plasma. The effect of this difference in viscosity is a slight increase in blood flow in the wall and a slight decrease in blood flow in the center; that is, a somewhat flattening of the parabolic shape of the velocity curve (see Figure 12-25A). Small blood vessels have a smaller hematocrit (percentage of red blood cell volume in the blood) than larger blood vessels. In small vessels, the plasma boundary layer occupies a larger portion of the vessel lumen at a given flow rate than in large vessels. This axial flow of red blood cells in small vessels results in the largest velocity changes in the plasma layer

close to the wall and explains why the apparent viscosity of blood flowing in these small vessels is close to that of plasma. Therefore, the Fahraeus-Lindqvist effect can be explained by a decrease in hematocrit in small vessels. A reduction in the apparent viscosity of blood in arterioles reduces the energy required to propel blood through the microcirculation. For very small blood vessels - those approximately 5 to 7 µm in diameter - a further reduction in diameter leads to a reversal of the Fahraeus-Lindqvist effect, an increase in the apparent viscosity of the blood. In such vessels, red blood cells completely fill the lumen and deform as they pass through. Because the red blood cell membrane is not firmly anchored to the underlying structures, it is able to move on its own cellular contents, acting like chain mail as it travels along the walls of blood vessels. Deformation of RBCs in small blood vessels results in a complex flow of RBC membranes and surrounding fluid as cells squeeze through narrow lumens. When the flow is laminar but pulsating, as in an artery, the velocity profile is flatter than in continuous laminar flow (see Figure 12-25B). Therefore, the velocity of blood flow is constant over most of the vessels, but drops sharply near the vessel walls. In turbulent flow, blood moves in different directions relative to the flow axis, so red blood cells rarely collect in the center of the vessel. As a result, there is little change in blood viscosity and flow rate throughout the vessels.

these fish? What modifications might have evolved to compensate for these low temperatures?

Compliance of the Circulatory System Another consideration when analyzing the relationship between pressure and flow in the circulatory system is that blood vessels contain elastic fibers that allow them to dilate. The container is not actually a straight rigid tube for which Poiseuille's law applies. Conversely, as the pressure in the container increases, the walls are stretched and the volume of the container increases. The ratio of volume change to pressure change is called the compliance of the system. A system's compliance depends on its size and the resilience of its walls. The greater the elasticity of the initial volume and walls, the greater the flexibility of the system. The venous system is very flexible; that is, small changes in pressure can result in large changes in volume. For this reason, the venous system can act as a volume reservoir, since large changes in volume have little effect on venous pressure (and thus cardiac filling during diastole or capillary flow). The arterial system is not at all as compliant as the venous system, which acts as

A pressure reservoir that maintains capillary blood flow. Nonetheless, the arterial system near the heart is partially elastic, both dampening pressure fluctuations from systole and maintaining blood flow in distal arteries during diastole. In summary, there are various factors that affect the relationship between pressure and flow in a circuit. The flow rate depends on the total cross-sectional area of flow; it is highest in arteries and veins and lowest in capillaries, since the sum of the cross-sectional areas of all capillaries is greater than that of arteries or veins (see Figures 12-24). Contraction of the heart creates pressure and flow. The highest pressures in the circulatory system are found mainly in the ventricles and blood vessels that emanate from the heart. The pressure is reduced due to energy loss and flow resistance in the vessel being overcome. As blood flow velocity changes, the change in kinetic energy is reflected in only very small changes in blood pressure. Although blood flow is high, there is little pressure drop in the arterial and venous system because the blood vessels are large and have low resistance to flow. The greatest pressure drop is seen in arterioles because this is where flow is high and vessels are small and resistance is high (see Figure 12-26). The blood flow pattern through this high-resistance pathway reduces the apparent viscosity of the blood (Fahraeus-Lindqvist effect), thereby reducing the resistance to flow; however, the strongest drop in blood pressure occurs in the arterioles. Capillaries are even smaller than arterioles, but there is much less flow in each capillary; therefore, the pressure drop in a capillary is much smaller than in an arteriole.

Peripheral circulation Blood pumped from the left ventricle of the mammalian heart transports oxygenated blood through the arterial system to capillary beds in tissues where oxygen is exchanged for carbon dioxide. The venous system returns deoxygenated blood to the right atrium (see Figure 12-3). Although all blood vessels share certain structural features, vessels in different parts of the peripheral circulation adapt to the functions they serve. Figure 12-27 shows the structure of arteries and veins of different sizes. A layer of endothelial cells called endothelial cells lines the lumen of all blood vessels. In larger blood vessels, the endothelium is surrounded by a layer of elastic collagen fibers, while the capillary walls consist of a single layer of endothelial cells. Circular and longitudinal smooth muscle fibers may be mixed with or surrounded by elastic and collagen fibers. The walls of larger blood vessels are composed of three layers: Adventitia: A restrictive fibrous outer layer. Tunica Media: middle layer, composed of circular and longitudinal muscles

Intima: The inner layer closest to the lumen, composed of endothelial cells and elastic fibers. Boundary between intima and media is ill-defined; tissue is fused together. The media of the arteries thickens due to stronger muscle tissue, and larger arteries closer to the heart are more elastic and have a wider intima. The thick walls of larger blood vessels require their own capillary circulation, the so-called vasa vasorum. In general, arteries have thicker walls and smoother musculature than veins of similar external diameter. Some veins lack muscle tissue. arterial system

The arterial system is a series of branching blood vessels with thick, elastic, and muscular walls—perfect for carrying blood from the heart to the tiny capillaries that carry blood into the tissues. Arteries have four main functions, as shown in Figure 12-28: 1. They serve as conduits for blood between the heart and capillaries. 2. They act as pressure reservoirs, pushing blood into small diameter arterioles. 3. They suppress pressure and pressure fluctuations 4. Control the distribution of blood to different capillary networks by selectively narrowing the terminal branches of the arterial tree

Precisely controlled arterial blood pressure depends on the volume of blood contained in the arterial system and the properties of its walls. If either value changes, the pressure will change. The volume of blood in the arteries depends on the filling rate caused by the contraction of the heart and the rate of emptying through the arterioles into the capillaries. If cardiac output increases, arterial blood pressure increases; when capillary flow increases, arterial blood pressure decreases. Typically, however, arterial blood pressure varies little because filling and emptying rates are evenly matched (ie, cardiac output and capillary flow are evenly matched). Blood flow through capillaries is directly proportional to the pressure difference between the arterial and venous systems. While venous pressure is low and invariant, arterial pressure primarily controls capillary blood flow velocity and is responsible for maintaining adequate tissue perfusion. Arterial pressure varies by species, usually between 50 and 150 mmHg. Pressure differences along large arteries are small (less than 1 mmHg), but pressure drops significantly along arterioles and arterioles because of increased resistance to flow as vessel diameter decreases. Due to the elasticity of the arterial walls, fluctuations in blood pressure and blood flow generated by heart contraction are suppressed in the arterial system. like blood

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Diaphragm Figure 12-27 In mammalian peripheral circulation, blood flows from the heart through progressively smaller arteries, then through the microcirculation, and finally through progressively larger veins back to the heart. A layer of endothelial cells, the endothelium, that fills the lumen of all blood vessels

In larger vessels, the endothelium is surrounded by a muscular layer (Tunlca Medla) and an outer fibrous layer (Tuncaadvent~t~a) [adapted from Mart ~n~ and T~mmons, 1995].

If it is drained into the arterial system, pressure increases and blood vessels dilate. When the heart relaxes, the elastic recoil of the vessel walls maintains blood flow to the periphery, resulting in a decrease in arterial volume (see Figure 12-28). If the arteries were just rigid tubes, the pressure and flow around them would stop and start the same at the outlet of the ventricles with each heartbeat. Although arteries are elastic, the more they stretch, the stiffer they become. As a result, they expand slightly at low pressure, but resist further expansion at high pressure. The response of the arterial wall to stretch is similar in a variety of animals and reflects similar structural and functional features (Figs. 12-29).

According to Laplace's law, the wall stress required to maintain a given transwall pressure within a hollow structure increases with increasing radius (see Equation 12-1). Thus, elastic blood vessels are unstable and prone to dilation; that is, they tend to dilate because they cannot develop high wall stresses with increasing pressure. In blood vessels, this instability is prevented by a collagen covering that limits their expansion. However, when the collagen sheath breaks down, blood vessel swelling (aneurysm) may develop. In general, the elasticity of the arterial wall and the thickness of the muscle layer decrease with distance from the heart. That is, the arteries become stiffer the farther they are from the heart and serve primarily as blood passages. For example, the dog's aorta

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Figure 12-28 The systemic arterial system acts as a conduit and pressure vessel; it also compensates for pressure fluctuations and controls flow distribution to capillaries. The conduction function (1) is performed by vascular channels along which blood flows to the periphery with minimal frictional pressure loss. Stretchable walls and high drainage resistance

Arterial resistance is responsible for the pressure storage function (2) and the suppression of pressure and flow fluctuations (3). Controlled hydraulic resistance in the peripheral vascular bed controls the distribution of blood to various tissues (4). [According to Rushmer, 1965a.l.]

It gradually stiffens and its diameter decreases with increasing distance from the heart (Fig. 12-30). In whales, the aortic arch at the outlet of the heart is very elastic and large in diameter, but the arterial system other than the aorta

The twitch bow quickly narrows and becomes much stiffer than a dog's. The elastic whale aortic arch expands with each heartbeat, absorbing approximately 50-75% of stroke volume; the remainder flows into the portion of the arterial system downstream of the aortic arch. Large whales can vary the volume of their ventricles by up to 35 liters per heartbeat, with a heart rate of about 12 to 18 beats per minute. The amount of elastic tissue in arteries varies according to the specific function of each vessel. For example, in fish, blood pumped by the heart is pushed into the elastic ball and the abdominal aorta (see Figures 12-15). The blood then passes through the gills and into the dorsal aorta, the main channel for distributing blood to the rest of the body. Efficient gas transfer requires steady, continuous blood flow in the gill capillaries. The bulb, abdominal aorta, and afferent branchial artery leading to the gills are very compliant and smooth and maintain blood flow in the gills despite the large oscillations caused by cardiac contraction. The dorsal aorta, which receives blood from the gills, is much less elastic than the abdominal aorta. If the dorsal aorta was more elastic than the abdominal aorta, blood would flow rapidly through the gills with each heartbeat. Such rapids would increase rather than decrease flow fluctuations through the gills. In this example, to ensure an even flow of blood through the gill capillaries, the greatest compliance must be placed in front of the gills rather than behind to dampen fluctuations in flow through the gills. Abdominal aorta must

Relative Expansion (PIP) Figure 12-29 Arterial elastic properties are surprisingly similar in many animals, with the exception of the nautilus and the octopus. This similarity is reflected in a plot of elastic modulus versus relative strain, expressed as pressure (P) divided by the species' resting blood pressure (P). [Adapted from Shadwick, 1992.1

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................................................... .Abdominal aortic blood flow (ml min-')

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Dog- _,,------ Figure 12-31 Blood flow in the abdominal aorta (A) of a fish is more pulsatile than in the dorsal aorta (B). The elasticity of the sphere and the abdominal, thoracic, and abdominal aorta helps dampen pressure and flow fluctuations. The recordings shown are from COD. [Jones et al., 1974.1

Relative position along the aorta Figure 12-30 The arterial system in dogs and whales becomes stiffer and smaller in diameter with increasing distance from the heart. In cetaceans, there is an abrupt decrease in diameter and increase in stiffness between the aortic arch and the thoracic artery. [Adapted from Gosline and Shadwick, 1996.1

The elastic and bulky dorsal aorta is relatively stiff to allow smooth flow through the gills (Fig. 12-31).

80 mm Hg). Blood is 12.9 times less dense than mercury, so a blood pressure of 120 mmHg is equivalent to 120 x 12.9 = 1550 mm (155 cm) of blood. In other words: If the vessel is suddenly opened, blood is ejected to a maximum height of 155 cm above the incision. To convert pressure in millimeters of mercury to kilopascals (kPa), multiply by 0.1333 kPa (eg 120 mm Hg x 0.1333 = 16 kPa). Pressure fluctuations generated by ventricular contraction and relaxation are reduced at the entrance to the capillary bed and absent in the venous system. The contraction of the heart causes tiny pressure fluctuations in the capillaries. The pressure pulse propagates at a velocity of 3-5 m-s-l. The velocity of the pressure pulse increases with decreasing arterial diameter and increasing arterial wall stiffness. In mammalian aortas, pressure pulses propagate at 3-5 msec, reaching velocities of 15-35 msec in arterioles. Both the peak blood pressure and the size of the pressure pulse in the aorta of mammals and birds increase with distance from the heart (Figure 12-32). This increase in heart rate can be strong during exercise. There are three possible explanations for this rather strange phenomenon. First, pressure waves are reflected from peripheral branches of the arterial tree; the initial and reflected waves add; where the peaks coincide, the pressure pulse and peak pressure are greater than where they are out of phase. When the initial and reflected waves are 180 inches out of phase, pressure fluctuations are reduced. It has been suggested that the heart is in a position where the initial and reflected waves are out of phase, thereby reducing peak arterial pressure in the aorta near the ventricle. With increasing distance from the heart, the initial and reflected pressure waves enter phase, and pressure maxima are observed in peripheral vessels. Second, the elasticity and diameter of arteries decrease with distance from the heart. Cardiac stress causes an increase in pulse strength. 3. The pressure pulse is a complex waveform consisting of the following

Blood pressure Blood pressure values reported in the arterial system are usually the transmural pressure (ie, the difference in pressure between the inside and outside of the vessel wall). The pressure outside the blood vessel is usually close to the ambient pressure, but changes in the extracellular pressure of the tissue can have a dramatic effect on the transmural pressure, which affects the diameter of the vessel and thus the blood flow. For example, systole increases the pressure around the coronary arteries, resulting in a significant decrease in coronary blood flow during systole. Inhalation is associated with a decrease in chest pressure and thus increases transmural pressure in the veins returning to the heart, increasing the pressure on the veins returning to the heart. In a heartbeat cycle, the maximum value of arterial pressure is called systolic pressure, and the minimum value is called diastolic pressure; the difference is the pressure pulse. Transmural pressure is usually reported in mm Hg; both systolic and diastolic pressure are usually indicated with a slash (eg, 1201).

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Time (ms) Figure 12-32 In the aorta of mammals and birds, both peak blood pressure and pressure pulses increase with distance from the heart. Shown are blood pressures recorded simultaneously at the aortic arch (2 cm from the heart) and aortic trigeminal (24 cm from the heart). Note that the average pressure in the aorta is slightly lower than the pressure in the aortic arch near the heart. [Adapted from Rangel, 1975. )

over the heart (Fig. 12-33A). If the arterial pressure of the blood flowing through the brain is to be maintained at about 98 mm Hg, the blood pressure of the aorta near the heart must be 195-300 mm Hg. Aortic blood pressure greater than 195 mmHg was recorded in an anesthetized giraffe with the head elevated approximately 1.5 m (Fig. 12-33B). The arterial pressure in the giraffe's legs is even higher than the aortic pressure; in order to prevent blood from pooling, the blood vessels in the giraffe's legs are surrounded by a large amount of connective tissue. When the giraffe lowers its head to the ground, arterial blood pressure at the level of the heart drops significantly, keeping blood flow to the brain relatively constant. When giraffes change head position, large fluctuations in aortic pressure can lead to either massive pooling of blood (head up) or reduced blood flow (head down) in arterioles outside the head. Vasoconstriction of these peripheral vessels during head elevation likely prevents accumulation. Conversely, when the head is down, there is extensive vasodilation of arterioles, resulting in capillary beds other than these

from several harmonics. Higher frequencies propagate at higher velocities, and it has been shown that the variation of the pressure pulse waveform with distance is due to the sum of various harmonics. The third interpretation is problematic because the distances are too small to allow harmonic summation.

Effects of Gravity and Body Position on Pressure and Flow When a person is lying down, the heart is at the same level as the feet and head, and arterial pressures in the head, chest, and extremities are similar. Once a person moves to a sitting or standing position, the relationship between the head, heart, and extremities changes relative to gravity, with the heart now standing three feet higher than the lower extremities. The result is an increase in arterial pressure in the lower extremities and a decrease in arterial pressure in the head. Due to gravity, the height of the blood column only causes blood pressure to rise. Gravity has little effect on capillary blood flow, which is determined by arteriovenous differential rather than absolute pressure. That is, gravity increases arterial and venous pressure by the same amount and therefore does not have a large effect on the pressure gradient across the capillary bed. However, since the vasculature is elastic, an increase in absolute pressure dilates blood vessels, especially compliant veins. Therefore, when the animal changes its position relative to gravity, blood pools, especially in veins in different parts of the body. This effect is only related to the elasticity of the blood vessel and does not occur if the blood flows in a rigid tube. Accumulation and maintenance problems of capillary flow are severe in long-necked species. For example, when a giraffe stands with its head held high, its brain is about 6 meters above the ground, more than 2 meters

lower body vasoconstriction

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Time (s) Figure 12-33 When a long-necked animal raises and lowers its head, the cardiovascular system must adjust to keep blood flowing to the brain and avoid pooling in the lower body. See discussion text. [Adapted from White, 1972.1

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.......................................... Head may maintain flow aorta despite lower upright pressure. The giraffe's ability to regulate peripheral vascular pressure and flow outside the head is particularly important for kidney function. If the renal tubules were exposed to the dramatic changes in blood pressure that accompany the raising and lowering of the giraffe's head, the glomerular filtration rate would be disrupted. Each time the animal raises its head, arterial blood pressure rises dramatically, resulting in a very high rate of ultrafiltrate formation in the kidneys; this in turn requires an equally high rate of fluid absorption. Without proper control, a giraffe may lower its head to drink, then lose the fluid filtered through its kidneys when it raises its head. Therefore, when a giraffe turns its head from the ground to a height of 6 m to feed, it must have mechanisms to regulate the peripheral resistance to flow in different capillary beds. Many other long-necked animals, such as dinosaurs and camels, had or had similar problems. A pool of blood that changes position relative to gravity does not pose a problem for animals in the water because water is only slightly less dense than blood, and air is much less dense than blood. In water, hydrostatic pressure increases with depth and effectively corresponds to the increase in blood pressure caused by gravity. Therefore, the transmural pressure does not change, so blood does not pool. Clearly, the circulatory problems of the large terrestrial dinosaurs were very different from those of the aquatic dinosaurs. Arterial Flow Velocity Flow and beat-to-beat variability are greatest at the ventricular outlet and decrease with distance from the heart (Figure 12-34). As mentioned earlier, at the base of the aorta, blood flow is turbulent and reverses during diastole as the aortic valve closes

Figure 12-34 The maximum vascular blood flow velocity and blood flow fluctuation gradually decrease with the distance from the heart. A regurgitation phase is observed in the aorta; in the ascending aorta, it may be associated with a transient regurgitation of blood through the aortic valve. The traces were obtained from arteries of dogs. Oscillating flow decays completely in the capillary. [Adapted from McDonald's, 1960.1

Substance in the blood that is expelled into the aorta during systole. In most other parts of the circuit, the flow is laminar, with velocity fluctuations dampened by the compliance of the aorta and proximal arteries. Based on a cross-sectional area of about 2.5 cm2 and a cardiac output of about 5 La min, the mean velocity in the aorta - the point of maximum blood flow velocity - is calculated to be about 33 cm -s-'-' in humans. If we assume that the maximum velocity in a vessel is twice the average velocity (only applies if the velocity curve is parabolic), then the maximum blood velocity in a human aorta would be 66 cm-s-l. If cardiac output is increased 6-fold during vigorous exercise, the maximum velocity increases to 3.96 m-s-l. In contrast, the pressure pulse associated with each heartbeat travels through the circulation at a rate of 3-35 m s-l; thus, the pressure pulse propagates faster than the flow pulse.

venous system

The venous system acts as a conduit for blood from the capillaries back to the heart. It is a high-volume low-pressure system composed of vessels with inner diameters larger than the corresponding arteries (see Figure 12-27). In mammals, approximately 50% of the total blood volume resides in veins (see Figure 12-3). Venous pressure rarely exceeds 11 mm Hg (1.5 kPa), which is about 10% of arterial pressure. Vein walls are thinner, have less smooth muscle, and are less elastic than arterial walls; vein walls also contain more collagen than elastic fibers. This allows the walls of veins to stretch more easily than arteries and recoil much less. The large diameter and low pressure of the veins allow the venous system to act as a reservoir for blood. According to Laplace's law (see Equation 12-1), if the venous pressure is high, very high wall stresses develop, requiring a strong wall to prevent rupture. With blood loss, venous rather than arterial blood volume decreases to maintain arterial pressure and capillary blood flow. Decreases in venous blood banks were offset by decreases in venous volumes. Many vein walls are lined with smooth muscle innervated by sympathetic adrenergic fibers. Stimulation of these nerves results in vasoconstriction and a decrease in venous reservoirs. This reflex allows some bleeding without a drop in venous pressure. Blood donors do lose part of their venous reservoir; however, this loss is temporary and the venous system gradually expands as fluid builds up to replace blood. Venous Blood Flow In addition to heart contraction, blood flow in veins is affected by many other factors. The contraction of the muscles in the extremities and the pressure the diaphragm exerts on the bowel can squeeze the veins in these parts of the body. Because veins contain pocket valves that only allow flow

In the direction of the heart, this squeezing increases the return of blood to the heart. Therefore, during exercise, the volume of venous blood returning to the heart increases as the contraction of the skeletal muscles squeezes the veins and forces blood towards the heart. An increase in venous return increases cardiac output. Activation of this skeletal muscle venous pump is associated with increased activity of sympathetic fibers innervating venous smooth muscle, thereby increasing smooth muscle tone. Increased venous tone causes skeletal muscle pumps to increase venous pressure, which returns to the heart, rather than just dilating another part of the venous system. If the skeletal muscles are not contracted, there may be a large pool of blood in the venous system of the extremities. Mammalian breathing also assists the return of venous blood to the heart. Expanding the chest reduces pressure in the chest and draws air into the lungs. This pressure drop draws blood from the veins in the head and abdomen into the large veins in the heart and chest. In sharks, the contraction of the ventricles reduces the pressure in the pericardial space, allowing blood to be drawn from the venous system into the atria (see Figures 12-14). Peristaltic contraction of venule smooth muscle (the small blood vessels that connect capillaries to veins) facilitates venous blood flow to the heart. This peristaltic activity has been observed in the venules of bat wings. Venous blood congestion Venous smooth muscle also helps regulate blood distribution in the venous system. When a person transitions from a sitting to a standing position, the change in the relative position of the heart and brain with respect to gravity activates the sympathetic adrenergic fibers that innervate the venous vessels, causing contraction of the venous smooth muscles, which leads to blood flow that promotes the distribution of accumulated blood. However, such vein structures are not enough; ensuring good posture for long periods of time without body movement, such as when soldiers stand motionless during parade. In this condition, venous return to the heart, myocardial output, arterial pressure, and blood flow to the brain are all reduced, leading to fainting. Similar problems affect bedridden patients trying to get out of bed after days of inactivity, and astronauts returning to Earth after prolonged periods of weightlessness. In these cases, other control systems involving baroreceptors (baroreceptors) and arterioles may also be disrupted. Without physical changes that change the relative positions of the heart and brain with respect to gravity, the correction system degrades and the result is pooling of blood. Reflex control of venous volume usually recovers during use. The organization of the venous system is influenced by the level of support provided by the medulla. A major reorganization of the system occurred when the vertebrate moved into the air and lost the support of water. As mentioned earlier, gravity affects blood circulation.

In aquatic animals, the distribution is not important because the density of water and blood do not differ much. Because of this, aquatic animals do not experience blood pooling due to gravity. Because the densities of air and blood vary widely, the accumulation of air became an immediate problem in the evolution of terrestrial forms. In addition to the changes needed to maintain the heart's separation of oxygenated and deoxygenated blood, changes are required in the venous system. While gravity has little effect in aquatic animals, swimming in fish increases venous return to the heart. As the fish moves forward, blood pools in the tail due to inertia and the pressure waves across the body associated with the fish's swimming motion. To reduce these problems, most of the veins returning to the heart pass through the midsection of the fish. Some fish also have an additional tail heart at the end of the tail that helps pump blood to the central heart (Fig. 12-35). The flow of water in the thoracic region of some fish can reduce the hydrostatic pressure in this region, promoting venous return to the heart as swimming speed increases. Countercurrent Exchangers Countercurrent exchangers are a common feature in animal designs (see Spotlight 14-2). Arteries and veins coexist with blood flow in many animals

Figure 12-35 Some fish have elevated caudal centers, causing deoxygenated blood to return to the central heart. The walls of the heart connect with skeletal muscles and beat rhythmically. [Adapted from Kampmeer, 19691

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499

................................................... ........... Movement in the opposite direction (i.e., countercurrent blood flow). In many of these cases, especially when the blood vessels are small, heat exchange occurs between opposing blood flows. Since heat is more easily transferred than gas, heat exchange with a small amount of gas transfer is possible. Counterflow heat exchangers are commonly found in the limbs of birds and mammals to regulate heat loss from the limbs. The opposite arrangement of arterioles and venules is called the miracle web. Before entering the tissue, the artery divides into a large number of small capillaries that run parallel to a series of venous capillaries that exit the tissue. "Arterial" capillaries are surrounded by "venous" capillaries and vice versa, creating an extensive exchange surface between inflowing and outflowing blood. These mesh-like capillaries are used to transfer heat or gas between arterial blood entering the tissue and venous blood leaving the tissue. In humans, this type of countercurrent exchanger is only found in the kidneys. Tuna have massive retinas that regulate the temperature of the brain, muscles, and eyes (see Figures 16-22 and 16-23). Miracle Net Leads to Physiological Swim Bladder

Other fish, such as eels, act as countercurrent carbon dioxide exchangers (see Figure 13-59). Capillaries and Microcirculation

Most tissues have such an extensive network of capillaries that each individual cell is no more than three or four cells from a capillary. This is important for the transport of gases, nutrients and waste, since diffusion is an extremely slow process. Capillaries are usually about 1 mm long and 15-10 microns in diameter, just large enough for red blood cells to pass through. However, large white blood cells can lodge in the capillaries and block blood flow. White blood cells are either displaced by increased blood pressure or slowly migrate along vessel walls until they reach larger vessels and enter the bloodstream.

Microcirculatory Bed Figure 12-36 shows the blood vessels that make up the microcirculatory bed. Small terminal arteries divide into arterioles, which in turn divide into posterior arterioles, which then divide into capillaries, which then rejoin venules and veins. smooth lining of arterioles

pericytes covered with

Figure 12-36: The microcirculatory bed is composed of arterioles (arterioles), sacs, and venules. The bursa consists of a single layer of endothelial cells surrounded by a basement membrane and occasionally encased in contracted pearl cells. DC

;

Blood from the arteriovenous system can pass through through channels, but most blood flows through the capillary network. The pre-capillary helps regulate the flow into the capillary bed. See also Figures 12-27 and 12-37

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Physiological system integration

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The muscle becomes discontinuous in the posterior arteriole and terminates in a ring of smooth muscle, the anterior capillary sphincter. Capillary walls are completely devoid of connective tissue and smooth muscle and consist of a single layer of endothelial cells surrounded by a collagen and mucopolysaccharide basement membrane. Capillaries are usually classified as arterial, medial, or venous capillaries, with the latter being slightly wider than the other two. Elongated cells with the ability to contract, called pericytes, wrap around capillaries. Venous capillaries flow into pericellular venules, which in turn connect with muscle venules and veins. Venules and veins are ventilated, and the muscular sheath emerges behind the first capillary posterior valve. Although capillary walls are thin and fragile, due to their small diameter, they require little wall stress to resist stretching under pressure (see Equation 12-1). The innervated smooth muscles of the arterioles, especially the smooth muscle sphincters at the arteriolar and arteriolar junctions, control the distribution of blood to each capillary bed. Most arterioles are innervated by the sympathetic nervous system; some arterioles, such as those in the lungs, are innervated by the parasympathetic nervous system. Different tissues have different numbers of capillaries open to blood flow and exhibit some differences in how blood flow is controlled through capillary beds. In some tissues, the anterior capillary sphincter is not innervated and is locally controlled, its opening and closing altering the distribution of blood in the capillary bed. By contrast, in other tissues, most, if not all, capillaries are either open (for example, in the brain) or closed (for example, in the skin) for long periods of time. The combined potential volume of all capillaries is approximately 14% of the animal's total blood volume. However, only 30-50% of capillaries are open at any given time, so only 5-7% of the total blood volume is contained in capillaries.

Material Transfer Across Capillary Walls Material transfer between blood and tissues occurs in capillary walls, pericellular venules, and to a lesser extent posterior arteriolar walls. The endothelial cells that make up the capillary walls are orders of magnitude more permeable than the epithelial cell layer, allowing substances to move in and out of the capillaries with relative ease. However, there are significant differences in the permeability of capillaries in different tissues. These permeability differences were accompanied by marked changes in endothelial structure. Capillaries are classified into three types according to their wall structure (Fig. 12-37): The least permeable continuous capillaries are found in muscle, nervous tissue, lung, connective tissue, and exocrine glands. Porous capillaries with moderate permeability are found in the glomerulus, intestine, and endocrine glands.

continuous capillary

(mucopolysaccharides and collagen fibers)

Second

windowed capillary

~ a g e m e nmembrane t

c sinusoidal capillary

Figure 12-37 Differences in capillary endothelial structure define the three types of capillaries found in characteristic tissues. A portion of the endothelial wall is shown here (A). Continuous capillaries with 4 nm gap, intact basement membrane, and many vesicles. (B) Capillary with holes, the capillary passes through a thin section of the wall, a few vesicles, and an intact basement membrane. (C) Sinusoidal capillaries with large paracellular spaces extending across a discontinuous basement membrane. In general, continuous capillaries are the least permeable, while sinusoidal capillaries are the most permeable.

The most permeable sinusoidal capillaries are found in the liver, bone marrow, spleen, lymph nodes, and adrenal cortex. In the well-studied continuous capillaries of skeletal muscle, the endothelium is approximately 0.2 to 0.4 µm thick and lies beneath a continuous basement membrane (see Figure 12-37A). Endothelial cells

Edition 501...................................

separated by gaps approximately 4 nm wide at their narrowest point. Most cells contain a large number of pinocytic vesicles, approximately 70 nm in diameter, mostly attached to the inner and outer membranes of endothelial cells; the rest are in the cellular matrix. Substances can pass through the endothelial cells or across the continuous capillary wall between endothelial cells. Fat-soluble substances diffuse through cell membranes, while water and ions diffuse through the water-filled spaces between cells. In addition, at least in the capillaries of the brain, there are transport mechanisms for glucose and some amino acids. Large molecules can pass through many capillary walls, but exactly how they are transferred is not always clear. There is some evidence that numerous vesicles in endothelial cells play a role in the transport of substances across the capillary wall. For example, electron microscopy studies have shown that when horseradish peroxidase is placed in the lumen of muscle capillaries, it appears first in vesicles close to the lumen and then in vesicles close to the adventitia, but Never occurs in the surrounding cytoplasm. This finding suggests that the substance is packaged in vesicles and transported through endothelial cells. This concept of vesicle-mediated trafficking is supported by the observation that endothelial cells from brain capillaries contain fewer vesicles and are less permeable than endothelial cells from other capillary beds. However, it has also been suggested that the reduced permeability of brain capillaries is due to tight junctions between endothelial cells. Microscopic observation of rat septal capillaries suggested another possibility, where vesicles were observed to coalesce and form pores through endothelial cells. It is therefore conceivable that substances diffuse through the pores formed by the coalescence of immobilized vesicles, rather than being packaged into vesicles, which then travel through the cell. The continuous capillaries in the lung are less permeable than capillaries in other tissues. In these less permeable capillaries, pressure pulses can play a role in enhancing the movement of substances, such as oxygen, across the endothelium. As pressure increases, fluid is pushed into the capillary walls, but as pressure decreases, fluid returns to the blood. This tidal flushing of the capillary walls should improve mixing at the endothelial barrier and effectively increase transfer. In the capillaries of the glomerulus and intestine, the inner and outer plasma membranes of endothelial cells are tightly spaced and perforated in some areas, resulting in endothelial fenestration (see Figure 12-37B). Not surprisingly, these porous capillaries are permeable to almost everything except large proteins and red blood cells. Kidney ultrafiltrate is formed across this endothelial barrier. The basement membrane of the fenestrated endothelium is usually intact and can provide an important barrier to the movement of substances through the fenestrated capillaries. These capillaries contain only a few vesicles, which are unlikely to play a role in transport.

Endothelial cells in sinusoidal capillaries are characterized by large paracellular spaces extending through the basement membrane and the absence of intracellular vesicles (see Figure 12-37C). Hepatic and bone capillaries always contain large paracellular spaces, and most of the material transferred through these capillaries occurs between cells. Therefore, the fluid surrounding the capillaries in the liver is very similar in composition to blood plasma. Cracks, pores, and paracellular gaps that allow free diffusion of substances through the capillary walls are approximately 4 nm wide, but only molecules much smaller than 4 nm pass through, suggesting another sieving mechanism. The diameter of these openings varies within the individual capillary network and is generally larger in pericellular venules than in arterial capillaries. This has an important function because blood pressure is the filtering force that moves fluid through the walls, decreasing from the arterial end to the venous end of the capillary network. Inflammation or treatment with various substances such as histamine, bradykinin, and prostaglandins dilates the openings at the venous end of the capillary network, making it highly permeable. Capillary Pressure and Flow Arterioles and venules are arranged such that all capillaries are within a short distance of each arteriole, so pressure and flow are fairly uniform across the capillary bed. Transmural pressures of approximately 10 mmHg were recorded in capillaries (Fig. 12-38). High pressure within the capillary causes fluid to filter from the plasma into the interstitial space. This filtration pressure is offset by the plasma colloid osmotic pressure, which is mainly due to the higher protein concentration in blood than in interstitial fluid. Due to their size, these plasma proteins remain in the blood and do not cross the capillary walls. To illustrate the relationship between these two pressures, consider the schematic in Figure 12-39. In general, blood pressure is higher than the oncotic pressure at the arterial end of the capillary bed, allowing fluid to enter the interstitial space (Zone 1). Blood pressure decreases steadily along the length of the capillary, while colloid oncotic pressure remains constant. Once the blood pressure falls below the oncotic pressure, the fluid in the interstitial space is drawn back into the blood through osmosis (zone 2). Thus, the net movement of fluid at any point on a capillary is determined by two factors: (a) the difference between blood pressure and oncotic pressure, and (b) the permeability of the capillary wall, which tends to increase toward the venous end. This concept is sometimes referred to as the Starling hypothesis, after its original proponent, Ernest Starling (1866-1927), whose prolific studies included studies of the relationship between ventricular work output and venous filling pressure (see Spotlight 12-1 ). In most capillary beds, the net loss of fluid from the arterial side is slightly greater than the net absorption from the venous side

502

Physiological system integration

1 second

arteriole

(17:00)

A，

to arterioles

(70~m)

large

1 minute C--l

small artery cancer

f'15

(25 watts)

; - 0

[ D

large

Capillary

1 minute

capillary. However, instead of accumulating in the tissues, fluid is carried away through the lymphatic system and returned to the circulatory system. Thus, fluid circulation typically occurs from the arterial end of the capillary bed into the interstitial space and back into the blood through the venous end of the capillary bed or through the lymphatic system. Due to this large fluid flow, the exchange of gases, nutrients, and waste products between the blood and tissues exceeds that expected by diffusion alone. The net filtration of fluid through capillary walls results in an increase in tissue volume, called edema, unless excess fluid is removed by the lymphatic system. In the kidney, capillary pressure is high and the filtration pressure exceeds the impingement osmotic pressure; thus, ultrafiltrate and ultimately urine form in the renal tubules. The kidneys are encapsulated to prevent tissue swelling during ultrafiltration. In most other tissues, there is little net movement of fluid through the capillary walls and tissue volume remains constant. Increased capillary pressure due to increased arterial or venous pressure leading to increased fluid loss from blood and tissue edema. Generally, however, arterial pressure is kept fairly constant to prevent large fluctuations in tissue volume. The decrease in colloid osmotic pressure may be caused by the loss of protein in plasma due to starvation or excretion, or the movement of plasma protein to the intercellular space due to increased permeability of capillary walls. A decrease in colloid osmotic pressure also leads to an increase in net fluid loss in the tissue space if the filtration pressure remains constant.

Figure 12-38 As blood flows through the capillary bed, the pressure pulse weakens and the mean blood pressure decreases. (A) Blood pressure traces recorded in the frog mesenteric capillary bed. As blood flows through the capillary bed, the pressure levels off and falls. (B) Diagram of blood pressure versus circulatory sites in the batwing subcutaneous layer. Shaded area represents 2 1 SE (standard error) from the mean indicated by the bold line. Typical tissue and lymphatic pressures are also plotted for comparison. [Part A of Weiderhielm et al., 1964; Part B of Weiderhielm and Weston, 1973.1

Capillary

end

filter

Record

9 3 (0 m

Second

Osmotic pressure

1 arterial end

Vein end capillary length

Figure 12-39 Net fluid flow through the capillary wall depends on the difference between blood pressure and the colloid osmotic pressure of the extracellular fluid. At the arterial end of the capillaries, blood pressure exceeds the oncotic pressure and fluid is filtered out of the plasma into the extracellular space (zone 1). On the venous side, the opposite is true and fluid is drawn back into the plasma from the extracellular space (region 2). In most capillary beds, zone 1 is slightly larger than zone 2; that is, the net loss of fluid from the circulation to the extracellular space is small. Normally, this interstitial fluid is drained through the lymphatic system and returned to the bloodstream.

503

transportation

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Lymphatic strain

10 seconds

1 minute

Lymphatic System Lymph—a clear, yellowish, or sometimes milky white fluid—collects from the interstitial fluid in various parts of the body and returns to the blood through the lymphatic system. Because this fluid contains many white blood cells and no red blood cells, it is almost colorless and it is difficult to see the lymphatic vessels. Thus, although the lymphatic system was first described about 400 years ago, it has not been studied as extensively as the cardiovascular system. The lymphatic system begins with blind-ended lymphatic capillaries that drain the plastid space. These lymphocapillary connections form a tree-like structure, with branches reaching all tissues. Larger lymphatic vessels resemble veins, channeling blood under low pressure. In mammals and many other vertebrates, lymphatic vessels drain through the thoracic duct into a very low-pressure region of the venous system, usually near the heart (see Figure 12-3). The lymphatic system serves to return excess fluid and protein to the blood that has entered the interstitial space through the capillary walls. Macromolecules, especially fats and possibly high-molecular-weight hormones taken up from the gut, enter the bloodstream via the lymphatic system. The walls of lymphatic capillaries are composed of a single layer of endothelial cells. The basement membrane is absent or ruptured, and large paracellular spaces exist between adjacent cells. This feature can be demonstrated by microscopic observation of horseradish peroxidase or quinine ink particles passing through the walls of lymphatic capillaries. Since the lymphatic pressure is usually slightly lower than that of the surrounding tissues, interstitial fluid can easily enter the lymphatic vessels. These blood vessels are fitted with valves that only allow flow out of the capillaries. The larger lymphatic vessels are surrounded by smooth muscle and sometimes contract rhythmically, generating pressures of up to 10 mmHg and draining fluid from the tissue (Fig. 12-40). Blood vessels are also squeezed by intestinal and skeletal muscle contractions, as well as whole-body movements, which promote lymphatic flow. Fats are absorbed from the gut through the lymphatic system, rather than directly into the bloodstream. Folds in the intestinal wall called villi each contain a lymphatic vessel (central milk duct) through which fats and fat-soluble nutrients (such as vitamins A, D, E, and K) pass from the intestinal lumen (see Chapter 15 ) . ). Intestinal contractions "squeeze" the milk fat from the milky fat lymph, forcing the lymph forward and eventually through the thoracic duct into the bloodstream. Lymphatic vessels are innervated, but it is unclear which type of innervation is involved and what the function of these nerves is.

Second

minute

Lymphatic capillaries (5 ~ r n )

1 minute Figure 12-40 The pressure in the lymphatic system is similar to the pressure in the venous system. These recordings are from lymphatic vessels (A) and lymphatic vessels (B) in unanesthetized bats. They were obtained by micropuncture without anesthesia, Prtor Surgical Intervention, Welderhlelm and Weston, 19731

Lymphatic flow is variable and 11 ml-h-' is an average for a person at rest. This corresponds to 1/3ooo of cardiac output over the same period. However, lymph flow, although small, is important for draining excess interstitial fluid from tissues. Severe edema occurs when lymph production exceeds lymph flow. In the case of the tropical disease filariasis, nematode larvae transmitted by mosquitoes to humans can penetrate the lymphatic system and block lymphatic channels; in some cases, the flow of lymph fluid to certain parts of the body is completely blocked. The resulting edema can cause parts of the body to swell so much that the condition is known as elephantiasis because of the swollen, hardened tissue that resembles an elephant's skin. Reptiles and many amphibians have a lymphatic heart that helps move fluid through the lymphatic system. Avian embryos have a pair of lymphatic hearts in the pelvic region; these hearts persist in some adult birds. Mammals lack these structures for lymphatic transport. Not only do frogs have multiple lymphatic hearts, but they also have a very large lymphatic space that serves as a reservoir of water and ions and acts as a fluid buffer between the skin and underlying tissues. The large lymphatic volume in amphibians arises from capillary plasma filtration and diffusion of water through the skin. The ratio of lymphatic flow to cardiac output in toads (approximately 1:60) is much higher than in mammals (approximately 1:3000), and the toad's lymphatic heart beats faster than the blood heart, although its stroke volume is much smaller. Fish appear to either have no lymphatic system or have a very rudimentary lymphatic system, although they do have a secondary circulatory system formerly known as the lymphatic system

system. This secondary circuit with low hematocrit is connected to the primary circuit by an arterial anastomosis and drains into the primary venous system close to the heart (see Figures 12-15). The secondary circuit provides nutrients to the skin and gut, but not much oxygen, and generally not distributed to other parts of the body. The skin exchanges gas directly with the surrounding water. Due to its narrow distribution, the secondary circulation is unlikely to perform the lymphatic function of maintaining interstitial fluid balance. It's unclear how fish maintain fluid balance in their tissues, but the lack of lymphatic vessels appears to be related to fish living in a medium with a similar density to their own body.

Circulatory and Immune Response Both the circulatory and lymphatic systems are involved in the body's defense against infection. A key player in the immune response is the lymphocyte, a type of white blood cell (white blood cell). A unique property of lymphocytes is their ability to "recognize" foreign objects.

(antigens), including surface-invasive pathogens, virus-infected cells, and tumor cells. There are two main types of lymphocytes: B lymphocytes (B cells) and T lymphocytes (T cells). The latter are divided into helper T (TH) cells and cytotoxic T (Tc) cells. Lymphocytes are supported by other white blood cells, especially neutrophils and macrophages. Under certain conditions, both neutrophils and macrophages can engulf microorganisms and foreign particles through phagocytosis. These phagocytes also produce and release various cytotoxic factors and antibacterial substances. The immune response is to recognize the invader, then mark and destroy it. Recognition occurs only by lymphocytes, whereas destruction can occur by both lymphocytes and scavenger cells (phagocytes). The lymphocyte recognition system must be able to distinguish between natural components of the body and foreign invaders, namely self and non-self. Failure to recognize yourself can lead to autoimmune diseases, some of which can be fatal. Lymphocytes respond to pathogen invasion in three ways (Figure 12-41). B cells develop into plasma cells, which secrete antibodies that bind and label pathogens.

Figure 12-41 Three types of lymphocytes: B cells, T helper cells (T1), and cytotoxic T cells (Tc).

Tc cells

develop into antibody-secreting plasma cells

receptor

Secretes cytokines that promote B cell and Tc cell growth and response

Antibodies bind to pathogens cleared by scavenging cells

Cytokines

Evolve into active CTLs that destroy altered autologous cells

Cells respond to antigens in different ways. Membrane-bound antibodies on B cells and T cell receptors on T cells specifically recognize and bind antigens. T cells and Tc cells can be distinguished by the presence of membrane molecules called CD4 and CD8. See discussion text. I adapted from Kuby, 1997.1

Induces cells to be decomposed by clear cells. T1 cells recognize tumor cells and tumor cells infected with pathogens; upon recognition of such cells, Tc are stimulated to mature into active cytotoxic T lymphocytes (CTL), which destroy altered autologous cells. Recognition of antigens by T cells stimulates them to secrete cytokines that promote the growth and response of B cells, T cells, and macrophages, thereby enhancing the strength of the Kn-Mune response to pathogens. White blood cells circulate in the blood and lymph. There are a large number of lymphocytes in the lymph nodes along the lymphatic vessels (see Figure 12-3). These nodes filter lymph and help bring antigens into contact with lymphocytes. In order to reach tissues invaded by pathogens, white blood cells must be able to leave the lymphatic and circulatory system. This process is called extravasation. Normally, white blood cells are naturally entrained in the bloodstream and do not pass through the blood vessel walls. However, the site of infection generates inflammatory signals that induce the synthesis and activation of adhesion proteins on the blood side of the endothelium. As leukocytes roll past inflamed endothelial cells, P-selectin on the blood-facing surface binds and slows the passing leukocytes (Figure 12-42). This interaction stimulates leukocytes to produce integrin receptors (such as LFA-I), which then bind to intracellular adhesion molecules (ICAMs) on the surface of endothelial cells. as

As a result of these and other interactions, cells adhere to endothelial cells. Once white blood cells are firmly attached, they can move between endothelial cells and migrate into infected tissue.

one

Cancer Regulation Circulatory regulation depends on the control of arterial blood pressure to meet three key priorities: Adequate blood supply to the brain and heart. Once the blood supply to the brain and heart is established, blood can be supplied to other organs of the body. Capillary pressure is controlled to maintain tissue volume and interstitial fluid composition within reasonable limits. The body uses a variety of receptors to monitor the state of the cardiovascular system. In response to sensory input from these receptors, both neural and chemical signals trigger appropriate adjustments to maintain adequate arterial pressure. In this section, we first discuss regulatory features affecting the heart and major vessels, then focus on the microcirculation.

Sectional view of the vascular endothelium

/ roll

stuck

Extravasation

White blood cells migrate into infected tissue

International Association for Migration

+ Selection combined with inflamed endothelium Figure 12-42 Leukocyte migration from cell to inflammatory cell (A) Leukocyte hyperattachment and extravasation through inflamed vascular endothelium (B) some interstitium

LFA-1 production

Integrin-ICAM Interaction

Interaction between cell surface molecules leading to leukocyte adhesion to inflamed endothelial cells [Adapted from Kuby, 1997 I

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Physiological system integration

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control central cardiovascular system

Baroreceptors monitor blood pressure at various points in the cardiovascular system. Information from baroreceptors, and from chemoreceptors monitoring CO,'. oh,'. and blood. transmitted to the brain. ~~~~l~ Or activation of afferent fibers embedded in muscle tissue in components of muscle extracellular fluid, which in turn causes changes in the cardiovascular system. In addition, inputs from cardiac mechanoreceptors and various thermoreceptors result in reflex effects on the cardiovascular system. In mammals, the integration of these sensory inputs occurs in a collection of neurons in the brain called the medullary cardiovascular center, at the level of the medulla and pons. The medullary cardiovascular center also receives input from other regions of the brain, including the medullary respiratory center, hypothalamus, amygdala, and cortex. Output from the medullary cardiovascular center feeds into sympathetic and parasympathetic autonomic motor neurons, the smooth muscles that innervate the heart and arterioles and veins, and other regions of the brain such as the medullary respiratory center. Sympathetic stimulation increases the rate and force of cardiac contraction, resulting in vasoconstriction; the result is a marked increase in arterial blood pressure and cardiac output. Generally speaking, when the parasympathetic nerve is stimulated, the opposite effect will occur, which will eventually lead to a decrease in arterial blood pressure and cardiac output. The medullary cardiovascular center can be divided into

Divided into two functional areas that have opposite effects on stress: Stimulation of the stress center leads to activation of the sympathetic nervous system and an increase in blood pressure.

Variety

me me

Stimulation of the inhibitory center leads to activation of the parasympathetic nervous system and a drop in blood pressure. In general, different sensory inputs affect the balance between pressor and depressive activity: some activate pressor centers and depress depressive centers; others have the opposite effect. This modifies and integrates various inputs that converge at the medullary cardiovascular center. The result is an output that activates the pressor or depressor centers and produces cardiovascular changes in response to changes in body demands or disturbances in the cardiovascular system. Figure 12-43 outlines this central circulatory control in mammals. Arterial baroreceptors Baroreceptors, which are ubiquitous in the vertebrate arterial system, display an increased firing rate with elevated blood pressure. Unmyelinated baroreceptors are located in the central cardiovascular system of amphibians, reptiles, and mammals. These unmyelinated baroreceptors respond only to pressures above normal pressure, triggering a reflex that lowers arterial blood pressure, thereby protecting the animal from harmful blood pressure increases. Myelinated baroreceptors found only in mammals respond to subnormal blood pressure, protecting animals from prolonged exposure

Other inputs Arterial blood components, lung inflation, body, etc. Other circulating mechanoreceptors

me me me me

riot

"set point"

Cardiovascular Center

effector

cardiopulmonary disease

heart and blood vessels

intravascular pressure

I - - - - - -

Figure 12-43 The mammalian circulatory control system consists of a series of negative feedback loops. Various receptors monitor changes in the state of the cardiovascular system and send signals to the medullary cardiovascular center. Integrate these inputs and combine them with

Barometer

This center sends signals through the autonomic nerves to maintain adequate arterial blood pressure. Arterial setpoints are altered by inputs from other brain regions, which in turn are affected by various peripheral inputs (dashed lines). [According to Korner, 1971]

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507

................................................... ................................... Blood pressure drops. Baroreceptors in the mammalian carotid sinus have been more extensively studied than those in the aortic arch or subclavian, common carotid, and pulmonary arteries. In mammals, there appear to be only minor quantitative differences between the baroreceptors of the carotid sinus and aortic arch. Birds have baroreceptors in the aortic arch. The carotid sinus in mammals is an extension of the origin of the internal carotid artery and has slightly thinner walls than the rest of the artery. Hidden within the thin walls of the carotid arteries are tiny nerve endings that act as baroreceptors. Under normal physiological conditions, these baroreceptors fire at rest. Elevated blood pressure stretches the walls of the carotid sinus, resulting in increased outflow from baroreceptors. The relationship between blood pressure and baroreceptor pulse rate is S-shaped, with the system most sensitive in the physiological blood pressure range (Fig. 12-44). Furthermore, the discharge rate of baroreceptors was higher under pulsating pressure than under constant pressure. Carotid sinus baroreceptors are most sensitive to pressure vibration frequencies between 1 and 10 Hz. Because arterial pressure rises and falls with each heartbeat, this frequency range is within the normal physiological range for arterial pressure fluctuations. Similar observations were made for the association between firing frequency and pressure of baroreceptors in the skin arteries of the toad lung (Fig. 12-45). Sympathetic efferent fibers terminate in the arterial wall near the carotid sinus baroreceptors; stimulation of these sympathetic fibers increases the release of these baroreceptors. under normal physiological conditions

20 40 60 80 Pressure increase gradually (mm Hg)

50 100 150 Karotissinusdruck (mmHg)

Figure 12-44 S-shaped increase in baroreceptor firing frequency with pressure. These receptors are most sensitive in the physiological pressure range and in pulsatile blood flow. These values were recorded from multifiber preparations of the carotid sinus nerve and plotted against mean carotid sinus pressure under pulsatile or constant flow. [According to Korner, 1971.I

The central nervous system (CNS) can use these efferent neurons to control the sensitivity of receptors. Signals from baroreceptors, in response to elevated blood pressure, are transmitted through the medullary cardiovascular center to autonomic motor neurons, resulting in a reflex decrease in cardiac output and peripheral vascular resistance (Table 12-1). The decrease in cardiac output is due to a decrease in heart rate and the force with which the heart contracts. The end result of the various autonomic actions is a reduction in arterial blood pressure. But when arterial pressure drops, so do baroreceptors

Figure 12-45 Baroreceptors are very sensitive to changes in pressure. The effect of increasing pressure on the firing rate of skin baroreceptors in toad lungs is shown immediately (A) and 45 s after the pressure increase (B). Each numbered black line represents an observation corresponding to the pressure increase shown on the horizontal axis. Fast initial peak response greater than

I I I I I I I 20 40 60 80 Pressure increase (mm Hg)

45 seconds to respond after pressure drop. [Adapted from Van Vilet and West, 1994.1

508

Physiological system integration

.....................................*...

Table 12-1 Reflex Effects Observed as a Function of Carotid Sinus Pressure Carotid Sinus Pressure* Autonomic Effector

Elevated

reduce

Herzvargos

++++

Sympathy

+++

visceral bed resistance vessels

capacity container

kidney bed

==0

++ ++ +

Changes in arterial pressure caused by peripheral vasoconstriction. Not surprisingly, there are many interactions between the respiratory and cardiovascular control systems. For example, the firing pattern of stretch receptors in the lung has a profound effect on the nature of cardiovascular changes induced by chemoreceptor stimulation. If the animal is breathing normally, changes in the gas content of the blood cause a series of reflex changes; however, when the animal is not breathing, chemoreceptor stimulation causes a very different series of cardiovascular changes, as we will discuss in their discussion of diving as seen.

muscle bed resistance vessels

capacity container

skin resistance vessels

capacity container

？

adrenal catecholamines

== oh

Ant ~ diuretic hormone

？

++++

*A indicates increased autonomic effect; a -, decreased autonomic effect; and 0, no autonomic effect. Source: Kona, 1971

The discharge rate results in a reflex increase in cardiac output and peripheral resistance, which tends to increase arterial pressure. Thus, the carotid sinus baroreceptor reflex is a negative feedback loop that tends to stabilize arterial blood pressure at a certain set point. Set points can be altered by interactions with other receptor inputs, or reset centrally in the medullary cardiovascular centers by inputs from other brain regions (see Figure 12-43). Arterial chemoreceptors Arterial chemoreceptors located in the carotid arteries and aortic body are particularly important in regulating ventilation (see Chapter 13), but also have some influence on the cardiovascular system. These chemoreceptors respond to increases in CO or decreases in 0 and pH in blood perfusing the carotid arteries and aortic body by increasing their firing frequency. When the animal is not breathing (e.g., during submersion), the increased firing rate results in peripheral vasoconstriction and slowing of the heart rate. Cardiac output is reduced during diving in birds and mammals; peripheral vasoconstriction then maintains arterial blood pressure, which maintains cerebral perfusion in the face of reduced cardiac output. Peripheral vasoconstriction results in an increase in arterial pressure, which then slows the cardiac reflex by stimulating systemic baroreceptors. However, even when arterial pressure is regulated at a constant level, stimulation of arterial chemoreceptors causes the heart to slow down. Thus, stimulation of chemoreceptors has a direct and indirect effect on heart rate

Cardiac Receptors Both mechanoreceptor and chemoreceptor afferent nerve endings are located in different regions of the heart. Information collected by these receptors about the state of the heart is transmitted through the spinal cord to the medullary cardiovascular centers and other regions of the brain. In addition, stimulation of certain cardiac receptors results in the release of hormones directly from the atria or other endocrine tissues in the body. Stimulation of cardiac receptors triggers a cascade of reflex responses, including changes in heart rate and contractility of the heart muscle, and, in extreme cases, the pain that may be associated with a heart attack.

Atrial Receptors The atrial wall contains many mechanoreceptive afferent fibers, divided into three types. Myelinated afferent type A and type B fibers are embedded in the atria. A fiber afferents respond to changes in heart rate and appear to relay heart rate information to central cardiovascular control centers. B fiber afferents respond to increases in atrial filling rate and volume. An increase in venous volume leads to an increase in venous pressure, which in turn increases atrial filling and thus increases the firing rate of B fibers. This increased activity is handled by the central cardiovascular center and has two main effects, one on the heart and one on the kidneys. Stimulation of atrial B fibers results in an increase in heart rate, which is mediated by increased sympathetic nerve flow to the heart's sinus node. Stimulation of these afferent fibers also results in a marked increase in urine output (diuresis), possibly due to decreased levels of antidiuretic hormone (ADH) in the blood. Thus, there is a negative feedback loop that regulates blood volume. Increased blood volume increases venous pressure and atrial filling; this stimulates the B fibers in the atria, which inhibits release of ADH from the pituitary gland. The resulting drop in ADH levels in the blood leads to diuresis, which reduces blood volume. A third type of atrial mechanoreceptor consists of unmyelinated type C afferent fibers innervating the venous and atrial junction. Stimulation of these C fiber afferent receptors affects heart rate and blood pressure. At low heart rates, stretching the area causes the heart rate to increase, while at high heart rates, stimulation causes the heart rate to decrease. Stimulation of C fibers

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................................... Also causes a drop in blood pressure. Both myelinated and unmyelinated sympathetic fibers innervate the atria. Atrial contraction and stretching reflexively stimulate these fibers, causing an increase in heart rate. The atrial wall also contains stretch-sensitive secretory cells that produce atrial natriuretic peptide (ANP). When these cells stretch, this hormone is released into the bloodstream and has a variety of endocrine effects. As the name suggests, ANP causes increased urine output and sodium excretion, which effectively lowers blood volume, thereby lowering blood pressure. ANP inhibits renal release of renin and adrenal cortex production of aldosterone. This weakens the renin-angiotensin-aldosterone system, which stimulates sodium absorption and increases blood volume (see Chapter 14). In addition to these actions, ANP also inhibits the release of ADH and acts directly on the kidneys to increase water and sodium excretion. ANP has been shown to be inhibitory, reducing cardiac output and blood pressure. In addition, ANP antagonizes the antihypertensive effect of angiotensin. Atrial natriuretic peptides belong to the natriuretic peptide family (A, B, C, and V-type natriuretic peptides), which share a 17-amino acid ring structure linked by disulfide bonds. Since ANP was first studied in the early 1980s, natriuretic peptides have been identified in a variety of tissues, including the central nervous system. In many cases, they can have autocrine or paracrine functions. For example, natriuretic hormone receptors are found in both the atria and ventricles of the hearts of several vertebrates. Binding of locally released natriuretic hormone to these receptors may reduce contractility, suggesting a paracrine function of the heart.

Ventricular Receptors The ends of both myelinated and unmyelinated sensory afferent fibers are embedded in the ventricles. Myelinated fibers are mechanosensitive and chemosensitive, with distinct ends for each mode. Stimulates mechanoreceptor terminals by stopping coronary blood flow. Chemoreceptor terminals are stimulated by substances such as bradykinin. At low levels of stimulation, these fibers cause increased sympathetic outflow and decreased vagal outflow to the heart, thereby increasing myocardial contractility and blood pressure. These fibers are necessary for the heart's pain perception at higher levels of stimulation. There are far fewer myelinated afferent fibers than unmyelinated afferent C-fiber terminals in the left ventricle. Stimulates C fiber events

At low concentrations, this causes dilation of peripheral blood vessels and a decrease in heart rate. Increased stimulation of these fibers causes the stomach to relax and, at a higher frequency, to vomit. Afferent Fibers of Skeletal Muscle Surprisingly, most nerves innervating skeletal muscle contain more afferent fibers than efferent fibers. Afferent fibers can be divided into four main groups. Groups I and I1 are sensory fibers from muscle spindles and Golgi tendon organs; these appear to have little role in cardiovascular control. In contrast, stimulation of group III fibers (myelinated "free nerve endings") or group IV fibers (unmyelinated sensory endings) appears to have cardiovascular effects. These fibers are activated by mechanical or chemical stimuli, and most fibers respond in only one way. Mechanical stimulation may be due to muscle contraction, pressure, or stretching. It is also thought that changes in extracellular fluid associated with muscle contraction stimulate chemoreceptive afferent muscle fibers and induce cardiovascular changes. Large changes in pH and osmolality increase the activity of group IV fibers, but it is unclear whether the pH or osmolality changes that occur in vivo are sufficient to mediate cardiovascular effects. Electrical stimulation of afferent muscle fibers can result in an increase or decrease in arterial blood pressure, depending on the fiber being stimulated or the frequency of stimulation for a particular group of afferent nerves. At low frequencies, stimulation of some afferent fibers leads to a drop in arterial blood pressure, while stimulation of the same fibers at high frequencies leads to an increase in blood pressure. Electrical stimulation of muscle afferents usually causes changes in heart rate in the same direction as blood pressure changes. This means that when blood pressure rises, so does heart rate, and vice versa. Where electrical stimulation of muscle afferents leads to increases in heart rate and cardiac output, blood distribution in the body also changes. Blood flow to the skin, kidneys, intestines, and inactive muscles is reduced to increase blood flow to active muscles. The cardiovascular response to muscle contraction has been shown to disappear after dorsal root transection, suggesting that the response may be of reflex origin and due to stimulation of afferent fibers in the muscle. The response varies depending on whether the muscle contraction is isometric (static training) or isotonic (dynamic training). Static training was associated with increases in arterial blood pressure with little change in cardiac output, whereas dynamic training resulted in large increases in cardiac output with little change in arterial blood pressure. Sensory input from afferent muscle fibers is processed at the central cardiovascular center and results in stimulation of the autonomic nerves innervating the heart and blood vessels, the efferent arm of the reflex arc.

510 Integration of Physiological Systems ................................................ ...... microcirculation control

Capillary blood flow adapts to tissue requirements. When there is a sudden change in demand, as in skeletal muscle during exercise, there are changes in capillary flow. When nutrient requirements fluctuate very little over time, as in the brain, capillary flow also rarely fluctuates. Regulation of capillary blood flow can be divided into two main types: neural control and local control. Neural Control of Capillary Blood Flow Neural control maintains arterial pressure by modulating resistance to blood flow in the peripheral circulation. The brain and heart of vertebrates must be perfused with blood at all times. Interruption of blood flow to the human brain can quickly lead to damage. The neural control of arterioles ensures that only a limited number of capillaries are open at any given time, because if all capillaries were open, arterial pressure would drop rapidly and blood flow to the brain would decrease. Neural control of capillary blood flow is subject to priority systems. When arterial pressure drops, blood flow to the intestines, liver, and muscles is reduced to maintain blood flow to the brain and heart. Most arterioles are innervated by sympathetic nerves, which release norepinephrine at their terminals. However, some arterioles are innervated by parasympathetic nerves, which release acetylcholine at their terminals. Sympathetic stimulation and circulating catecholamines. Binding of the catecholamine norepinephrine to alpha-adrenergic receptors in arteriolar smooth muscle normally causes vasoconstriction, resulting in a decrease in arteriolar diameter. This reduction in diameter results in increased resistance to flow, thereby reducing blood flow through the capillary bed. The general effect of sympathetic stimulation is peripheral vasoconstriction and subsequent increase in arterial blood pressure. This general response is mediated by the binding of norepinephrine from nerve endings to alpha-adrenergic receptors in vascular smooth muscle, resulting in increased smooth muscle tone. However, stimulation of beta-adrenergic receptors in arterial smooth muscle typically results in muscle relaxation and an increase in arteriolar diameter (i.e., vasodilation), which reduces resistance to flow and increases blood flow through the capillary bed. Since p-adrenergic receptors are rarely located near nerve endings, they are usually stimulated by circulating catecholamines. Catecholamines are released into the blood by adrenergic neurons of the autonomic nervous system and chromaffin cells in the adrenal medulla. The predominant circulating catecholamine is epinephrine, which is released from the adrenal medulla (see Chapter 8). Epinephrine reacts with alpha and beta adrenergic receptors, causing vasoconstriction and vasodilation, respectively. Although alpha-adrenoceptors are less sensitive to epinephrine, when activated they override beta-adrenoceptor-mediated vasodilation. The result is that high levels of circulating epinephrine cause vasoconstriction, which increases peripheral vascular

Drug resistance through alpha-adrenoceptor stimulation. However, at lower circulating levels of epinephrine, P-adrenoceptor stimulation dominates, leading to systemic vasodilation and decreased peripheral resistance. Even at vasodilation levels of epinephrine, it causes an increase in arterial blood pressure through stimulation of p-adrenoceptors in the heart, resulting in a significant increase in cardiac output. p-adrenergic receptors can be divided into two subgroups: PI-adrenergic receptors, stimulated by circulating catecholamines (epinephrine) and adrenergic nerve stimulation (norepinephrine), and β,-adrenergic receptors that respond only to circulating catecholamines. Only p1-adrenergic receptors are present in the peripheral circulation, whereas PI-adrenergic receptors are present in the heart and coronary circulation, where both circulating catecholamines and neuronal-released norepinephrine can have significant effects. We can summarize these effects as follows: Sympathetic stimulation generally results in peripheral vasoconstriction and an increase in arterial blood pressure. An increase in circulating catecholamines results in a decrease in peripheral resistance and an increase in arterial pressure due to simultaneous stimulation of the heart and increase in cardiac output. The response of any vascular bed depends on several factors: the type of catecholamine, the type of receptor involved, and the relationship between receptor stimulation and changes in muscle tone. Although stimulation of alpha-adrenergic receptors is usually associated with vasoconstriction and stimulation of beta-adrenergic receptors with vasodilation, this is not always the case. Another complicating factor is that not all sympathetic fibers are adrenergic. In some cases, they may be cholinergic, releasing acetylcholine from nerve endings. Stimulation of sympathetic cholinergic nerves results in vasodilation of the skeletal muscle vasculature. The actions of catecholamines are primarily mediated by several substances, including neuropeptide Y and adenosine. Neuropeptide Y was first isolated from porcine brain in 1982 and is structurally related to mammalian pancreatic polypeptide and peptide YY. Neuropeptide Y is distributed throughout the animal kingdom and has been found in many vertebrates and insects. Neuropeptide Y is localized with norepinephrine in sympathetic ganglia and adrenergic nerves; it is also present in many non-adrenergic fibers. Atrial and ventricular myocardium and coronary arteries are surrounded by nerve fibers containing neuropeptide Y. Furthermore, cardiomyocytes appear to synthesize and secrete neuropeptide Y themselves. In general, neuropeptide Y reduces coronary blood flow and myocardial contraction by reducing levels of the intracellular second messenger inositol triphosphate (InsP) (see Chapter 9).

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................................... Neuropeptide Y Appears to Increase Catecholamine Effects on Cardiac Improvement and Coronary Artery Cycling, mediated by InsP. The role of neuropeptide Y in the peripheral circulation is unknown, but it appears to attenuate the increase in blood pressure caused by α-adrenoceptor-mediated norepinephrine-induced peripheral vasoconstriction. ATP and neuropeptide Y are stored and released along with catecholamines. ATP and its breakdown product adenosine inhibit the release of catecholamines. Adenosine is released by many tissues in response to hypoxia, but has only paracrine or autocrine effects due to rapid inactivation. Hypoxia tends to promote the release of catecholamines from chromaffin tissues into the blood, but this effect is mediated by the local release of adenosine. Parasympathetic stimulation The arterioles in the blood flow to the brain and lungs are innervated by parasympathetic nerves. These nerves contain cholinergic fibers that release acetylcholine from the nerve endings when stimulated. In mammals, stimulation of the parasympathetic nerves leads to vasodilation in arterioles. Some parasympathetic neurons release ATP and other purines at their terminals. Some of these purinergic neurons may be involved in the control of capillary blood flow. For example, ATP causes blood vessels to dilate.

Local Control of Capillary Blood Flow Tissues require basal capillary blood flow to provide nutrients and oxygen and to remove waste products. Active tissues have higher demands, so capillary blood flow must increase during activity. In addition to the neural control of the central cardiovascular system, there are multiple mechanisms that control the local microcirculation. For example, when a vessel is stretched by increased inlet pressure, the smooth muscle of the vessel responds by constricting and counteracting the increase in vessel diameter. This tendency to keep the vessel diameter within tight limits prevents large changes in flow resistance, thereby providing a relatively constant baseline flow through the capillary bed. Local tissue heating, which may be accompanied by inflammation, is accompanied by marked vasodilation, while a drop in temperature causes vasoconstriction. Therefore, ice packs reduce blood flow, which reduces swelling associated with tissue damage. Many compounds also affect capillary blood flow within tissues. These fall into three categories: compounds produced by the vascular endothelium; various vasoconstrictors and vasodilators released by other cells; and metabolites associated with increased activity. Compounds Produced by Endothelial Cells Endothelial cells are not only a barrier between blood and underlying tissue, but are living tissues that produce many compounds. Some of these, such as nitric oxide, endothelin, and prostacyclin, affect vascular smooth muscle and thus capillary blood flow.

The vascular endothelium continuously produces and releases nitric oxide, which causes vascular smooth muscle to relax. Nitric oxide-mediated vasodilators regulate blood flow and pressure in mammals and other vertebrates. The observation of endothelium-dependent vascular relaxation led to the discovery of endothelium-derived relaxing factor (EDRF). This phenomenon is now known to be primarily due to the production and release of nitric oxide, which activates guanylate cyclase and leads to an increase in the intracellular second messenger cGMP (cyclic 3',5'-guanosine monophosphate). This connection, in turn, regulates muscle relaxation. A family of enzymes called nitric oxide synthases oxidize L-arginine in the endothelium to nitric oxide and L-citrulline. Several nitric oxide synthases are calcium-dependent, and entry of calcium into endothelial cells has been shown to result in the production and release of nitric oxide and the relaxation of surrounding smooth muscle. The finding that some calcium channels in endothelial cells are stretch-sensitive suggests that nitric oxide production in response to blood vessel stretching may be due to increased calcium entry into endothelial cells. Multiple chemicals (eg, acetylcholine, ATP, and bradykinin) stimulate nitric oxide release, as does Hy;,xis, pH changes and increased vessel shear stress. This is evidence that nitric oxide production increases with the stress that accompanies each heartbeat. h L-oxygen synthase has been found in a variety of animals, including horseshoe crabs, blood-sucking beetles Rhodnius, lampreys, and humans. Nitric oxide has been shown to have many functions in addition to maintaining a vasodilator, so its presence in animals without vascular tone or in nonvascular tissues is not surprising. For example, nitric oxide released in the central nervous system through stimulation of N-methyl-D-aspartate receptors is involved in the regulation of synaptic activity. Nitric oxide may also be involved in nonspecific immune responses, relaxation of nonvascular smooth muscle in the gastrointestinal and genitourinary tracts, and regulation of the release of certain hormones. In addition, nitric oxide released by endothelial cells, platelets, and leukocytes regulates cell adhesion and aggregation and inhibits thrombus formation. The vascular endothelium releases endothelin and prostacyclin as well as nitric oxide. Endothelins are small vasoconstrictor proteins of 21 amino acid residues. Prostacyclin causes vasodilation and acts as an anticoagulant. Thus, it acts as an antagonist of the prostaglandin thromboxane A, promoting blood clotting and causing vasoconstriction. Inflammation and other mediators Thromboxane A is formed in plasma from arachidonic acid, which is released by platelets upon attachment to damaged tissue. Local injury in mammals is associated with marked vasodilation of blood vessels in the injured area, mainly due to local release of histamine, although thromboxane levels in injured tissue are elevated and cause vasoconstriction. Histamine is not released from endothelial cells, but

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But there is some connective tissue and white blood cells in the damaged tissue. Antihistamines reduce this inflammatory response, but don't eliminate it completely. Another group of potent vasodilators, plasma kinins, are also activated in damaged tissue. Tissue damage results in the release of proteolytic enzymes that cleave kininogen, an α2-globulin, into kinins. Hypoxia also stimulates kinin formation. Vasoconstrictors acting on arterioles include norepinephrine and angiotensin IT released from the sympathetic nerves. Angiotensin is mainly formed in the lungs from angiotensinogen circulating in the blood (see Chapter 14). Finally, serotonin can act as a vasoconstrictor or a vasodilator depending on the vascular bed and dose level. It is found in high concentrations in the gut and in platelets. Histamine, bradykinin, and serotonin lead to increased capillary permeability. Consequently, large proteins and other macromolecules tend to distribute more evenly between the plasma and the intercellular space, reducing the osmotic pressure differential across the capillary walls. As a result, filtration increases and tissue edema occurs. On the other hand, norepinephrine, angiotensin II, and vasopressin tend to promote the absorption of fluid from interstitial fluid into the blood. This absorption can be achieved by reducing the filtration pressure and/or permeability of the capillaries.

Although low 0, values indicate tissue activity, leading to vasodilation and increased blood flow in systemic capillaries, the pulmonary capillary bed exhibits the opposite behavior. That is, low levels in the lungs cause local vasoconstriction rather than vasodilation. The functional significance of this difference is related to the direction of gas transport. In the pulmonary capillaries, 0, is absorbed by the blood, so blood flow should be greatest in areas where 0, is high. In contrast, in systemic capillaries, 0, that carry blood to tissues, the highest blood flow should go to the areas that need it most, represented by low 0, areas. If blood flow to an organ is stopped by a squeezed artery or severe narrowing of a blood vessel, much more blood flow to that organ after the blockage is cleared than before the blockage. This phenomenon is called reactive hyperemia. It is hypothesized that during ischemia (time without blood flow), O2 levels drop and CO2, H+, and other metabolites are formed, causing local vasodilation. The result is a much higher blood flow than normal after the occlusion is removed.

Cardiovascular responses to extreme conditions In the previous chapters we have described the general organization of the circulatory system and its regulation under normal conditions. The cardiovascular system responds in unique ways during physical exertion, diving and bleeding to meet the physiological challenges of these extreme conditions. a practice

Activity-Related Metabolic Disorders When activity in tissues increases, blood flow necessarily increases. Local control of capillary flow ensures that the most active tissues have the most dilated vessels and therefore the greatest blood flow. The degree of dilation depends on local conditions in the tissue, with conditions associated with high levels of activity often causing dilation of blood vessels. The term hyperemia means increased blood flow to tissue; ischemia means cessation of blood flow. Active hyperemia refers to increased blood flow following increased tissue (especially skeletal muscle) activity. Active tissue with aerobic metabolism is characterized by a decrease in 0, an increase in CO, H+, various other metabolites (e.g. adenosine, other ATP breakdown products), and heat. Extracellular K+ in skeletal muscle also increases after exercise. All of these activity-related metabolic changes, along with nitric oxide and prostacyclin, have been shown to lead to vasodilation and increased local capillary blood flow. This means that the most active tissues have the most dilated blood vessels and therefore the highest blood flow.

Regulation of the cardiovascular system during exercise is clearly a complex process involving central nervous control mechanisms, peripheral reflex mechanisms (especially those involving skeletal muscle afferent fibers), and local control. Many of the cardiovascular changes observed during exercise may occur without neural mechanisms, suggesting the importance of local control systems in increasing blood flow to active skeletal muscle. However, central nervous control mechanisms and reflexes of muscular afferent mechanosensory and chemosensory inputs clearly play a role, the exact form of which varies by movement type. For example, the reflex effect of afferent muscle stimulation on the cardiovascular system depends on the type of exercise: isometric muscle contractions tend to increase blood pressure without affecting cardiac output. Isotonic contraction increases cardiac output but hardly changes arterial blood pressure. During exercise, blood flow to skeletal muscles increases in proportion to muscle activity. Blood flow to muscles increases by up to 20

Just; at the same time, the transfer of oxygen from the blood to the muscles can be tripled, resulting in a 60-fold increase in muscle oxygen utilization. Active hyperemia is primarily responsible for increased blood flow to the muscles; a decrease in peripheral resistance results in a sympathetic-mediated increase in cardiac output. At the same time, blood flow to the intestines, kidneys, and, under high pressure, blood flow to the skin is reduced (Fig. 12-46). Cardiac output can increase up to 10 times the resting level due to large increases in heart rate and small changes in stroke volume. Most of the increase in cardiac output is attributable to a decrease in peripheral resistance to approximately 50% of the resting value and to an exercise-related increase in venous return due to pumping of the veins by skeletal muscle and increased respiration. Increased sympathetic activity but decreased parasympathetic activity in the nerves innervating the heart has the effect of increasing heart rate and contraction force, thereby keeping stroke volume at a relatively constant level. In fact, exercise increases stroke volume in mammals by about 1.5-fold, despite a dramatic increase in heart rate and the associated shorter time available for inflation and deflation. However, after sympathetic stimulation, blood is ejected from the ventricles more rapidly with each heartbeat, maintaining stroke volume at a higher heart rate. The relative roles of stroke volume and heart rate variability in increasing cardiac output during exercise vary among animals. For example, in fish, stroke volume varies much more than heart rate, and in birds, the epidermis of the heart, brain, etc. is very large.

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0, uptake (L . r n i n - I ) Figure 12-46 During exercise, total cardiac output increases and blood flow is diverted to active muscles. Shown is the approximate distribution of cardiac output in a normal young adult at rest and at various exercise levels up to maximal oxygen consumption (Max Vo2). A gradual decrease in absolute blood flow and the percentage of cardiac output distributed to the viscera (visceral regions and kidneys) increases muscle blood flow. The skin also shrinks under short-term stress and high oxygen consumption. [Adapted from Rowell, 1974.1

Changes in heart rate and small changes in stroke volume during exercise. Exercise is associated with only small changes in arterial blood pressure, pH, and gas tension. PCO2 and POI fluctuate slightly with respiration, as does the arterial pressure pulse. Increased pressure pulses are partially attenuated due to increased arterial wall elasticity due to increased circulating catecholamines. Arterial chemoreceptors and baroreceptors may play only a minor role in exercise-related cardiovascular changes. Motor neurons innervating skeletal muscle are activated by higher brain centers in the cerebral cortex (see Chapter 10) at the onset of exercise; this activation system may also induce changes in lung ventilation and blood flow. Muscle proprioceptive feedback may also play a role in increasing pulmonary ventilation and cardiac output (see Chapter 13). Many other changes improve gas transport during exercise; for example, in many animals, red blood cells are released from the spleen, increasing the oxygen-carrying capacity of the blood. Thus, exercise is responsible for a complex series of integrated changes that lead to adequate oxygen and nutrients for the muscles in motion.

diving

Many air-breathing vertebrates can stay underwater for long periods of time. During any period of submersion, all air-breathing vertebrates cease breathing, leaving the animal dependent on available blood oxygen reserves (see Chapter 13). The cardiovascular system is designed to distribute limited oxygen stores to the organs least able to withstand hypoxia - the brain, heart and some endocrine structures. Much of the information on responses to submersion comes from studies of animals forced to dive, sometimes simply by submerging the animal's head. Because naturally occurring diving varies widely in depth, duration and skill level, the information obtained on mandatory diving is not always directly applicable to natural diving. Whales and dolphins spend their entire lives in the water before surfacing to breathe, while seals may stay out of the water for extended periods of time on land. Other animals may spend most of their time on land, diving only occasionally. Oxygen storage varies in animals, so metabolism may be entirely aerobic in some dives, but largely anaerobic in others. Figure 12-47 shows typical cardiovascular changes that occur when a seal dives and remains underwater. In mammals, but not in other vertebrates, stimulation of the face receptors that inhibit respiration leads to overt bradycardia. Although the initial pressurization

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Figure 12-47 When a seal dives, the cardiovascular system undergoes numerous adaptations. Heart rate, cardiac output, and blood O2 levels decrease during a dive, but blood CO2 levels increase. During post-dive recovery, blood lactate levels rise sharply; other parameters initially overshoot and then gradually return to pre-dive values.

The lungs cause temporary increases in O2 and CO2 levels in the blood. The continued use of oxygen during a dive causes a gradual drop in oxygen and carbon dioxide in the blood and an increase in carbon dioxide in the blood. This zero blood drop stimulates arterial chemoreceptors and, in the absence of lung stretch receptor activity, results in peripheral vasoconstriction and a drop in heart rate and cardiac output. This reduces blood flow to many tissues in order to maintain blood flow to the brain, heart, and some endocrine organs. The lack of activity of the stretch receptors in the lungs is due to the lack of breathing and compression of the lungs as the animal descends into the water column. The increase in peripheral resistance is caused by a marked increase in sympathetic output and is accompanied by considerable arterial narrowing. Reduced renal blood flow has been observed in Weddell seals during dives. In some cases, blood flow to muscles can be reduced, but this depends on the level and type of physical activity while diving. Sometimes, arterial pressure increases during diving, which stimulates arterial baroreceptors. During such dives, increased chemoreceptor and baroreceptor firing rates maintain bradycardia. Bradycardia is caused by increased parasympathetic activity and, to some extent, decreased sympathetic activity in the fibers innervating the heart. Seals have shown that the development of diving bradycardia may involve some form of associative learning. In some trained seals, bradycardia occurs before the dive begins and therefore before peripheral receptors are stimulated. This psychological effect on heart rate can have a major impact on heart rate variability in many animals while diving. Generally speaking, when the heart rate

If your heart rate was low before the dive, your heart rate may vary little or not at all during the dive. When the heart rate is high, there may be marked bradycardia and reduced activity of lung stretch receptors due to facial moistness. The "water" receptors present in birds are not directly involved in the cardiovascular changes associated with submersion. No decrease in heart rate was observed in submerged ducks breathing air through a tracheostomy tube or in submerged ducks after carotid body denervation (Fig. 12-48). Activation of the "water" receptors thus leads to a cessation of breathing (apnea); a subsequent drop in Pol and pH in the blood and an increase in PCOl leads to stimulation of the chemoreceptors, which then reflexively produce cardiovascular changes. Stimulation of mammalian lung stretch receptors alters reflex responses to chemoreceptor stimulation. There is no breathing and thus no stimulation of the stretch receptors in the lungs, and stimulation of the chemoreceptors elicits a different reflex response than when the animal breathes. In the absence of breathing, lung inflation tends to suppress reflex cardiac arrest and peripheral vasoconstriction induced by stimulation of arterial chemoreceptors. As submerged animals ascend in the water column, the lungs expand, possibly activating stretch receptors in the lungs and causing the heart to speed up. Stimulation of arterial chemoreceptors leads to a marked increase in pulmonary ventilation when the animal breathes. In this case, low levels of O in the blood and/or high levels of CO in the blood cause peripheral blood vessels to dilate. This vasodilation results in an increase in cardiac output to maintain arterial pressure in the face of increased peripheral blood flow. Therefore, hypoxia (lack of oxygen) caused by apnea during diving is associated with bradycardia and decreased oxygen levels.

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Figure 12-48 A common drop in heart rate (bradycardia) in underwater ducks depends on intact carotid body innervation. Recordings showing heart rate and brachiocephalic oxygen pressure (PO2) during head immersion, indicated by inward and outward arrows. (A) Controlling a six-week-old duck with nerves intact. (B) The same duck three weeks after carotid artery denervation. [Jones and Purves, 1970a.l

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................................... Effects on Cardiac Output. Conversely, the hypoxia that occurs when animals breathe (for example, at high altitudes) is associated with increases in heart rate and raw meat production. Bleeding Normally, stimulation of arterial and atrial baroreceptors inhibits vasopressin release and sympathetic outflow into the peripheral circulation. Bleeding reduces venous and arterial blood pressure and reduces the firing rate of atrial and arterial baroreceptors. This relieves baroreceptor inhibition of sympathetic outflow, leading to narrowing of arteries (vasoconstriction) and veins (venoconstriction) and increased cardiac output. Peripheral vasoconstriction and increased cardiac output increase arterial blood pressure, while venous constriction maintains venous return to the heart. Reduced baroreceptor inhibition by bleeding also promotes vasopressin release. In addition, renin/angiotensin/aldosterone activity is increased, which is caused by a drop in blood pressure and an associated decrease in renal blood flow. Both vasopressin and aldosterone reduce urine formation, thereby preserving plasma volume. Has a pronounced thirst-stimulating effect and helps restore plasma volume. Reduced blood flow to the kidneys increases the production of erythropoietin by the kidneys, which stimulates the bone marrow to produce red blood cells. So, in the days (weeks) after the bleeding, the lost red blood cells are replaced by increased production. The liver is also stimulated to increase the production of plasma proteins. Returns the blood to its original state by increasing the production of red blood cells and plasma proteins, as well as reducing urine production and increasing the frequency of drinking water.

summarize

Circulatory systems can be divided into two categories: open circuit systems and closed circuit systems. In an open circulatory system, the transmural is depressed and the blood pumped by the heart is emptied into the spaces where the blood directly infiltrates the cells. In a closed circulatory system, blood flows from arteries to veins through capillaries. Transmural pressure is high, and fluid that slowly passes through the capillary walls to the extracellular space is then returned to circulation through the lymphatic system. The heart is a muscular pump that ejects blood into the arterial system. The heart is stimulated by a pacemaker, and the pattern of stimulation of the remaining muscle groups is determined by the type of contact between cells. The connections between muscle fibers in the heart are low-resistance, allowing electrical activity to be transmitted from one cell to the next. The initial phase of each systole is isometric; this is followed by an isotonic phase in which blood is drained into the arterial system. Cardiac output depends on venous flow and, in mammals, on changes in the heart

Performance has to do with changes in heart rate, not stroke volume. Blood flow is generally streamlined (continuous laminar flow), but because of the complex relationship between pressure and flow, Poiseuille's law applies only to flow in smaller arteries and arterioles. The arterial system acts as a pressure accumulator and conduit for blood between the heart and capillaries. Elastic arteries dampen pressure and flow fluctuations caused by heart contraction, while muscular arterioles control the distribution of blood to capillaries. The venous system acts both as a blood conduit between the capillaries and the heart, and as a blood reservoir. In mammals, 50% of the total blood volume is in the veins. Capillaries are the site of material transfer between blood and tissues. Only 30-50% of all capillaries are open to blood flow at any given time, but no capillary stays closed for long because they all open and close continuously. Capillary blood flow is controlled by nerves that innervate the smooth muscles surrounding the arterioles. Changes in the composition of blood and extracellular fluid in the region of the capillary bed lead to narrowing or widening of blood vessels, thereby altering blood flow. Capillary walls are typically an order of magnitude more permeable than other cell layers. The transfer of substances between blood and tissues occurs through or between the endothelial cells that form the walls of capillaries. Endothelial cells contain numerous vesicles that coalesce to form channels for the transport of substances through the cell. In addition, some endothelial cells have specific carrier mechanisms to transfer glucose and amino acids. The size of intercellular spaces varies by capillary bed; brain capillaries are closely connected, whereas hepatic capillaries have large gaps between cells. Arterial pressure is regulated by central control mechanisms to maintain capillary blood flow and can be further adjusted locally to meet specific tissue needs. Arterial baroreceptors monitor blood pressure and reflexively alter cardiac output and peripheral resistance to maintain arterial pressure. Atrial and ventricular mechanoreceptors monitor venous pressure and systolic leads to ensure that cardiac activity correlates with blood inflow from the venous system and outflow from the arterial system. Arterial chemoreceptors respond to changes in blood pH and gas concentrations. All of these sensory receptors feed information to the medullary cardiovascular center, where inputs are integrated to ensure that the circulatory system responds appropriately to the animal's changing needs, such as during exercise. Natriuretic peptides, vasopressin, and the renin-angiotensin-aldosterone system work in conjunction with neural reflexes to maintain blood volume after alcohol consumption or bleeding. In general, stimulation of the sympathetic nerves that innervate vascular smooth muscle results in peripheral vasoconstriction and increased arterial blood pressure, while increases in circulating catecholamines (particularly epinephrine) are associated with decreased peripheral resistance

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Increase in arterial pressure due to concomitant increase in cardiac output. The vascular endothelium releases various compounds (eg, nitric oxide, endothelin, and prostacyclin) that cause local vasoconstriction or vasodilation, thereby regulating blood flow to meet tissue needs. Inflammatory mediators, including histamine and kinins, increase blood flow to sites of tissue injury. Finally, when there is increased aerobic metabolism in tissues, there is a local increase in capillary blood flow, called active hyperemia. This ensures that the most active tissues generally have the highest capillary blood flow.

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Describe the properties of myogenic pacemakers. Describe the transmission of impulses through the mammalian heart. Describe the changes in pressure and flow during a single beat of the mammalian heart. Discuss factors that affect stroke volume. What is the nature and function of the innervation of the mammalian heart? What is the effect of rigid and compliant pericardium on cardiac function? What is the functional significance of partially divided ventricles in some reptiles? Discusses the cyclic changes that occur in mammalian fetuses at birth. Discuss the applicability of Poiseuille's equation to the relationship between pressure and flow in a circuit. What function does the arterial system perform? Describe the factors that determine capillary blood flow. Describe the location of various baroreceptors and/or mechanoreceptors in the mammalian circulatory system and their role in cardiovascular regulation. Comparing and contrasting mammalian cardiovascular responses to inhalation of deoxygenated air and diving. To describe cardiovascular changes associated with exercise in mammals. What is the effect of increased or decreased arterial blood pressure on cardiac function and capillary wall exchange?

16. Discuss the relationship between capillary structure and organ function and compare it to that of different organs of the body. 17. Describe the manner in which substances are transferred between blood and tissues through capillary walls. 18. What are the functions of the venous system? 19. Describe the effect of gravity on blood circulation in land mammals. How do these effects change when the animal is in the water? 20. Define Laplace's Law. This law is discussed in the context of the structure of the cardiovascular system. 21. Discuss the role of the lymphatic system in the circulation of body fluids. Discuss how and why the character looks different in different parts of the body.

Recommended Reading Bundgaard, M. 1980. Transport routes in capillaries: looking for pores. install. Physiology Pastor. 42:325-326. Crone, C. 1980. Ariadne's topic: An autobiographical essay on capillary permeability. capillaries. Resolution 20:133-149. Hessel; N., ed. 1995. Systemic regulatory mechanisms: respiration and circulation. Adu. Compare the environment. Physiol., Vol. 21. Hoar, W.S., D.J. Randall and A.P. Farrell, eds. 1992. Fish Physiology: Volume XIIIA & B. New York: Academic Press. Johansen, K. and W. Burggren, eds. 1985. Cardiovascular Shunt. (Alfred Benzon Seminar 21.) Copenhagen: Munksgaard. Kooyman, G.L. 1989. Various miscellaneous items. Animal Physiology. Volume 23. New York: Springer-Verlag. Kuby, J. 1997. Leukocyte migration and inflammation. In Immunology, 3rd ed. New York: W.H. Freeman. Lewis, D.H., ed. 1979. Lymphatic circulation. Physiological Acta. Scand., Suppl. 463. Radomski, M.W. and E. Salas. 1995. Biological significance of nitric oxide. 4. Interpretation. Congress. Comparative Physiology. biochemistry. physiology. zoo. 68:33-36. Schmidt-Nielsen, K. 1972. How animals work. New York: Cambridge University Press. Van Vilet, B.N. and N.H. West. 1994. Phylogenetic trends in baroreceptor control of arterial blood pressure. physiology. Zool. 67(6):1284-1304.

Just 200 years ago, Antoine Lavoisier showed that animals use oxygen and produce carbon dioxide and heat (Spotlight 13-1). It was later shown that this process occurs at the mitochondrial level (see Chapter 3). Animals obtain oxygen from the environment and use it for cellular respiration. The carbon dioxide produced is eventually released into the environment. For cellular respiration to occur, a constant supply of oxygen must be guaranteed, and waste carbon dioxide must be continuously removed. As carbon dioxide builds up in the body, the pH drops and the animal dies. Although oxygen and carbon dioxide are transported in opposite directions, the two processes have many things in common. When gas transport is impaired, animals die from hypoxia rather than carbon dioxide accumulation, because metabolism requires oxygen and carbon dioxide is a product of aerobic metabolism. Air contains about 21 percent oxygen, but almost no carbon dioxide, and the remainder is mostly nitrogen. Carbon dioxide introduced into the environment by animals is removed by oxygen-producing photosynthetic bacteria, plants and algae. This cycle of O and CO is part of the great interdependence that exists between plants and animals. In this chapter, we examine the transport of 0 and C 0 2 in the blood, and the systems that animals have evolved to facilitate the movement of these two gases between the environment and blood, and between blood and tissues. The focus is on systems found in vertebrates, especially mammals, as these have been most thoroughly studied. Of particular interest are the many systems that transport oxygen between the environment and tissues, including systems that deliver oxygen into the swim bladder of fish, with gradients that can be several atmospheres. This is described at the end of the chapter as an example of one of the many interesting problems of gas transfer in animals.

General considerations Oxygen and carbon dioxide are passively transferred from the environment to body surfaces (ie, skin or specialized airway epithelium) by diffusion. related constitution

The laws governing gas behavior and some terms used in respiratory physiology are discussed in Spotlight 13-2. To facilitate gas transmission rates for a given concentration difference, the airway epithelial surface area should be as large as possible and the diffusion distance should be as small as possible. The C0 requirement and C02 production of an animal increase with mass, but the rate of gas transport across the body surface depends primarily on the surface area. The surface area of a sphere increases as the square of its diameter, while the volume increases as the cube of its diameter. In very small animals, the diffusion distance is small and the surface-to-volume ratio is large. Therefore, diffusion alone is sufficient to transport gases in small animals such as rotifers and protozoa with a diameter of less than 0.5 mm. An increase in size leads to an increase in diffusion distance and a decrease in surface area to volume ratio. Larger animals can maintain a larger surface area to volume ratio by creating specialized regions for gas exchange. In some animals, the entire body surface is involved in gas transport, but in large, active animals, there are specialized respiratory surfaces. This surface consists of a thin layer of cells, the respiratory epithelium, which is 0.5 to 15 µm thick. This surface accounts for most of the entire body surface. In humans, for example, the respiratory area of the lungs is between 50 and 100 square meters and varies with age and alveoli; the remaining body surface area is less than 2 square meters. Gas transfer between the environment and eggs, embryos, many larvae, and even some adult amphibians occurs by simple diffusion. As long as gas transport is by diffusion only, a liquid boundary layer with low oxygen content and high carbon dioxide content will appear. The thickness of this hypoxic (low oxygen) layer increases with decreasing animal size, oxygen uptake, and temperature. In most animals, stagnation of the medium near gas exchange surfaces is avoided by the movement of air or water during respiration. In larger animals, the circulatory system has evolved to carry oxygen and carbon dioxide via an electric current

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Early experiments in gas exchange in animals

was burned. According to this theory, coal contains a large amount of phlogiston, which is released into the air when burned, leaving behind ash. That is, when substances burn, they lose phlogiston, which reduces weight. However, Antoine Lavoisier (1743–1794) discovered that phosphorus gains weight when burned in air, while some other substances gain weight when heated in air but not in vacuum weight. In other words, something in the air is charged with—

Two eminent scientists in the field of gas exchange, Poul Astrup (1915–) and John Severinghaus (1922–), described many of the most important experiments of our time

Summarizes when certain substances are heated. This is the end of the phlogiston theory. Lavoisier called this substance

Learn about gas transfer in animals in her 1986 book "A History of Blood Gases, Acids, and Alkalis." study

Consumed while burning, and needed to keep animals alive. Oxygen, of Greek origin, means "acid-forming".

Gas exchange in animals begins in the 17th century as an extension of the work of Robert Boyle (1627-1691) on the properties of gases

Lavoisier repeated some experiments of Henry Cavendish (1731-1810), who found that combustible gases

Air. He showed that both animals and fire would die in a vacuum,

Metals are added to the acid, which can combine with oxygen to form water. Lavoisier called this gas hydrogen, which comes from the Greek word meaning "hydrogen".

This suggests that something in the air is needed to sustain life and keep the candle burning.

ing "to form water". He also repeats and expands on some of these

Joseph Priestley (1733–1804), who lived near the brewery

Priestley's experiments, found it to be mercury oxide

fascinated by the large amounts of gas produced

When heated with charcoal, solid air (carbon dioxide) is formed. solid air

brewing process. Priestly continued Boyle's experiments in a modified form, heating various chemicals and collecting the resulting gases.

Joseph Black (1728-1799) previously described that he made it by adding acid to chalk.

on water or mercury, and then determine whether mice can survive these gases. He noticed a mouse lived longer

Knowing that exhaled air contains some fixed air, Lavoisier took the next step. He realizes that both burn—

The flame burns brighter in the gas produced when mercury is heated

The mining of coal and animals consumes oxygen and produces heat and carbon dioxide. He then measured oxygen uptake and heat production.

Oxides produce gases more than other chemicals. He also observed that mice lived longer when the containers had plant material. Priestley's observation prompted Benjamin

Sensing in animals to detect heat produced

Franklin points out that the practice of felling trees is nearby

The combustion of coal, although these processes are much slower in animals.

Houses should be stopped because plants can restore the air damaged by animals. So Priestley showed plants, how

is related to the animal's oxygen uptake, and

Some chemicals may generate gas when heated

Lavoisier was also a tax collector. Such people are usually not respected, and this brilliant scientist was no exception.

Keep animals and flames alive. He thought the gas might

Severe punishment: He was guillotined in 1794.

absorb phlogiston, something that is released from a material

Blood between tissues and airway epithelium. Blood flows through an extensive network of capillaries and diffuses in the membrane beneath the respiratory surface, shortening the diffusion distance needed to diffuse the gases contained within. Gases are transported between breathing surfaces and tissues by blood flow in the circulatory system. The gas diffuses between the blood and tissues through the capillary walls. Likewise, to facilitate gas transfer, the diffusion area is large and the diffusion distance between one cell and the next capillary is small. Graham's law states that the rate of diffusion of a substance along a given gradient is inversely proportional to the square root of its molecular weight (or density). Because oxygen and carbon dioxide molecules are similar in size, they diffuse through air at similar rates; they are also used (O2) and produced (C02) by animals in approximately the same amounts. Therefore, it can be assumed that a delivery system that meets the animal's oxygen requirements will also ensure adequate CO2 removal.

- Figure 13-1 schematically shows the components of the gas transport system in many animals, which consists of four basic steps:

1. The breathing movement that ensures a continuous supply of air or water to the breathing surface (eg lungs or gills). 2. Diffusion of O and C 0 2 in airway epithelial cells. 3. Massive transport of gas through the blood. 4. 0 and C 0 2 diffuse through the capillary wall between blood and mitochondria in tissue cells. The capacities at each of these steps are matched because natural selection tends to remove metabolically costly untapped capacities. This adjustment of capabilities in a series of linked events is called co-morbidity. Presumably, the capacity of elements in the chain is determined by the capacity of the rate-limiting step. However, capabilities in chains of events; do not always match and occur homomorphically

Gas Exchange and Acids - B A S E B A L A N C E

519

................................................... ................................. Spread i

diffusion

Figure 13-1 The gas transport system of vertebrates consists of two pumps and two diffusion barriers alternately connected in series between the external environment and tissues. [Adapted from Lahn, 1967.1

air or water

liquid pump

blood pump

Note these seemingly uneconomical design features. One explanation for overcapacity or undercapacity is that a single element can be a link in multiple chains; thus, its capacity may be sufficient for one chain of events but excess for another, which explains Obvious overcapacity. Gas flow rates vary widely among different animals, ranging from 0.08 ml.g-l-h-l in earthworms to 40 ml.g-l hpl in soaring hummingbirds. Both the concentration of aerobic enzymes (eg, pigment oxidase) and the cristae area per mitochondria increased with increasing metabolic rate. Hummingbirds and some insects, however, may have reached an upper limit for oxygen availability in animals. Clearly, the volume and density of mitochondria in muscle cannot increase indefinitely without affecting muscle contractility; that is, there must be some relationship between the energy-providing structures (mitochondria) and energy-consuming structures (myofilaments) . Mitochondria never occupy more than 45% of the total muscle volume, even in mammals, birds and insects. The animal with the highest oxygen uptake. Mitochondrial design must also limit the number of cristae per unit mitochondrial volume, and the ultimate miniaturization depends on the minimum volume required for the enzymes involved in energy production. It appears that hummingbirds, and possibly some other small mammals and some insects, have reached the structural limits that determine maximum oxygen uptake. Insects are usually much smaller than the smallest birds and mammals. Some large insects seem to have been replaced by smaller birds, just like monoplanes replaced biplanes before WW11. The miniaturization of vertebrates may be limited by the nature of their gas delivery systems. Insects have a tracheal system that exchanges gases directly between media and tissues, allowing very small animals to absorb large amounts of oxygen.

Oxygen and Carbon Dioxide in the Blood When considering the movement of oxygen and carbon dioxide in the environment and between cells, we will first discuss how these gases are transported in the blood, rather than starting with the environment or cells. We took this approach because the mechanisms by which oxygen and carbon dioxide are transported in the blood affect their transport between the environment and the blood and between the blood and the tissues. respiratory pigment

As oxygen diffuses through the airway epithelium into the blood, it binds to respiratory pigments, which give the blood its distinctive color. The most famous respiratory pigment, hemoglobin, is red. By binding oxygen, respiratory pigments increase the oxygen levels in the blood. Without respiratory pigments, O levels in the blood would be low. The Bunsen solubility coefficient of oxygen in blood at 37°C is 2.4 ml per 100 ml of blood per atmosphere of oxygen pressure 0. Thus, at normal arterial Po2, the concentration of 0 in physical solution (i.e. not bound to respiratory pigments) in human blood is only 0.3 mL of 0 per 100 mL of blood, or 0.3% by volume. In fact, the total content of human arterial blood in normal arterial blood is 20% (volume). The 70-fold increase in wages is due to the binding of oxygen to hemoglobin. In most animals that use hemoglobin as a respiratory pigment, the 0 content in the physical solution is only a small fraction of the total 0 content in the blood. An exception among vertebrates is the Antarctic icefish. The blood of this fish lacks respiratory pigments, so oxygen levels are low. It compensates for the lack of hemoglobin by increasing blood volume and cardiac output, but its uptake rate is reduced compared to species with hemoglobin from the same habitat. Low temperatures may be a factor in the development of fish that lack hemoglobin. Low temperatures are associated with low metabolic rates in ectotherms, and oxygen, like all gases, exhibits higher solubility at low temperatures. Airway pigments are complexes of proteins and metal ions, each with a unique color. this

520

integrated physiological system

................................................... .................... Spotlight 13-2

water, but when the temperature drops, the water condenses, and this condensation also reduces the volume of the gas if you exhale it

natural gas law

Air pressure is 760mm~Gand, water vapor pressure

More than 300 years ago, Robert Boyle discovered that the product of pressure times volume is constant for a given number of gas molecules at a given temperature. Gay-Lussac's law states that the pressure or volume of one gas is proportional to the absolute value of the temperature at which the other gas is kept constant

47 mm Hg at 37" and 17 mm Hg at 20 °C, then convert the measured gas volume of 500 ml at 20 °C to the BTPS volume explored as follows

500mlX

(760 - 17) (760 - 47)

(273 (273

+ 37) + 20)

551 ml

Taken together, these laws are expressed by the equation of state: Under the above conditions, the gas volume is 551 ml

For gas:

Due to the drop in temperature of the gas and the condensation of water, the volume in the lungs is reduced to 500 ml after exhalation. where P is pressure, Vis volume, n is the number of gas molecules, R is the universal gas constant (0.08205 L. atm. K-I. mot-' or 1.987 cal. K-' . mol-I) and

Dalton's law of partial pressure states that the partial pressure of each gas in a mixture is independent of the other gases present, so that the total pressure is equal to the sum of the partial pressures.

Ki is the absolute temperature. For accurate use, the equation should be

Protect all existing gases. The partial pressure of the gas in A

It can be modified using the van der Waals constant.

The composition of a mixture depends on the number of molecules present in the mixture

or 8.314 times

lo7ergs. OK - '.mol-',

The equation of state for a gas states that the volumes are equal

A given volume at a given temperature. Typically, these are oxygen accounts

Composed of different gases at the same temperature and pressure

20.94% of all gas molecules in dry air; so if one were to-

The number of molecules is equal (Avogadro's law). At 0°C and 760 mm Hg, one mole of gas occupies approximately 22.414 liters. perhaps-

With a total pressure of 760 mm Hg, the partial pressure of oxygen Po* is 760 x 0.2094 = 159 mm Hg, but the air usually contains moisture.

The reason is that the number of molecules per unit volume

Three vapors contribute to the total pressure. When the air is 50%

Pressure and temperature should always be the condition

Saturated with water vapor at 22°C, this is the water vapor pressure

It is given together with the gas quantity. gas volume in physiology

18 mm Hg. When the total pressure is 760, it is the partial pressure of oxygen

Usually given at body temperature, in the atmosphere

gen is (760 - 18) x 0.2094 = 155 mm Hg. if part

pressure and saturated water vapor (BTPS); at ambient temperature and pressure saturated with water vapor (ATPS); or

If the pressure of CO in a gas mixture is 7.6 mm Hg and the total pressure is 760 mm Hg, then 1% of the molecules in air are CO.

Standard temperature and pressure (O°C, 760 mm Hg) and dry,

Gas is soluble in liquid. The amount of gas dissolved at a given temperature is proportional to the partial pressure.

or Zero Water Vapor Pressure (STPD). The gas volume measured under specific conditions (e.g.

Make sure the gas is in the gas phase (Henry's Law). Lots of

ATPS) can convert one gas to another (eg BTPS) using the equation of state. For example, is the volume of outgoing air

A gas in solution is equal to a P where Pi is the partial pressure of the gas and a is the independent Bunsen solubility coefficient.

Radiation from the lungs of mammals with a body temperature of 37 °C (273 + 37 = 310 K) is usually measured at room temperature.

depends on P. The Bunsen solubility coefficient varies with the gas, temperature and type of liquid involved, but different

20 °C (273 20 = 293 K). The drop in temperature reduces the amount of exhaled gas. gas turns into water

is constant for any gas in a given liquid at constant temperature. The Bunsen solubility coefficient of oxygen decreases with increasing ionic strength and temperature of water.

Saturated with water vapor. The water vapor pressure at 100% saturation varies with temperature. Exhaled air saturation

The color of respiratory pigments changes with their O content. Thus, hemoglobin is bright red when saturated with O2 and turns dark maroon when degraded. Vertebrate hemoglobin (excluding that of ring-stouted animals) has a molecular weight of 68,000 and contains four iron-containing porphyrin prosthetic groups, called heme, linked to globulin, a tetrameric protein (Figure 13-2A ). The globin molecule consists of two dimers, alp and alp, each forming a closely related unit. The two dimers are more loosely connected by a salt bridge, except that the two p-strands do not touch. The supply of oxygen alters these bridges, causing a conformational change in the hemoglobin molecule. Hemoglobin can dissociate

Divided into four subunits of approximately equal weight, each subunit contains a polypeptide chain and a heme group. Myoglobin, a respiratory pigment that stores O in vertebrate muscle, corresponds to a hemoglobin subunit and shares considerable sequence homology with the hemoglobin alpha chain. In the hemoglobin molecule, ferric iron (Fe2+) binds to the porphyrin ring of heme and forms coordination bonds with four pyrrole nitrogen atoms (Figure 13-2B). The remaining two coordination bonds are used to attach the heme group to the O molecule and the imidazole ring of the histidine residue in globin (Fig. 13-2C). When O2 binds, the molecule is called

OOC Ham

L-Histidine (His)

red lotus-

+H,N-C-H

Figure 13-2 Hemoglobin is the major respiratory pigment of vertebrates and consists of four globin subunits, each containing one heme molecule. (A) Schematic diagram of the hemoglobin molecule showing the relationship of the a-chain and p-chain. Two of the four heme units (red) are visible at the fold of the polypeptide chain. (B) The heme structure formed by the combination of iron ions (Fez+) and protoporphyrin IX. (C) Schematic representation of the heme in the pocket formed by the globulin molecule. The side chain of the histidine (His) residue in globin acts as an additional ligand for the iron atom in heme. When oxygen binds, it displaces the remaining H,O ligands. [Adapted from McGilvery, 1970.1

As oxyhemoglobin; if 0 is missing, it is called deoxyhemoglobin. By combining 0, with hemoglobin to form oxyhemoglobin, ferrous iron is not oxidized to ferric iron. Oxidation of ferrous iron in hemoglobin to the ferric state produces methemoglobin, which is not bound to O and therefore has no function. Although methemoglobin formation occurs normally, red blood cells contain methemoglobin reductase, which reduces methemoglobin to its functional iron form. Certain compounds (eg, nitrite and chlorate) oxidize hemoglobin or inactivate methemoglobin reductase, increasing methemoglobin levels and impairing oxygen delivery. Hemoglobin has an affinity for carbon monoxide approximately 200 times greater than its affinity for oxygen. As a result, even at very low partial pressures of carbon monoxide, carbon monoxide displaces oxygen and saturates hemoglobin, resulting in a significant reduction in oxygen delivery to tissues. Hemoglobin saturated with carbon monoxide is called carboxyhemoglobin. The effect of this saturation on oxidative metabolism is similar to hypoxia, which is why carbon monoxide produced by cars or improperly fueled coal or wood stoves is so toxic. Even values measured in urban traffic impair brain function due to local hypoxia. Hemoglobin is present in many groups of invertebrates, but other groups of invertebrates also have other respiratory pigments, including hemoglobin (Priapulida, brachiopods, annelids), chlorocruorin (annelids), and hemocyanin (molluscs, arthropods) . Many invertebrates do not have respiratory pigments. Hemocyanin is a large, copper-containing respiratory pigment that has many properties similar to hemoglobin: it binds oxygen when the partial pressure is high and releases it when the partial pressure is low. Hemocyanin binds oxygen in a ratio of 1 mole of 0 to about 75,000 g of respiratory pigment. In comparison, 4 moles of O2 bind 68,000 g of hemoglobin at full saturation. Unlike hemoglobin, hemocyanin is not packaged in cells and is not associated with high levels of carbonic anhydrase in the blood. In its oxygenated form, it is light blue; in its anaerobic state, it is colorless. Oxygen Transport in Blood Each hemoglobin molecule can bind four oxygen molecules, and each heme can bind one oxygen molecule. The extent to which O binds to hemoglobin varies with the partial pressure of gas Po2. When all the positions of the hemoglobin molecule are occupied by 0 2 , the blood is 100% saturated, and the oxygen content of the blood is equal to its oxygen capacity. One millimole of heme can bind one millimole of O, equivalent to 22.4 ml of O, in volume. Human blood contains about 0.9 millimoles of hemoglobin per 100 milliliters of blood. Therefore, the oxygen capacity is 0.9 x 22.4 = 20.2% by volume. The oxygen content in a unit volume of blood includes the oxygen in the physical solution and the oxygen combined with hemoglobin, but in most cases, the oxygen in the physical solution only accounts for a small part of the total oxygen content.

522

Integrated Physiological Systems

...................................................

Since the oxygen content of the blood is directly proportional to its hemoglobin concentration, the oxygen content is usually expressed as a percentage of the oxygen content, ie H. As a percent saturation. This makes it possible to compare the oxygen content in blood with different hemoglobin levels. The oxygen dissociation curve describes the relationship between percent saturation and partial pressure of oxygen. The oxygen dissociation curves of myoglobin and lamprey hemoglobin are hyperbolic, whereas those of other vertebrate hemoglobins are S-shaped (Fig. 13-3). This difference occurs because myoglobin and lamprey hemoglobin have only one heme group, while other hemoglobins have four. The S-shaped dissociation curve exhibited by hemoglobin with multiple heme groups is the result of subunit cooperation; that is, oxidation of the first heme group promotes oxidation of subsequent heme groups. The steeper part of the curve corresponds to oxygen levels where at least one heme group has been occupied by an oxygen molecule, increasing the affinity of the remaining heme group for oxygen. When a hemoglobin molecule is oxidized, it undergoes a conformational change from a tense (T) state to a relaxed (R) state. Oxygen enrichment is associated with changes in the tertiary structure near the heme, which weakens or disrupts the link between a,PI and a,p, dimers, resulting in a dramatic change in the quaternary structure from the T state to the R state. These conformational changes also lead to changes in the dissociation of acidic side chains, thus releasing protons (H+ ions) when hemoglobin is oxidized. An important property of -I airway pigments is that they are reversibly associated with 0 in some regions

large

Figure 13-3 Hemoglobin with multiple heme groups exhibits a characteristic oxygen dissociation curve, while myoglobin with only one heme group exhibits a hyperbolic dissociation curve. Lamprey hemoglobin, which has a single heme group, exhibits a dissociation curve similar to that of myoglobin. P is the partial pressure at which the respiratory pigment is 50% saturated with oxygen, a measure of its oxygen affinity.

Animals are usually under stress. When the hips are lower, only a small amount of O binds to the respiratory pigments. However, at high Po>, a large number of 0s are bound. Due to this property, respiratory pigments can act as oxygen carriers, charging at the respiratory surface (high Po> regions) and discharging at the tissue (low Po> regions). In some animals, the primary role of respiratory pigments may be to act as an oxygen sink, releasing O to tissues only when O is relatively unavailable. In many resting animals, venous blood entering the lungs or gills is approximately 70% oxygenated; that is, most of the oxygen bound to hemoglobin is not removed as it is transported through the tissues. When tissue oxygen demand increases during exercise, this venous oxygen pool is pumped and venous saturation drops to 30% or less. Hemoglobin with a high oxygen affinity is saturated at low oxygen partial pressures, whereas hemoglobin with a low oxygen affinity is fully saturated only at relatively high oxygen partial pressures. Affinity is expressed as P, the partial pressure of oxygen at which the hemoglobin reaches 50% oxygen saturation; the lower the P, the higher the oxygen affinity. As the curve in Figure 13-3 shows, myoglobin has a much higher affinity for oxygen than hemoglobin. Differences in oxygen affinity between hemoglobins are related to differences in the protein globulins and not to differences in the heme moieties. Each a and p chain of the globin molecule consists of 141 to 147 amino acids, depending on the chain and hemoglobin. The amino acid sequences of the alpha and beta chains of different hemoglobins share many similarities, but also some differences. While most amino acid substitutions are neutral, some amino acid substitutions have a pronounced effect on function. For example, a genetic defect that causes valine to replace glutamic acid at position 6 of the p-chain causes human hemoglobin to form large aggregates that twist red blood cells into a sickle shape, resulting in sickle cell anemia. Because these sickle cells are unable to pass through small blood vessels, the oxygen supply to the tissue is compromised. Individuals with normal and sickle cell hemoglobin were only mildly debilitated but showed greater resistance to malaria, ensuring the persistence of the sickle cell gene in the population. Certain amino acids in globin bind different ligands, and exchanging these residues results in changes in the oxygen affinity of hemoglobin. The rate of oxygen transport into and out of the blood is proportional to the difference in Po across epithelial cells. Hemoglobin with high oxygen affinity facilitates the entry of 0 into the blood from the environment as 0 binds to hemoglobin at low Po; IE. H. 0 entering the blood immediately binds to hemoglobin, thus removing 0 from solution and keeping Po2 low. Thus, a large PO2 differential is maintained throughout the airway epithelium - and thus the rate at which oxygen enters the blood - until the hemoglobin is fully saturated. Only then will the Po2 in the blood rise. Hemoglobin with high oxygen affinity

Gas exchange and acid-base balance

523

................................... releases O2 into tissues only when the hips are up Low. In contrast, hemoglobin, which has a low affinity for oxygen, helps release oxygen into tissues, thereby maintaining a large polarity difference between blood and tissues and a high rate of oxygen transfer into tissues. Thus, hemoglobin with a high affinity for oxygen facilitates the uptake of O2 by the blood, whereas hemoglobin with a low affinity for oxygen facilitates the transport of O2 to the tissues. Therefore, from a functional point of view, hemoglobin should have a low O2 affinity in tissues and a high O2 affinity at respiratory surfaces. In this context, it is important to note that the oxygen affinity of hemoglobin is influenced by changes in the chemical and physical factors in the blood that favor oxygen incorporation in airway epithelial cells and oxygen release in tissues. The hemoglobin-oxygen affinity is unstable and depends on the conditions inside the red blood cell. For example, the hemoglobin-oxygen F f i t y is reduced by:

Hemocyte organophosphates vary by species. For example, mammalian red blood cells contain high levels of 2,3-diphosphoglycerate (DPG); in fact, hemoglobin and DPG are nearly equimolar in human red blood cells. DPG binds to specific amino acid residues in the p-chain of deoxyhemoglobin, but DPG binding decreases with increasing pH. An increase in DPG levels is accompanied by a decrease in blood zero or hemoglobin concentration, an increase in pH, or both. Low levels of O2 in the blood may be the result of increasing to higher levels

artery

Elevated temperature Binding of organophosphate ligands including 2,3-diphosphoglycerate (DPG), ATP or GTP pH decrease (increase in H+ concentration) CO increase. When the hemoglobin molecule is in a T-deficient state, it has a higher affinity for the ligand. An increase in H+ concentration (decrease in pH) results in a decrease in the oxygen affinity of hemoglobin, a phenomenon known as the Bohr effect or Bohr shift (Figure 13-4). Carbon dioxide reacts with water to form carbonic acid, and reacts with -NH2 groups on plasma proteins and hemoglobin to form carbamido compounds. Thus, an increase in PCO2 reduces the oxygen affinity of hemoglobin in two ways: by lowering blood pH (Bohr effect) and by promoting the direct binding of CO to hemoglobin to form carbamido compounds. Therefore, when C 0 2 enters the blood through tissues, it facilitates the excretion of O from hemoglobin, and when CO leaves the blood at the lungs or gills, it facilitates the uptake of O by the blood. Compared to hemoglobin, the oxygen dissociation curve of myoglobin is relatively insensitive to changes in pH. Hemocyanins from Dungeness crabs, Cancer crabs, and some other invertebrates show a Bohr shift similar to that of hemoglobin (Figure 13-5). However, hemocyanins from several species of snails and horseshoe crabs showed stronger oxygen affinity with decreasing pH. This phenomenon, known as the reverse Bohr effect, may boost oxygen uptake in conditions where oxygen supply is insufficient when the blood pH of these animals drops for prolonged periods of time. As noted above, the binding of organophosphate compounds to hemoglobin reduces the oxygen affinity of most vertebrate hemoglobins, with the exception of ring-stouted, crocodile and ruminant hemoglobins. Advantage cherry red

“

Post-90s

Po, (mm Hg) Figure 13-4 The oxygen affinity of hemoglobin decreases with decreasing pH. Changes in blood PCO, which affects blood pH, indirectly affect hemoglobin-oxygen affinity due to this phenomenon known as the Bohr effect. Experimental blood oxygen dissociation curves for humans at three pH values are shown. Give the Po value for mixed venous and arterial blood. [Based on Bartels, 1971]

Post-90s

Po, (mm Hg) Figure 13-5 Some hemocyanins, such as hemoglobin, exhibit a Bohr shift. The blood oxygen dissociation curve for Magister cancer shown here shows that the hemocyanin of this cancer exhibits a Bohr shift. [Unpublished data provided by D.G. McDonald]

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Physiological system integration

.....................................

Altitude, since both air pressure and partial pressure in air from 0 decrease with altitude. The increase in DPG in humans in response to high altitude is complete within 24 hours, with a half-life of approximately 6 hours. At an altitude of 3000 meters, the concentration of DPG in red blood cells is 10% higher than at sea level. Low O2 levels at high altitudes cause O2 levels in the blood to drop, which stimulates breathing. The resulting increase in ventilation (i.e., the exchange of air between the lungs and the surrounding air) lowers blood carbon dioxide levels and increases blood pH, which increases hemoglobin's affinity for oxygen. The increase in DPG with altitude counteracts the effect of the decrease in CO levels and keeps the hemoglobin-oxygen affinity close to sea level. Other phosphorylated compounds are present in higher concentrations than DPG in the erythrocytes of some vertebrates and thus play a more important role than DPG in regulating the oxygen affinity of hemoglobin. In most fish, ATP and/or GTP serve this function, while in birds, inositol pentaphosphate (InsP, ) is the major erythrocyte organophosphate. In the Amazonian fish Arapazrna gzgas, ATP is the predominant erythrocyte organophosphate in juvenile aquatic forms, whereas InsP predominates in obligate air-breathing adults. Phosphorylated compounds in red blood cells not only affect the oxygen affinity of hemoglobin, but also enhance the strength of the Bohr effect and affect subunit interactions. It appears that the functional importance of increased DPG levels in mammals is the maintenance of hemoglobin-oxygen affinity under hypoxic (low oxygen) conditions, such as high altitude. Conversely, hypoxia reduces red blood cell organophosphate levels in fish. In these animals, however, hypoxia is usually associated with a drop in blood pH (acidosis) rather than the elevated pH (alkaliosis) seen in high-altitude mammals. The effect of reducing fish ATP (or GTP) is to counteract the effects of this hypoxia-related acidosis, thereby maintaining blood oxygen affinity. Thus, functionally, the effects of altering erythrocyte organophosphate levels are similar in fish and mammals; in either case, the result is maintenance of hemoglobin-oxygen affinity. The reaction of oxygen binding to hemoglobin is rapid and generally does not limit the rate of oxygen transfer. However, the rate at which oxygen binds to hemoglobin also depends on the hemoglobin concentration. The higher the hemoglobin concentration, the more oxygen can be combined per unit time. The more oxygen bound per unit time, the longer the large diffusion gradient of oxygen across the airway epithelium and thus the higher the rate of oxygen transfer. The presence of respiratory pigments also increases oxygen transport through the blood, as oxygenated pigments diffuse along concentration gradients with oxygen. This means that there is a gradient in the same direction for oxygen and oxygenated pigments

By solution; the gradient for anoxic pigments is in the opposite direction to the gradient for oxygen and oxygenated pigments. Therefore, oxygen-containing pigments diffuse in the same direction as oxygen, while oxygen-deficient pigments diffuse in the opposite direction. For example, pigments like hemoglobin facilitate the mixing of gases in the blood, while myoglobin can play a similar role in tissues.

In some fish, cephalopods and crustaceans, an increase in CO2 or a decrease in pH not only causes a decrease in the oxygen affinity of hemoglobin but also a decrease in oxygen capacity, known as the root effect or root shift (Fig. 13 )-6). For those hemoglobins that show root shift, low pH reduces oxygen binding to the hemoglobin, so even at high Po2 only some binding sites are oxidized; that is, 100% saturation is never reached. Increased temperatures can exacerbate oxygen supply problems in ectothermic aquatic animals such as fish. An increase in temperature not only reduces the solubility of oxygen in water, but also reduces the oxygen affinity of hemoglobin, making the transfer of oxygen between water and blood more difficult. Unfortunately, this decrease in affinity occurs at a time of increased tissue oxygen demand, which is also a consequence of increased temperature. It is generally believed that a specific hemoglobin evolved to meet the unique needs of animals for gas transfer and H+ buffering. Differences in the properties of hemoglobin are due to changes in the amino acid sequence of the globin partial peptide chain

Nur plasma

Figure 13-6: Lowering pH reduces blood oxygen capacity in some teleost fish hemoglobin (root effect). Oxygen balance curves for the blood of these eels were obtained at 14°C, with blood pH ranging from 6.99 to 8.20. The figure below depicts the plasma content [from Steen, 1963.1].

Gas Exchange and Acids - B A S E B A L A N C E

525

.............................................. Hemoglobin is the same molecule as all hemoglobins. Hemoglobin levels not only vary between species, but also change during development. In humans, for example, several genes encode plike globin chains, the relative expression of which differs prenatally and postnatally (Fig. 13-7). Human fetal hemoglobin contains gamma chains instead of adult beta chains with an affinity higher than 0.0 for adult hemoglobin. The higher the 0, the affinity of fetal hemoglobin improves oxygen transfer from mother to fetus. After birth, when the proportion of fetal hemoglobin decreases and adult hemoglobin increases, the oxygen affinity of the blood decreases (Figure 13-8). Other mammals show similar differences between fetal and adult hemoglobin. It is important to remember that hemoglobin is contained in the red blood cells of most animals, but blood parameter values are usually related to the conditions in the plasma rather than the red blood cells. Differences in these parameters exist inside and outside cells, including red blood cells. For example, mammalian arterial blood typically has a pH of 7.4 at 37°C. This is the pH of arterial plasma; the pH inside red blood cells is lower, around 7.2 at 37°C. transport of carbon dioxide in the blood

Carbon dioxide diffuses from tissues into the blood, where it is transported, and diffuses through respiratory surfaces into the environment. Carbon dioxide reacts with water to form carbonic acid, a weak acid that breaks down into bicarbonate and carbonate ions: CO,

+hydrogen

----TH,CO,

H + + C0;-

HC0,-

1 me

H + + HC0,-

Fetal hemoglobin (y-chain)

(p chain)

: embryo : hemoglobin : (€ chain)

A，

(&chain)

back,

Figure 13-8 In humans, blood oxygen affinity decreases around the first trimester of life as adult hemoglobin replaces fetal hemoglobin (see Figure 13-7). These oxygen dissociation curves were measured at pH 7.40. [According to Bartels, 1971.1

Carbon dioxide also reacts with hydroxyl ions to form bicarbonate:

=H+ + OHCO, + OH- =HCO, H,O

The ratio of CO, , HCO, - and CO in the solution: depends on the pH value, temperature and ionic strength of the solution. In mammalian blood, at pH 7.4, the ratio of CO to H2CO3 is approximately 1000:1 and the ratio of CO to bicarbonate ions is approximately 1:20. Therefore, bicarbonate is usually the predominant form of CO in blood pH. Carbonate levels in birds and mammals are usually negligible; however, in ectothermic animals, due to low temperature and high blood pH, carbonate levels can be as high as 5% of total blood CO2, but bicarbonate remains It is the main form of CO2. Carbon dioxide also reacts with -NH2 groups on proteins, especially with -NH2 groups on hemoglobin to form carbamido compounds. protein-NH,

Pregnancy time (months)

the birth

age (months)

Figure 13-7 Hemoglobin changes during human development. The relative amounts of the different hemoglobin chains synthesized by the fetus vary throughout pregnancy. Fetal hemoglobin, which contains two a-chains and two y-chains, has a higher affinity for oxygen than adult hemoglobin [adapted from Young, 1971].

(mmHg)

+ carbon monoxide,

H+ + protein--NHCOO-

The extent of urethane formation depends on the number of available terminal NH groups and increases with blood pH and CO levels. The terminal NH groups of the alpha and beta chains of mammalian, avian and reptile hemoglobin can be used to form carbamido groups. However, the terminal NH groups of the a-chains of fish and amphibian hemoglobins are acetylated and thus cannot be used for carbamate formation. because

Organophosphates bind to some of the same amino acids involved in carbamate formation. Organophosphate bonds reduce carbamate formation. However, high pH reduces organophosphate incorporation, thereby increasing carbamate formation by providing more NH groups. Since fish erythrocytes typically contain high levels of organophosphates and acetylated alpha-chains, fish are less dependent on carbamate formation for CO2 transport than mammals. The sum of all forms of CO in the blood—that is, the molecules C 0 2 , H, CO, ,HCO,-, COj2-, and carbamyl compounds—is called the total CO content in the blood. The CO content as a function of PCO2 content can be graphically described in the form of a CO dissociation curve (Figure 13-9). As Pco2 levels increase, the greatest changes occur in blood bicarbonate levels. Bicarbonate formation is of course pH dependent. The relationship between plasma HCO, concentration, and plasma pH at the three PCo2 values is shown in Figure 13-10. The drop in pH at constant PCo2 is related to the drop in bicarbonate. Red blood cells have a lower pH than plasma, but PCo2 maintains equilibrium on the cell membrane. Therefore, bicarbonate levels in red blood cells are lower than those in blood plasma. Red blood cells typically make up less than 50% of blood volume (ie, plasma volume is greater than red blood cell volume), and plasma has a higher bicarbonate concentration than red blood cells; thus, most of the bicarbonate in blood is present in plasma.

deoxygenated blood,

Figure 13-9 Whole blood CO increases with Pco, but only molecular CO volume increases linearly. Note that in glven Pco, oxygenated blood contains less C 0 2 than deoxygenated blood (Haldane effect). A and V refer to arterial and venous blood levels, respectively.

HCO, - or reacts with -NH, hemoglobin groups and other proteins to form carbamido compounds. The opposite process occurs when CO is eliminated from the blood. The largest changes occur in HCO,- concentrations; changes in CO and carbamate compounds typically account for less than 20% of total CO2 excretion. The reaction of CO and OH to form HCO is slow, taking seconds without catalysis. But in the presence of carbonic anhydrase, the reaction reaches equilibrium in less than a second. Although the total CO content of plasma is higher than that of erythrocytes, most of the CO entering and leaving plasma is carried by erythrocytes because carbonic anhydrase is present in erythrocytes but not in plasma. Thus, the formation of HCO, ions in tissues, and CO in the lungs occurs primarily in red blood cells; a

Transport of gases into and out of the blood

Concentrations of CO, HCO, and carbamate compounds change when CO is added to blood in tissues and removed at respiratory surfaces. Carbon dioxide enters and leaves the blood as molecular CO rather than bicarbonate ions because CO molecules diffuse through the membrane much faster than HCO ions. In tissues, CO enters the bloodstream where it is hydrated

40 -

PCOp

48,0 40 0 35,0

-zm

at 30 °C

Figure 13-10 Human plasma pH, B-carbonate concentration, and PCo are correlated, usually within a narrow range (indicated by the red box) at specific values. As indicated by the full body buffer line [Adapted from Davenport,

(mmHg)

-28

19741

%.-

-23

Mimi

HC0,-

20-

Full body cushioning l ~ n e I

Ha ha

710

7.2

7,4 Plasma pH

7.6

7.8

Gas Exchange and Acids - B A S E B A L A N C E

527

................................................... ................................... HCO, ions and CO formed are then transported or transported from the plasma into the plasma. After entering the blood from the tissue, CO diffuses into the erythrocytes, and HCO is rapidly formed in the presence of carbonic anhydrase, - (Fig. 13-11A). When HCO,- levels in red blood cells increase, HCO, ions migrate from the cells into the plasma. The electrical balance within the cell is maintained by anion exchange; as HCO,- ions leave the red blood cells, there is a net influx of C1 ions from the plasma into the cells, a process known as chloride transfer. Unlike many other cells, erythrocytes are highly permeable to both C1 and HCO because the cell membrane contains a high concentration of a specialized anion carrier protein, the band III protein. This transporter binds C1 and HCO and transports them across the red blood cell membrane in opposite directions. Anion exchange is passive, relying on a concentration gradient to drive the process.

police officer

organize

This can occur in either direction, with bicarbonate flowing from the RBC into the tissue and into the RBC on the respiratory surface (Fig. 13-11B). Band I11 proteins are present in all vertebrate erythrocytes except lampreys and hagfish. In these animals, bicarbonate is retained in the erythrocytes and there is no anion transfer between erythrocytes and plasma. A second reason why most of the CO entering or leaving the blood passes through red blood cells is that oxidation of hemoglobin (Hb) leads to the release of H+, which acidifies the cell interior; conversely, deoxygenation causes H+ to bind to Hb. Thus, the binding of O2 to Hb at the respiratory surface promotes the formation of CO, while the release of O2 from Hb in tissues promotes the formation of HCO, (Fig. 13-12). Due to proton binding to and

Ton

capillary wall

(slowly) + H2O = H2CO3L=; HCO, + H+

carbon dioxide

Ton

Figure 13-11 Most of the carbon dioxide that enters the blood and leaves the blood in the lungs passes through the red blood cells (A). Carbon produced in tissues rapidly forms carbon dioxide (HCO,-) in red blood cells. Because the hydration reaction is catalyzed by carbonic anhydrase present in the cells. The B-carbon leaves the red blood cell in exchange for chloride ions, and the excess proton is bound by deoxygenated hemoglobin (Hb) (B). These responses were reversed in the lungs. Oxygen entering red blood cells displaces protons in Hb, while carbon d~ox~d enters plasma. Carbonic anhydrase (induced by solid particles). In the lung membranes, endothelial cells convert part of the plasma carbonate to carbon. Enhanced movement of carbon content across the respiratory surface by diffusion of carbonate and its conversion back to carbon dioxide at the outer surface, a process called accelerated diffusion

528

integrated physiological system

................................... Kapillares Endothel

airway epithelium

oh-

lco2+>

Figure 13-12 Changes in pH accompanying tissue and airway surface blood Pco changes are balanced by the binding and release of H+ ions from deoxygenated and oxygenated blood. For example, CO transfer to blood in tissues results in a drop in pH due to bicarbonate formation; simultaneous deoxygenation of hemoglobin releases proton acceptors that bind excess H+ ions. The opposite response occurs on airway epithelial cells.

When hemoglobin is deoxygenated or oxygenated, protons are released from hemoglobin. For example, when PC increases in tissue, the subsequent formation of HCO or carbamido compounds releases H+ ions. At the same time, the release of oxygen produces deoxygenated hemoglobin that binds protons. However, as deoxygenation progresses, more proton acceptors appear on the hemoglobin molecule. In fact, saturated hemoglobin is completely deoxygenated, releasing 1 mole of O, resulting in the binding of 0.7 moles of H+ ions. Thus, if the ratio of CO production to 0 consumption (known as the respiratory quotient) is 0.7, CO transport can occur without changing blood pH. (As discussed in Chapter 16, the respiratory quotient depends on the type of diet.) Even with a respiratory quotient of 1, the extra 0.3 mol H+ is buffered by blood proteins including hemoglobin, and blood pH changes only slightly. For a given PCo1, deoxyhemoglobin binds more protons, promotes the formation of HCO, and reacts with CO to form carbamoylhemoglobin more readily than oxyhemoglobin. Therefore, at a given Pco, deoxygenated blood has a higher total CO content than oxygenated blood (see Figure 13-9). Thus, deoxygenation of hemoglobin in tissues reduces changes in PCol and pH when CO enters the blood; this is the so-called Haldane effect. The lungs have two mechanisms that they use to move carbon dioxide from the blood. As mentioned previously, carbonic anhydrase is absent in plasma, so the interconversion of CO and HCO in plasma occurs at a slow, uncatalyzed rate. (Any carbonic anhydrase released from the breakdown of red blood cells is excreted through the kidneys.) However, in the endothelial cells of the pulmonary capillaries, carbonic anhydrase becomes embedded on the cell surface and becomes accessible to plasma CO and HCO3-. Therefore the conversion of HCO is also

CO is present in plasma at a catalytic rate as blood permeates the pulmonary capillaries (see Figure 13-11B). In addition, oxygenation of hemoglobin acidifies red blood cells in the pulmonary capillaries, facilitating the conversion of HCO2 to CO, which then diffuses into the plasma and across the lung epithelium. A drop in bicarbonate levels in red blood cells results in an influx of HCO- ions in the plasma, accompanied by an outward movement of C1- ions. The relative amount of HCO converted to CO2 in the erythrocytes and plasma of blood perfused by the airway epithelium depends on the level of proton production associated with hemoglobin oxygenation and the level of carbonic anhydrase activity in the affected airway epithelial cell wall. For example, in teleosts, plasma flowing through the gills is not exposed to carbonic anhydrase. In these animals, CO excretion occurs predominantly through erythrocytes and is closely linked to oxygen uptake through hemoglobin oxidation to generate protons. Carbonic anhydrase activity has also been found on the endothelial surface of many capillary beds throughout the body, including capillary beds in skeletal muscle. Carbonic anhydrase-catalyzed formation of HCO can occur in these capillaries in the absence of red blood cells. Therefore, some of the CO that enters the skeletal muscle blood does not pass through the red blood cells. Carbonic anhydrase also facilitates carbon dioxide transfer, termed facilitated diffusion of CO (see Figure 13-11B), due to the simultaneous diffusion of bicarbonate and protons across the epithelium, the latter also enhanced by release from the buffer. In this facilitated-diffusion process, carbonic anhydrase catalyzes the rapid interconversion of CO and HCO, with CO entering and leaving the cell. There are at least seven forms of carbonic anhydrase, designated CA-I through CA-VII. They both have a similar structure and catalyze the interconversion of carbon dioxide and bicarbonate. Carbonic anhydrase I (CA-I) and carbonic anhydrase II (CA-11), found in human red blood cells, have a molecular weight of approximately 29,000 and contain approximately 260 amino acid residues. CA-11 is an extremely efficient catalyst for the carbon dioxide-bicarbonate hydration-dehydration reaction and is present in a variety of tissues including the brain, eyes, kidneys, cartilage, liver, lungs, pancreas, gastric mucosa, skeletal muscle and Wall pituitary gland and erythrocytes. This form is involved in a variety of functions and increases the supply of bicarbonate and/or protons to a range of cellular and metabolic processes. Some people have hereditary CA-I1 deficiency, and the pattern of inheritance is autosomal recessive. Although CA-11 was undetectable in these individuals, they had normal levels of CA-I in their red blood cells. In addition to impairing the gas exchange process, CA-I1 deficiency can lead to a number of other symptoms, including metabolic acidosis, renal tubular acidosis, and sometimes intellectual disability. Since CA-I1 is also involved in the production of protons, which are required for osteoclastic bone resorption, its absence leads to osteoporosis, which is often associated with multiple fractures. wide range of associated symptoms

T.'

Congenital CA-I1 deficiency reflects a broad function of CA-I1 in increasing proton and/or bicarbonate release. The rate of movement of CO and O into and out of red blood cells depends on the diffusion distance and diffusion coefficient of these substances through the cell. As expected, differences in diffusion, as well as the rate of oxygenation of RBCs, are related to cell size, which varies widely among vertebrates. For example, the red blood cells of the amphibian Necturus are 600 times larger than those of goats. Previous studies have shown that microerythrocytes oxidize more rapidly than macrocytes in vitro (Fig. 13-13), but this finding may have little relevance in vivo. Recent experiments using whole blood thin-layer techniques similar to the in vivo situation have shown that oxygen uptake rates are independent of cell size. An explanation for this may lie in the flattened shape of the red blood cells. If the large planes of cells face the respiratory medium as they migrate individually through the respiratory capillaries, their diffusion distances will be very similar even though the cells are very different in volume. Therefore, in vitro results may not be extrapolated to in vivo situations. It is believed that CO excretion is limited by the rate of bicarbonate-chloride exchange across the erythrocyte membrane. The surface area to volume ratio of red blood cells and the transport capacity of band I11 protein-mediated bicarbonate-chloride exchange may be important in determining the rate of carbon dioxide excretion. To understand the relevance of these parameters, we compared red blood cells from trout and humans (Table 13-1). Trout erythrocytes are larger and have a higher concentration of I11 protein in their membranes than human erythrocytes. Higher concentrations of band I11 proteins likely compensated for the increased cell volume and, at least in part, the effect of lower body temperature in trout compared with humans on anion exchange rates. However, anion exchange between trout erythrocytes at 15°C was slower than that between human erythrocytes at 38°C. However, the transit time of red blood cells

Table 13-1 Comparison of bicarbonate-chloride exchange systems in trout and human erythrocytes Prolsertv Cell surface area (cm2) Band III molecules per cell Band III molecules per square centimeter

trout

2,67 ×

Humanity

1,42 x 10-6

lo6

1×lo6

30 × 10"

7 × 10"

3.42

17.2

CI ion exchange half-life (seconds):

switch

38/C Source: Romano and Passow, 1984.

0,05

.-0

d-4

2 (you

100

~Bear

' 1 Hound

#Humanity

rabbit

disaster

bullfrog

Red blood cell volume (p m 3 )

Figure 13-13 Microerythrocytes oxidize faster than large cells in vitro. However, cell size is unlikely to correlate with in vivo oxygenation rates. [From Holland and Forster, 1966.1

The path through the gills is longer than in the lungs, allowing more time for anion exchange through the red blood cells. Despite these considerations, it remains unclear why different species would evolve red blood cells of different sizes. Animals with macrocytic cells often also have macrocytic cells. Thus, cell size may have been chosen for reasons other than gas transport and may be largely independent of gas transport rates. For example, triploid salmon, whose red blood cells are 1.5 times the size of their diploid cousins but have the same hemoglobin concentration, can swim just as fast as their diploid cousins, suggesting comparable gas transfer efficiency. It is important to remember that gas transfer in the body is a dynamic process that occurs as blood moves rapidly through capillaries. When analyzing this process, the diffusion rate, reaction rate, and steady-state conditions of gases in the blood must be considered. For example, Bohr shifts (e.g., the decrease in hemoglobin-oxygen affinity with decreasing pH) are of little significance if they occur after blood leaves capillaries to supply active tissue. In fact, the Bohr shift is so fast that human red blood cells have a half-life of 0.12 seconds at 37°C. Although lowering the temperature will always reduce the rates of reactions involved in gas transfer among species, these rates do not change and are not adjusted to regulate the rate of gas transfer at constant temperature. However, changes in concentration are used to adjust the rate of gas transmission over hours or days. For example, oxygen content. Blood depends on hemoglobin concentration, which in many vertebrates is elevated by lack of oxygen. Rapid changes in the rate of gas transmission in vertebrates are achieved by adjusting the rate and volume of breathing and/or by adjusting the flow rate and distribution of blood across tissues and respiratory surfaces.

Body pH Regulation The body pH of animals is on the neutral alkaline side; that is, there is less hydrogen gas than hydroxide ions in the body. Due to water, the concentration of hydrogen and hydroxide ions in aqueous solution is very low

530

Integrated Physiological Systems

.................................................. .....................................

Only weakly dissociated. Human plasma has a pH of 7.4 or a hydrogen ion activity of 40 nanomoles per liter (1 nM = M) at 37°C. Mammals can maintain normal function, H at 37°C, within a plasma pH range of 7.0-7.8. Keep between 100 and 16 nM H+. This is indeed a large deviation from the normal 40 nM H+ concentration compared to the body's much lower tolerance for fluctuations in Na+ or K+ levels. However, it is important to remember that the absolute concentration varies very little, as does the actual concentration of H+ ions in the body. The blood pH of vertebrates lies between the pKa of the carbon dioxide-bicarbonate and ammonia-ammonium reactions (Figure 13-14A). Most cell membranes are not very permeable to HCO and NH,+ ions but are very permeable to CO and NH. Some membranes have relatively low NH permeability, but this is the exception rather than the rule. Body pH values between these pK values provide adequate rates of elimination by diffusion of the two major end products of metabolism, carbon dioxide and ammonia. As these pK values vary with temperature, the pH of the blood also changes, ensuring adequate clearance over a range of temperatures (Fig. 13-14B). Changes in pH in vivo alter the dissociation of weak acids and thus the ionization of proteins. The net charge of a protein determines enzymatic activity and subunit aggregation, affects membrane properties, and influences the osmolarity of body compartments. Osmolarity is affected because the charges on proteins are the major contributors to the total fixed charge inside the cell. Changing the fixed charge changes the Donnan equilibrium of the ions and therefore affects the osmolarity. Any differences in osmotic pressure between body compartments disappear quickly because the membrane is permeable to water, and the movement of water causes volume changes in the different body compartments. Animals thus regulate their internal pH in the face of continuous metabolic release of hydrogen ions to stabilize volume and regulate enzyme activity. Cells also undergo pH changes due to cellular function or regulation and control. For example, pH plays a central role in the activation of sea urchin sperm and insulin-stimulated glycolysis in frog muscle. The pH of cells can also change due to external influences. For example, cells become acidotic during periods of hypoxia due to an imbalance between the protons produced by the hydrolysis of ATP to ADP and the protons consumed by NAD in tissues undergoing anaerobic metabolism. Production and excretion of hydrogen ions

Hydrogen ions are produced through metabolism or ingested through food (such as citric acid in oranges), and then continuously excreted from the body. The largest pools and fluxes are generally due to the metabolism of CO, which reacts with water at the pH of the body to form H+ and

carbon monoxide,

6,0

+ oh-=

7,0

pka = 6,08

sodium bicarbonate;

8,0

9.0

10.0

7,6 7,4

6 5

look

Temperature (“C”) Figure 13-14 In vertebrates, plasma pH is between the pK values of the ammonia/ammonium and carbon dioxide/bicarbonate reactions. (A) Effect of different pH values on [CO,]/[HCO,-] and [NH,]/[NH,+] ratios in trout plasma at 15 °C. The dashed lines mark the pH values (i.e., pK values) at which the ratio equals 1. (6) The effect of temperature on the plasma pH value of several fishes. Red triangles are calculated pH values where CO,/HCO and NH,/NH,+ ratios are the same at different temperatures. Therefore, plasma pH is maintained at a level that ensures excretion of NH and CO. [Adapted from Randall and Wright, 1989.1

HCO,- ion (see Figure 13-11A). At the respiratory surface, HCO,- is converted to CO, which is then excreted (see Figure 13-11B). Therefore, when CO production and excretion are in balance, the overall effect of CO flux on body pH will be zero. If less carbon monoxide is excreted than produced so that it builds up, the body becomes acidic; when the opposite happens, the body's pH increases. However, terrestrial vertebrates can alter CO excretion rates to maintain body pH. Eating meat generally results in a net acid intake, while eating plant-based foods generally results in a net base intake. In general, there is little net production of hydrogen ions due to diet and metabolic activity. Therefore, the overall effect of food intake and metabolism is the continued production of small amounts of acid. The body's pH is maintained by the excretion of this acid through the earth's kidneys.

An area of an experimental vertebrate or body surface, such as the gills of a fish or the skin of a frog. Changes in blood pH also occur due to the movement of acids between compartments. For example, a large amount of acid produced in the stomach after a large meal can cause an alkaline flush in the blood as acid is transferred from the blood to the stomach. Likewise, the production of large amounts of alkaline pancreatic juice can cause a surge of acid in the blood.

As described in Chapter 3, the relationship between pH and the degree of dissociation of the weak acid HA is described by the Henderson-Hasselbalch equation:

pK'

[A-I + Daily [HA1

When the pH of a weak acid solution is equal to the pK' of the acid, 50% of the acid is in the undissociated form (HA) and 50% is in the dissociated form (H+ + A-). The ratio of undissociated to dissociated forms ranges from 10% to 90% at 1 pH unit above pK and from 1% to 99% at 2 pH units above pK. For the acid-base pair C 0 , / H C 0 3 , the Henderson-Hasselbalch equation can be rewritten as [HCO,-]

PH value

pK'

+ record "Pco2

where PCO2 is the partial pressure of CO in blood, a is the Bunsen solubility coefficient of CO, [HCO,-] is the concentration of bicarbonate, and pK' is the apparent dissociation constant. The term "apparent" is used because this pK' is a combination of the combined reaction of CO with water and the subsequent formation of bicarbonate, rather than a true pK. From this equation we can see that a change in pH affects the ratio of HCO,- to PCO7 and vice versa. The pK' for the CO,/HCO,- reaction is about 6.1 and the pK' for the HCO,-/CO,2 reaction is about 9.4. At the pH value of the human body, about 95% of CO exists in the form of HCO,-, and the rest consists of carbon dioxide and carbonic acid; the amount of C032- is negligible. Weak acids have the greatest buffering effect when pH = pK. Since plasma proteins and hemoglobin have pK values close to blood pH, these compounds are important physical buffers in blood. The CO 2 /HCO 3 pair, whose apparent pK' is lower than blood pH, is not as important as hemoglobin or protein in providing a physical buffer system. the meaning of

The CO and bicarbonate system is that the increase of respiration can rapidly increase the pH value, and by reducing the CO, concentration and HC03- in the blood, it can be excreted through the kidneys to lower the blood pH value. Although bicarbonate is not an important chemical buffer in living systems, it is often called a buffer because it can be excreted to adjust the ratio of CO2 to bicarbonate, thereby regulating pH. The most important true buffers in blood are proteins, especially hemoglobin. Phosphate is also an important buffer in many cells. The importance of buffers in mitigating pH changes can be seen in the effects of acid infusions on mammalian blood. To lower the pH from 7.4 to 7.0, approximately 28 millimoles of hydrogen ions must be added to the blood. In fact, only 60 nmol (approximately 0.2%) is required to change the pH of an aqueous solution to this extent; however, in blood, most of the added 28 mmol H+ is converted by converting HCO3- to buffered CO (18 mmol), Hemoglobin (8.0 mmol), plasma protein (1.7 mmol) and phosphate (0.3 mmol) were eliminated. Therefore, almost 500,000 times the number of buffer hydrogen ions is required to change the pH from 7.4 to 7.0. Clearly, when lung ventilation is reduced and CO excretion is lower than CO production, CO levels in the body rise and pH falls. This decrease in body pH is called respiratory acidosis. The opposite effect, an increase in pH due to increased lung ventilation, is called respiratory alkalosis. The term "respiration" is used to distinguish these pH changes from those caused by changes in metabolism or kidney function. For example, anaerobic metabolism results in the production of net acid, which lowers the body's pH; this change is called metabolic acidosis. Like other solutions, body fluids are electrically neutral, meaning that the sum of anions corresponds to the sum of cations. The normal electrolyte status of human plasma is shown in Figure 13-15. The sum of bicarbonate, phosphate, and protein anions is called the buffer base. The remaining cations and anions are called strong ions (i.e. ions that are completely dissociated in physiological solution); the difference between the total number of strong cations and the total number of strong anions is called the strong ion difference (SID), which reflects the size of the buffer base . Since changes in blood pH often result in changes in the buffer base, the SID must also change to maintain electrical neutrality. In this case, changes in SID usually affect sodium or chloride, since these are the main ions in the blood. For example, a decrease in bicarbonate must be accompanied by an increase in chloride or a decrease in sodium. Conversely, changes in the sodium-to-chloride ratio are accompanied by changes in the buffer base and, therefore, changes in blood pH. Vomiting of stomach contents results in a loss of chlorine and a decrease in blood chloride levels; thus, bicarbonate levels and blood pH increase, while P o l does not change. This is called metabolic alkalosis. However, vomit from the duodenum rather than the stomach results in a loss of bicarbonate more than chloride, resulting in metabolic acidosis.

532

Integrated Physiological Systems

.................................................. ................................... (

meq.1-I

Normal plasma electrolyte status Figure 13-15 All body fluids are electrically neutral and contain equal amounts of positive and negative charges. The graph shows the equivalent concentrations (meq. Lk') of major electrolytes in human plasma at normal pH. The concentration of buffer bases (non-respiratory acid-base exchange) depends on pH. Therefore, an increase or decrease in pH that changes the base concentration of a buffer must be accompanied by a corresponding change in the concentration of one or more strong ions, usually sodium or chloride. [Adapted from Siggaard-Andersen, 1963.1

Hydrogen ion distribution between compartments

The cell membrane separates the intracellular and extracellular compartments and the cell layer between the two body compartments, and carbon dioxide is more permeable than hydrogen or bicarbonate ions. Most cell membranes are generally less permeable to H+ ions but generally more permeable to K+, Cl, and HCO- ions; a notable exception is the red blood cell membrane, which is highly permeable to HCO3 and C1 ions but more permeable to H+ ions are not very permeable. In the collecting duct of the mammalian kidney there are red blood cells and cells

High levels of Band 111 protein were present in their plasma membranes, but not in other cells. As previously mentioned, the I11 protein band mediates the high-speed exchange of HCO,- and Cl- ions. Thus, although all cell membranes are permeable to CO, only a few membranes can transfer HCO3- at high rates through an I11-banded anion exchange mechanism. An increase in extracellular PCo2 leads to an increase in the concentration of bicarbonate and hydrogen ions, creating gradients of CO, HCO, - and H+ across the cell membrane. In cells that are highly permeable to CO but not very permeable to H+ or bicarbonate, this situation results in rapid movement of CO into the cell; as CO is converted to HCO3, the intracellular pH drops dramatically. Acidification associated with elevated Pco: Acidification that occurs in the intracellular compartment usually occurs faster than in the extracellular compartment because carbonic anhydrase, which catalyzes the conversion of CO to HCO, is present in the cell but not always is present in the extracellular fluid. Even when Pm2 remains elevated, intracellular pH slowly returns to its original value due to the slow extrusion of acid (or base uptake) across the cell membrane (Fig. 13-16A). When Pcol returned to its original value, the increase in intracellular pH resulted in a higher cellular pH than the initial value. That is, there is a small pH excess. As mentioned earlier, most cell membranes are much more permeable to molecular ammonia (NH3) than to ammonium ions (NH4+). When NH4Cl levels in the extracellular fluid are elevated, ammonia enters cells much faster than ammonium ions. The result of course is that the ammonium levels in the battery also rise faster. Ammonia equilibrates across the membrane and combines with hydrogen ions to form ammonium ions inside the cell, raising the pH of the cell (Figure 13-16B). After reaching a maximum, the pH begins to decrease with prolonged NH4Cl exposure due to slow passive NH4 influx+ and other acid-base regulation mechanisms in the membrane. When NH3 diffuses out of the cell, the restoration of the external NH4Cl content to its original value causes a sharp drop in the intracellular pH. However, due to the accumulation of intracellular NH,+, the cellular pH drops below baseline, but it slowly returns to baseline as NH4+ diffuses out of the cell. These pH-regulating mechanisms are activated by a decrease in intracellular pH or an increase in extracellular pH. In mammalian cells, acid excretion decreases to lower levels when the extracellular pH drops below 7.0 or when the intracellular pH rises above 7.4. When acid is injected into a cell, it is expelled from the cell at a rate proportional to the drop in cell pH. While some H+ efflux may be related to H+ diffusion out of the cell, some H+ efflux is related to sodium influx. This coupling of sodium and proton transport may be due to a cation exchange mechanism in the membrane or an electrogenic proton pump, which increases the membrane potential, thereby providing an electrochemical gradient for the diffusion of Na+ ions through sodium-selective channels. For exam-

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extracellular fluid OH

+ carbon monoxide,

two

20 minutes

Extracellular CO increases,

overshoot

7.5 CO, in CO, out 7.0 -

slow H' flow

Elevated NH, Cl in extracellular fluid Figure 13-16 Changes in extracellular carbon dioxide and ammonium chloride levels lead to changes in tissue intracellular pH. (A) When the level of CO in the extracellular fluid suddenly increases, CO rapidly diffuses into the cell, forming bicarbonate, and causing a sharp drop in intracellular pH. The subsequent slow efflux of H+ ions (dashed line) leads to this

For example, some cells can actively pump out protons through proton ATPases in the membrane; this proton efflux results in a sodium influx. Acid excretion is often accompanied by chloride efflux, possibly in exchange for extracellular HCO3-, which has been shown to be necessary for cellular pH regulation. For example, the drug SITS (4-acetamido4'-isothiocyanostilbene-2,2'-disulfonic acid) blocks chloride-bicarbonate exchange in red blood cells and also inhibits pH regulation in other cells. Therefore, the proton exchange and anion exchange mechanisms in the cell membrane play an important role in the regulation of intracellular pH. Acid load in cells is associated with H+ efflux, which is associated with Na+ influx, and HCO influx is associated with C1 efflux. The movement of HCO into the cell corresponds to the movement of H+ out of the cell, as HCO ions entering the cell are converted to CO, releasing hydroxide ions and raising the pH. The CO formed in this way leaves the cell and is converted to bicarbonate with the release of protons. This CO and HCO3- cycle, known as the Jacobs-Stewart cycle, serves to transfer H+ ions from the cell under intracellular acid stress, such as that produced by anaerobic metabolism (Fig. 13-17). .In the red blood cells of most vertebrates, unlike most other cells, hydrogen ions are passively distributed throughout the blood

The intracellular pH gradually increased. (B) When extracellular NH, Cl levels rise sharply, NH rapidly diffuses into the cell and combines with hydrogen ions to form ammonium ions, which slowly diffuse across the cell membrane (dashed line). This increases the pH inside the cells.

Figure 13-17 The Jacobs-Stewart cycle is the cycle in which carbon dioxide and bicarbonate transport acid between the extracellular and intracellular compartments. As shown, in red blood cells, this cycle is normally used to transport acid from the plasma to the interior of the cell. Since carbonic anhydrase is only present intracellularly, the rate of acid transfer is determined by the slow, uncatalyzed interconversion of CO and HCO in the extracellular fluid.

Membrane, the membrane potential keeps the pH inside the red blood cell lower than the pH in the blood plasma. Sudden addition of acid to plasma (such as after anaerobic H+ production) can cause red blood cell counts to drop

534

integrated physiological system

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pH value. Acid transfer from plasma to the interior of red blood cells is not by diffusion of H+ ions but by bicarbonate-chloride exchange (see Figure 13-17). Addition of H+ to plasma results in an increase in PCo1 due to the conversion of HCO,- to CO, which diffuses into red blood cells and converts to HCO3-, resulting in a decrease in intracellular pH. Bicarbonate then diffuses out of the cell through the chloride-bicarbonate exchange mechanism. The Jacobs-Stewart cycle in red blood cells results in the transport of acids from the plasma to the interior of the cells.

In fact, catecholamines released into the blood during metabolic acidosis activate the erythrocyte Na+/H+ exchanger, which transports H+ out of the cell and Na+ ions into the cell. In muscular fish, paddling swims can lead to marked acidosis. This decrease in plasma pH, if transmitted to red blood cells, impairs oxygen binding to hemoglobin and reduces the fish's ability to swim aerobically. This does not happen because the pH of red blood cells is regulated in these fish and remains high during post-shock acidosis.

Factors Affecting Intracellular pH

Factors Affecting Body pH

Intracellular pH remains stable when the rate of acid loading by metabolism or into the cell is equal to the rate of acid removal. Any sudden increase in cellular acidity is counteracted by the various mechanisms discussed earlier:

In order for the pH in the body to stabilize, acid production must accommodate acid excretion. In mammals, this symmetry is achieved by regulating the pulmonary excretion of CO and the renal excretion of acid or bicarbonate so that acid excretion balances production, which depends largely on the metabolic needs of the animal. In the collecting ducts of mammalian kidneys there are type A (acid-secreting cells) and type B (base-secreting) cells whose activity can be altered to increase acid or base excretion. In aquatic animals, the outer surface is capable of excreting acids, similar to the collecting ducts in mammalian kidneys (see Chapter 14). For example, frog skin and freshwater fish gills have proton-secreting proton-ATPases on the apical surface of epithelial cells. Gills also have HCO,/CI exchange mechanism. When these mechanisms are inhibited by drugs, the body's pH is affected. Temperature can have a major impact on your body's pH. The dissociation of water varies with temperature, with a neutral pH (i.e. [H+] = [OH-]) of only 7.00 at 25°C. The dissociation of water decreases, so the neutral pH (pN) increases with decreasing temperature. At 37 °C, the pN value is 6.8 and at 0 °C it is 7.46. At 37°C, human plasma has a pH of 7.4, which means it is slightly alkaline. At pH, the ratio of OH- to H+ concentration is 1. This ratio increases with alkalinity; at pH 7.4 and 37 °C it is about 20. Alkalinity relative to pH remains nearly the same in many tissues of most animals regardless of their body temperature (Figures 13-18). Fish have a plasma pH of 7.9-8.0 at 5°C; sea turtles have a plasma pH of approximately 7.6 at 20°C; and mammals have a plasma pH of 7.4 at 37°C. Therefore, they all have similar relative alkalinity and the same ratio of OH- to H+ ions in plasma (approximately 20). Tissues are generally less alkaline than plasma; for example, red blood cells have a pH about 0.2 pH units lower than plasma, and muscle cells have a pH of about 7.0. Temperature also had a significant effect on the pK' of plasma proteins and the CO,/HCO,- system, with pK' increasing with decreasing temperature. According to the Henderson-Hasselbalch equation, a change in pK' results in a change in pH or dissociation of weak acids. However, the temperature-dependent change in plasma pH (see Figures 13-18) counteracts the temperature-dependent change in plasma protein pK' such that

Buffering by physical buffers found inside cells such as proteins and phosphates. HCO,- reacts with H+ to form CO, which then diffuses out of the cell. H+ ions are passively diffused or actively transported out of the cell. Cation exchange (Nat/H+) or anion exchange mechanism (HCO,-/CI-) or both in the cell membrane. In addition, protons can be produced through metabolism by adjusting the pH. Many enzymes are inhibited by low pH, so inhibition of glycolysis (and possibly some other metabolic pathways) at low pH could regulate intracellular pH by reducing the net production of protons during periods of increased cellular acidity. In some cases, cellular pH can be adjusted to control or limit other cellular functions. It is not always clear whether these pH changes are the result or regulation of related cellular activities. In many cells, intracellular pH (pHi) and calcium levels are inversely or positively correlated. In other cells, they are sequential and not directly connected; in these cases, changes in pH can modulate calcium activity and thus its many effects on cellular function. For example, when frog eggs are fertilized, there is a brief increase in intracellular calcium levels, followed by a sustained increase in pH. Evidence suggests that this alkalinization of cells can prolong the effects of increased calcium. In some cases, regulation of intracellular pH (pHl) has a dramatic effect on cellular function. For example, the erythrocytes of many teleosts have a Na+/H+ exchanger and an HCO,-/Cl- exchanger in the membrane. As blood pH decreases, the hemoglobin of these animals shows a root shift, i.e. the blood becomes less oxygenated (see Figure 13-6). Apparently, in the absence of counter mechanisms, this effect impairs red blood cell oxygen delivery during metabolic acidosis. exist

Gas Exchange and Acid-Base Equilibrium 535 ................................................ .....

Airborne Gas Transfer: Lungs and Other Systems

Temperature ("C") Figure 13-18 Neutral pH (pN) and plasma pH decrease with increasing temperature, but the relationship between the two is constant in most animals. The figure shows the effect of temperature on the plasma pH of various turtles, frogs and fish compared to the change in pN. [According to Lahn, 1967.1]

The degree of dissociation of plasma proteins remained unchanged. Since the pK' of the CO hydration-dehydration reaction varies less with temperature than with blood pH, the animal must adjust the ratio of CO to HCO,- in the blood. In general, air-breathing ectothermic vertebrates appear to keep bicarbonate levels constant but decrease molecular CO2 levels as temperatures decrease. In aquatic animals, on the other hand, the CO content remained constant while the bicarbonate content increased with decreasing temperature. This process results in the same adjustment of the CO2 to bicarbonate ratio in aquatic and air-breathing vertebrates, as well as an adjustment in pH. An important point is that if the body's pH changes with temperature in the same way as the protein's pK', the Henderson-Hasselbalch equation predicts that the protein's charge should remain constant. When the protein's net charge changes little or not at all, the function remains the same over a wide range of temperatures. The body's ability to redistribute acid between body compartments serves an important function, as some tissues are more susceptible to changes in pH than others. The brain is particularly sensitive, while muscles can and do tolerate greater pH swings. Thus, the brain has extensive, though poorly understood, mechanisms for regulating the pH of cerebrospinal fluid (CSF). When there is a sudden acid load in the blood, the hydrogen ions are absorbed by the muscles, reducing vibrations in the blood and protecting the brain and other more sensitive tissues. The hydrogen ions are then slowly released from the muscles into the blood and excreted through the lungs as CO or through the kidneys as acidic urine. Therefore, when the body experiences a sudden acid load, the muscles can act as a temporary H+ reservoir, reducing the extent to which the rest of the body will experience pH fluctuations.

The previous sections have looked at the properties of oxygen and carbon dioxide, how these gases are transported in the blood, and how they affect the body's pH. In this section we will examine the way O2 and C 0 2 are transported between air and blood. The focus is on the vertebrate lung, but other gas transport systems are also considered. In the next section, gas transport between water and blood through gills will be discussed. The design of the gas delivery system is influenced by the properties of the medium and the requirements of the animal. For example, the lungs of mammals are constructed very differently than the gills of fish, and ventilate in a different way. This difference exists because although water is approximately 1000 times denser and more viscous than air, water contains only one-thirtieth the molecular oxygen of air. Additionally, gas molecules diffuse 10,000 times faster in air than in water. Thus, air respiration generally consists of the reciprocating movement of air in and out of the lungs, whereas aquatic respiration consists of a unidirectional flow of water through the gills (Fig. 13-19A). The design goal of fish gills is to minimize the distance the gills spread in the water, creating a thin layer of water on the breathing surface. These changes in environment, respiratory system structure, and ventilation patterns result in differences in gas partial pressures, particularly Pco, in the blood and tissues of air- and water-breathing animals (figure). 13-19B).

Functional Anatomy of the Lung The vertebrate lung develops as a diverticulum of the gut, consisting of a complex network of tubes and sacs, the actual structure of which varies from species to species. In the lungs of amphibians, reptiles, and mammals (in that order), the size of the terminal air spaces gradually decreases, but the total number of air spaces per unit of lung volume increases. The lung structure of amphibians is variable, ranging from the smooth-walled sacs of some pods to the lungs of frogs and toads separated by diaphragms that fold into many interconnected air sacs. Increased compartmentalization of RiGiles, more so in mammals, leads to an overall increase in respiratory surface area per unit lung volume. In general, mammalian airway surface area increases with body weight and oxygen uptake rate (Figs. 13-20). The breathing area of bony fishes is generally smaller than that of mammals of the same weight. The lungs of mammals are composed of millions of interconnected blind sacs called alveoli. The trachea divides into repeatedly branching bronchi leading eventually to terminal bronchioles and finally to respiratory bronchioli, each connected to a set of terminal alveolar ducts and alveolar sacs (Figures 13-21). lump sum

Figure 13-19. The different gas transport systems of aspirating and hygroscopic animals are related to the characteristic distribution of respiratory gases in the blood and tissues. (A) Schematic of O and CO fluxes in aspirating and hygroscopic animals. (B) Relative values of Po2 and Pcq in the inspiratory medium, blood, and tissues of aspirated (top) and hygroscopic (bottom) animals.

airflow

luft

\ Blood

+==r Capillary

c02

tissue water

exist

mammal

1000

slope = 1.0

orangutan

100

1,000 10,000100,000 Weight (grams)

Figure 13-20 Respiratory surface area increases with body size (A) Relationship between respiratory surface area and body weight for selected mammals and teleosts. (6) The relationship between the alveolar surface

(S.A.) and mammalian oxygen uptake. [Part A adapted from Randall, 1970; Part B of Tenney and Ternmers, 1963.1

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Bronchioles

Figure 13-21 In the mammalian lung, a series of branching, tapering ducts carry air to the respiratory portion, which consists of terminal and respiratory bronchioles and alveolar dilobular ducts and alveoli. Gas transfer occurs through the airway epithelium shown in red.

The cross-sectional area of the airways increases rapidly due to extensive branching, although the diameter of each trachea decreases from the trachea to the terminal bronchioles. The terminal bronchioles, respiratory bronchioles, alveolar ducts, and alveolar sacs make up the respiratory portion of the lungs. Gases are transported across the thin-walled alveoli, called acini, located in the region distal to the terminal bronchioles. The airways leading to the terminal bronchioles form the non-respiratory part of the lungs. Alveoli in adjacent acini are connected by a series of holes called pores of Kohn that allow collateral air flow, which may be a major factor in gas distribution during lung ventilation (Fig. 13-22A). The trachea, which leads to the breathing part of the lungs, contains cartilage and some smooth muscle and is lined with cilia. Epithelial cells secrete mucus, which is transported to the oral cavity by cilia. This "slime escalator"

Keep your lungs clean (see Chapter 8). In the breathing part of the lungs, smooth muscle replaces cartilage. The contraction of this smooth muscle can have a major impact on the size of the airways in the lungs. The resting uptake rate per body weight is higher in small mammals than in large mammals because of their greater alveolar surface area per body weight. The increase in respiratory surface area is achieved by reducing the size but increasing the number of alveoli per unit lung volume. In humans, the number of alveoli increases rapidly after birth, reaching about 300 million in adults by eight years of age; the subsequent increase in respiratory area is achieved by increasing the volume of each alveoli. Children have higher resting O2 intake per body weight than adults; here too, there is a link between intake per body weight and alveolar area per body weight. The mammalian diffusion barrier is composed of an aqueous surface membrane, alveolar epithelium, interstitial layer, capillary endothelium, plasma, and erythrocyte walls (Fig. 13-22B). The lung epithelium is composed of several cell types. The most common type I cells make up the majority of the lung epithelium. They are squamous cells with a lamellar structure, a single cell extending into two adjacent alveoli, with the nucleus hidden in one corner. Type I1 cells are characterized by intracellular lamellar bodies and surface villi; type I1 cells produce surfactant, which is discussed later. Type III cells are mitochondria-rich cells with a brush border. These rare cells appear to be involved in the uptake of NaCl from lung fluid. In addition to these cells, many alveolar macrophages migrate across the surface of the airway epithelium. It is generally assumed, but not confirmed, that the diffusion coefficients of gases in the lungs of different animals do not vary. The only structural variables are lung area and the diffusion distance between air and blood. The following terms are used to describe the different types of breathing and lung ventilation: Normal breathing, at rest.

calm breathing, typical of animals

Hyperventilation and hypoventilation - increases or decreases in the volume of air moving in and out of the lungs due to changes in breathing rate and/or breathing depth, causing ventilation to be out of sync with CO2, production, and CO2 values in the blood

Hyperpnea - increased ventilation of the lungs due to increased CO2 production, such as during exercise

Figure 13-22 During mammalian lung ventilation, breathing gases flow in and out of the alveolar spaces and blood in the pulmonary capillaries. (A) The three alveolar septa of the canine lung meet at the junction. Connective tissue fibers lie in the midplane and form a continuous stretched network, with which the capillary network interweaves. Type I endothelial and epithelial cells line the thin air-blood barrier. Kohn pores connect the alveoli. (B) Dimensions and structure of the alveolar-capillary membrane. [After Weibel, Part A, 1973; Hildebrandt and Young, Part B, 1965.1

Alveoli

Capillary

Alveoli

Second

Alveoli

Lining (0.01 ~ r n )

Alveolardurchmesser (50–300 μm)

5~m

Air exchanged between the alveoli and the environment must pass through a series of tubes (trachea, bronchi, non-respiratory bronchioles) not directly involved in gas transport. At the end of exhalation, the air in these tubes is expelled from the alveoli, low in oxygen and high in carbon dioxide. This air is the first to return to the alveoli on the next breath. At the end of inhalation, the non-breathing tube is filled with fresh air, and this volume is exhaled first on the next breath. Therefore, this volume does not participate in gas transfer and is therefore called anatomical dead space volume. Some air may enter non-functioning alveoli, or some alveoli may ventilate at too high a rate, increasing the amount of air not directly involved in gas exchange. This volume of air is called the physiological dead space and is usually greater than but includes the anatomical dead space (Spotlight 13-3). The volume of air that moves in and out of the lungs with each breath is called tidal volume. The volume of fresh air entering and leaving the alveolar sacs is equal to the tidal volume minus the anatomical dead space volume and is called the alveolar ventilation. Only this volume of gas directly participates in gas transport. Even with maximum exhalation, the lungs are not completely emptied.

Space (12:02 am - 2:00 pm)

The residual air volume remains in the lungs. The maximum amount of air that can be moved in or out of the lungs is called vital capacity. Figure 13-23 shows these and other terms used to describe various volumes and volumes in relation to lung function. Alveolar gas has less O and more CO than ambient air because only a portion of the lung volume changes with each breath. The human alveolar ventilation is about 350 ml, while the remaining functional volume of the lung is more than 2000 ml. During inspiration, the ducts leading to the alveoli lengthen and widen, resulting in an increase in acinar volume. During respiration, air moves in and out of the acini and can also move between adjacent alveoli through the pores of Kohn. Gas mixing in the ducts and alveoli occurs by diffusion and convection induced by respiration (Fig. 13-24). In the alveolar ducts, O diffuses into the alveoli and CO diffuses away from the alveoli. The partial pressures of O and CO in the alveoli are likely to be fairly equal because of the rapid diffusion in the air and the small distances. The partial pressure of gas in the alveoli changes in phase with respiratory movement, and its magnitude depends on the degree of tidal ventilation. ~

539 Gas Exchange and Acids - B A S E B A L A N C E ...................................... .... ……………………

Maximum Inspiration Level Reserve

Figure 13-23 A number of terms are used to describe the various volumes and volumes related to lung function. Tidal volume is the volume of air that normally moves in and out of the lungs, while vital capacity is the maximum volume.

Vital capacity resting inspiratory level

End-expiratory disease Ruheniveau

~Function

maximum

total expiratory volume

Primary subdivision of lung volumes

vital capacity

Eben

Pulmonary function test specialist

Alveolar gas O2 and CO2 levels are determined by the rate of gas transport through the respiratory epithelium and the rate of alveolar ventilation. Alveolar ventilation depends on respiratory rate, tidal volume, and anatomical dead space. Fluctuations in the size of the anatomical dead space alter the gas tension in the socket unless the tidal volume changes.

Artificial enlargement of the anatomical dead space occurs when breathing through a tube, resulting in increased C 0 2 and decreased O 2 in the lungs. As discussed in later sections, these changes activate chemoreceptors, leading to increased tidal volumes. Long-necked animals such as giraffes and trumpeting swans have greater tracheal length and anatomical dead space than short-necked animals (Fig. 13-25). To maintain adequate gas tension in the lungs, long-necked animals have increased tidal volumes. Animals vary widely in respiratory rate and tidal volume. Humans breathe about 12 times per minute, and the tidal volume at rest is about 10% of the total lung volume. This relatively fast, shallow breathing produces a small

Figure 13-24 The direction of airflow (large arrows) in the breathing portion of the lung changes during inspiration and expiration, but the diffusion of oxygen (small arrows) is always towards the alveolar walls.

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Figure 13-25 The extremely long trachea of Trumpeter swan leads to a huge anatomical dead space. For comparison, see Figure 13-29, which shows the length of a human trachea. 1 from Banko, 19Ml.l

Pulmonary blood bipolar fluctuations. In contrast, Amphiurna, a fully aquatic but air-breathing amphibian that lives in swampy waters, rises to the surface to breathe about once an hour; however, his tidal volume exceeds 50% of his lung volume. This large tidal volume combined with infrequent breathing produces large, slow oscillations in the hips, lungs, and blood that are more or less synchronized with the respiratory movement (Fig. 13-26). Amphiuma are preyed upon by snakes and are most vulnerable while standing up to breathe. Because it lives in water with low oxygen levels, aquatic breathing is not a viable option. The risk of being eaten while surfacing to breathe may have affected the development of their very slow breathing rate, large tidal and lung volumes, and cardiovascular adaptations that help maintain oxygen delivery to tissues despite fluctuating blood gas levels . Carbon dioxide levels in Amphiuma do not fluctuate like oxygen levels because carbon dioxide is lost through the skin and is not dependent on lung ventilation. In summary, O and CO levels in alveolar gas are determined by ventilation and gas transfer rates. Ventilation of the respiratory epithelium is determined by respiratory rate, tidal volume, and anatomical dead space volume. Ventilation type and volume also affect the magnitude of blood O and CO fluctuations during the respiratory cycle. pulmonary circulation

The lungs, like the heart, receive blood from two sources. The primary blood flow consists of deoxygenated blood from the pulmonary arteries, which seeps into the lungs, absorbs O and releases CO; this is called the pulmonary circulation. A second, smaller supply, the bronchial circuit, originates from the systemic (body) circulation and provides oxygen and other substrates to lung tissue for growth and maintenance. Our discussion here is limited to the pulmonary circulation.

Time (minutes) Figure 13-26 Respiratory rate tends to be inversely proportional to tidal volume and amplitude of oscillation period 2. The Amphiurna is an aquatic, air-breathing amphibian with regular breathing, with large variations in tidal volume and rump. Shown here are pbb blood pressure, heart rate, P021 and Pco2 for the 515-9 Amphiuma over two breathing dive cycles. Vertical arrows indicate the time when the animal surfaced and underwent lung ventilation. Note that blood pressure, heart rate, and Pco are nearly constant between breaths, while Po has large, slow fluctuations in the lungs and blood. [Adapted from Toews et al., 1971]

In birds and mammals, the pressure in the pulmonary circulation is lower than the pressure in the systemic circulation. This pressure difference reduces the filtration of fluid into the lungs. Extensive lymphatic drainage of the lung tissue also helps prevent fluid from accumulating in the lungs (see Chapter 12). These features are important because any fluid that collects on the lung surface increases the diffusion distance between blood and air and reduces gas transport. Blood flow through the pulmonary circulation is best described in terms of lobar flow, ie. H. As a liquid flows between two parallel surfaces. This is in contrast to the laminar nature of flow through pipes and system loops. The pulmonary capillary endothelium resembles two parallel surfaces connected by columnar structures, between which blood flows. As the pressure increases, the parallel surfaces move apart, causing the blood layer to increase in thickness. That is, the pressure increases the thickness of the blood layer instead of distributing the flow in other directions. The mean arterial pressure in the human lungs is about 12 mmHg, and the pressure varies between 7.5 mmHg and 22 mmHg with each systole

Gas Exchange and Acids - B A S E B A L A N C E

541

................................................. ................................. Lit 13-3

and

lung volume

PEC02X VT = (PACOZX VT) - (PAC02X V ),

The alveolar ventilation V corresponds to the difference between the trough ventilation VT and the dead space volume VD: VA = VT -

+ (PICOPx V),

by rearranging

five,

When f denotes respiratory frequency, the volume of air that enters and exits the lungs per minute, VAf, is called the alveolar minute ventilation or minute ventilation, and is symbolized as a dot above Vin as a function of frequency. ,,, is the volume of the non-respiratory portion of the lung; physiological dead space .V,ol,,, is the volume of the lung not involved in gas transfer. If the partial pressure of CO in exhaled air is expressed in PECO, in alveolar air in PACOP, and in inspired air in PICOP, then

G. But PICO tends to zero, PACO is the same as partial pressure of CO in arterial blood, P,CO,. So the last expression can be written as follows:

PEC02X VT = PACOZ(VT - VD) + (PICO2X V),

Therefore, the physiological dead space of the lung can be calculated from measurements of tidal volume V and CO, arterial partial pressure P, CO and exhaled gas PEC02.

Heart. In a vertical (upright) human lung, arterial pressure is just high enough to push blood to the top of the lung; therefore, flow is smallest at the top and increases toward the bottom of the lung (Figure 13-27). Blood is more evenly distributed to different parts of the horizontal lungs.

The blood vessels of the lungs are very elastic and deform with the movement of breathing. Small blood vessels within the alveolar septa are particularly sensitive to changes in alveolar pressure. The diameter of these thin-walled collapsible capillaries is determined by the transwall pressure

,, so if we substitute this equation we get But VA = V, - V

top notch

goodbye > goodbye

> P"

blood flow (volume)

Figure 13-27 In the upper part of the vertical lung, the diameter of the alveolar capillaries and the blood flow through the capillaries depend on the difference between the arterial pressure Pa and the alveolar pressure PA. In this diagram of blood, the lungs of the human body flow vertically; the boxes represent the state of blood vessels in the alveolar septa in different parts of the lungs, and at the apex of the lungs, PA often exceeds Pa; as a result, capillaries collapse and blood flow stops. PV is the venous pressure. [Adapted from West, 1970.1

(arterial blood pressure in capillaries, Pa, minus alveolar pressure, Pa). When transmural pressure is negative (ie, PA > Pa), these capillaries collapse and blood flow ceases. This collapse may occur at the lower Pa vertical human lung roof (see Figure 13-27). Alveolar pressure determines the diameter of the capillaries in the alveolar septa and acts like a gate to control blood flow through the capillaries. As long as alveolar pressure exceeds venous pressure, venous pressure does not affect flow into the venous reservoir. Thus, flow in the vertical upper lung may be determined by the difference between arterial blood pressure and alveolar pressure. Arterial blood pressure (and therefore blood flow) increases with distance from the lung apex. In the lower half of the vertical lung, venous pressure exceeds alveolar pressure and blood flow is determined by the difference between arterial and venous blood pressure. This pressure difference does not vary with location, although both arterial and venous pressures increase toward the base of the lung. An increase in absolute pressure causes dilation of blood vessels, which results in a decrease in flow resistance. Therefore, flow to the base of the lung increases even though the arteriovenous pressure difference does not change (see Figure 13-27). Therefore, the position of the lungs relative to the heart is an important factor affecting pulmonary blood flow. The lungs surround the heart, minimizing the effect of gravity on pulmonary blood flow as the animal transitions from a horizontal to a vertical position. This proximity of the lungs and heart in the chest cavity is also important for cardiac function: the reduced pressure in the chest cavity during inspiration supports venous return to the heart. This is often called a chest-abdominal pump. Although the mammalian pulmonary circulation lacks well-defined arterioles, both sympathetic-adrenergic and parasympathetic cholinergic fibers innervate the smooth muscles surrounding the pulmonary vessels and bronchioles. However, pulmonary circuits are much less innervated than systemic circuits and are relatively unresponsive to neural stimulation or injected drugs. Sympathetic nerve stimulation or norepinephrine injections caused a slight increase in blood resistance, whereas parasympathetic nerve stimulation or acetylcholin

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