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F O U R T H E D I T I O N 
E C K E R T 
ANIMAL 
PHYSIOLOGY 
M E C H A N I S M S A N D A D A P T A T I O N S 
D A V I D R A N D A L L 
U N I V E A S ~ T W O F B R I T I S H C O L U M B I A 
1 W A R R E N B U R G G R E N 
K A T H L E E N F R E N C H 
U N I V E R S I T Y O F C A L I F O R N I A , S A N D L E G O 
W I T H C O N T R I B U T I O N S B Y 
R U S S E L L F E R N A L D 
S T A N F O R D U N I V E R S I T Y 
W. H. Freeman and Company 
New Ynrk 
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L~brary of Congress Cataloging-in-Publication Data 
Randall, Dav~d J., 1938- 
Eckert an~mal phys~ology: mechanisms and adaptations/Dav~d Randall, Warren 
Burggren, Kathleen French.-4th ed. 
P. cm. 
Includes blbl~ograph~cal references and ~ndex. 
ISBN 0-71 67-2414-6 (hardcover) 
1. Physiology. I. Burggren, Warren. 11. French, Kathleen. 111. Tltle. 
QP31.2.R36 1997 
591.1-dc20 96-31713 
CIP 
Copyright O 1978, 1983, 1988, 1997 by W. H. Freeman and Company. All rights 
reserved. 
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process, or in the form of a phonographic recording, nor may it be stored in a retrieval 
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Printed in the United States of America 
Second printing, 1997, RRD 
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A B O U T T H E A U T H O R S 
DAVID RANDALL 
A prominent fish physiologist and a leading expert in 
respiratory and circulatory physiology, David Randall 
collaborated with the late Roger Eckert on the earlier 
editions of Animal Physiology and continues his con- 
tribution in the fourth. A faculty member at the 
University of British Columbia in Vancouver, Canada, 
since 1963, and full professor since 1973, Randall was 
appointed Associate Dean of Graduate Studies in 1990. 
Elected a fellow of the Royal Society of Canada in 1981, 
Randall has been both a Guggenheim and a Killam fel- 
low, and was awarded the prestigious Fry Medal for re- 
search contributions to zoology by the Canadian Soci- 
ety of Zoology in 1993. In 1995, he received the Award 
of Excellence from the American Fisheries Society for 
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 
WARREN BURGGREN 
Warren Burggren has taught in physiology for 23 years, 
and has been a professor of biological sciences at the Uni- 
versity of Nevada at Las Vegas since 1992. Courses he 
has taught at UNLV and at the University of Massachu- 
setts, where he was Professor of Zoology from 1987 
through 1991, include Human Anatomy and Physiology, 
Bioenergetics, Introductory Zoology, and Comparative 
Physiology. Burggren's research interest include develop- 
mental physiology, comparative animal physiology, and 
environmental and ecological physiology. In particular, 
his research focuses on the ontogeny of respiratory and 
cardiovascular systems, and how the systems that regu- 
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 
KATHLEEN FRENCH 
A neurobiologist at the University of California at San 
Diego since 1985, Kathleen French has for 10 years 
taught upper division courses in embryology, mammalian 
physiology for premedical students, and cellular neurobi- 
ology. In addition, at UCSD, French participates in a 
training program to instruct science teaching assistants in 
the techniques and philosophy of teaching. She also serves 
on the faculty of the Neuroscience and Behavior Course 
at the Marine Biological Laboratories in Woods Hole, 
Massachusetts, an intensive course designed primarily for 
graduate students and post-doctoral fellows. French 
. brings her expertise in-and love of-teaching to her 
contributions to the field of fish physiology. A frequent 
symposium lecturer on fish physiology and other sub- 
jects, most recently in Brazil, France, Germany, Italy, the 
People's Republic of China, Russia, and the United 
States. He has worked with both the World Health Or- 
ganization and the United States Environmental Protec- 
tion Agency in developing ammonia criteria. Widely 
published as author and co-author in leading journals, 
Randall is co-editor of the noted series Fish Physiolog?, 
(Academic Press), of which 15 volumes are in print. Vol- 
ume 16, subtitled "Deep-sea Fish," will appear in 1997. 
Along with his other duties, Randall co-teaches third 
year courses in vertebrate physiology and environmental 
physiology. His research interests concern the interac- 
tions between gas and ion exchange across fish gills. 
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 
late them change over the course of development. 
Burggren has been actively involved in symposia, semi- 
nars, and formal extramural researchltraining activities 
in many countries. A co-author of The Evolution of Air 
Breathing in Vertebrates (Cambridge University Press, 
1981), Burggren has been a frequent contributor since 
1980 to edited collections of physiology, including 
Presser's Comparative Animal Physiology, Fourth Edi- 
tion (Wiley-Liss, 1991). Burggren co-edited Environ- 
mental Physiology of the Amphibia (University of 
Chicago Press, 1992), and more recently co-edited De- 
velopment of Cardiovascular Systems; Molecules to Or- 
ganisms (Cambridge University Press, 1997). 
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 
role as co-author of the current edition of Animal Physi- 
ology, along with a lifelong interest in the nervous systems 
of organisms from a broad range of phyla. As an Associ- 
ate Project Scienrist at UCSD, French's research focuses 
on the control of neuronal development, a topic that she 
has studied in various invertebrate species. Her current re- 
search concerns the cellular events that control differenti- 
ation of identified neurons in the medicinal leech, with an 
emphasis on the cellular physiology of embryonic neu- 
rons and the effects of cell-cell contacts. She has been the 
author and co-author of numerous published research 
and review articles in journals including the Journal of 
Neuroscience and Journal of Neurophysiology. 
PART I PRINCIPLES OF PHYSIOLOGY 
1 STUDYING ANIMAL PHYSIOLOGY 
2 EXPERIMENTAL METHODS FOR EXPLORING PHYSIOLOGY 
3 MOLECULES, ENERGY, AND BIOSYNTHESIS 
4 MEMBRANES, CHANNELS, AND TRANSPORT 
PART II PHYSIOLOGICAL PROCESSES 
5 THE PHYSICAL BASIS OF NEURONAL FUNCTION 
6 COMMUNICATION ALONG AND BETWEEN NEURONS 
7 SENSING THE ENVIRONMENT 
8 GLANDS: MECHANISMS AND COSTS OF SECRETION 
9 HORMONES: REGULATION AND ACTION 
10 MUSCLES AND ANIMAL MOVEMENT 351 
11 BEHAVIOR: INITIATION, PATTERNS, AND CONTROL 405 
PART Ill INTEGRATION OF PHYSIOLOGICAL SYSTEMS 465 
12 CIRCULATION 467 
13 GAS EXCHANGE AND ACID-BASE BALANCE 517 
14 IONIC AND OSMOTIC BALANCE 571 
15 ACQUIRING ENERGY: FEEDING, DIGESTION. AND METABOLISM 627 
16 USING ENERGY: MEETING ENVIRONMENTAL
CHALLENGES 665 
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1 
I 
i C O N T E N T S 
I 
I I 
Preface ix 
Acknowledgments xvii 
PART I PRINCIPLES OF 
PHYSIOLOGY 
CHAPTER 1 STUDYING ANIMAL 
PHYSIOLOGY 3 
THE SUBDISCIPLINES OF ANIMAL PHYSIOLOGY 4 
WHY STUDY ANIMAL PHYSIOLOGY? 4 
Sc~ent~fic Curlos~ty 4 
Commerc~aYAgr~cultural Appl~cat~ons 4 
Inslghts into Human Physiology 4 
CENTRAL THEMES IN ANIMAL 
PHYSIOLOGY 4 
Structure-Funct~on Relat~onsh~ps 5 
Adaptation, Accl~mat~zat~on, and Accl~mat~on 5 
Homeostas~s 7 
Feedback-Control Systems 8 
Confo rm~t~ and Regulation 9 
LITERATURE OF PHYSIOLOGICAL SCIENCES 10 
SPOTLIGHT 1-1 THE CONCEPT OF FEEDBACK 12 
ANIMAL EXPERIMENTATION IN PHYSIOLOGY 13 
Summary 13 
Rev~ew Quest~ons 14 
Suggested Readlngs 14 
CHAPTER 2 EXPERIMENTAL METHODS 
FOR EXPLORING PHYSIOLOGY 
FORMULATING AND TESTING HYPOTHESES 
The August Krogh Prlnc~ple 
Experimental Deslgn and Phys~olog~cal Level 
MOLECULAR TECHNIQUES 
Traclng Molecules w ~ t h Radlolsotopes 
Traclng Molecules wlth Monoclonal Antlbod~es 
Genetic Englneerlng 
CELLULAR TECHNIQUES 
Uses of M~croelectrodes and Mlcroplpettes 
Structural Analys~s of Cells 
Cell Culture 
BIOCHEMICAL ANALYSIS 
Measurlng Composit~on: What Is Present 
Measurlng Concentration: How Much IS Present 
EXPERIMENTS WITH ISOLATED ORGANS 
AND ORGAN SYSTEMS 
OBSERVING AND MEASURING ANIMAL 
BEHAVIOR 
The Power of Behav~oral Experiments 
Methods In Behav~oral Research 
IMPORTANCE OF PHYSIOLOGICAL STATE 
IN RESEARCH 
Summary 
Revlew Quest~ons 
Suggested Readlngs 
CHAPTER 3 MOLECULES, ENERGY, 
AND BIOSYNTHESIS 
ORIGIN OF KEY BIOCHEMICAL MOLECULES 
ATOMS, BONDS, AND MOLECULES 
THE SPECIAL ROLES OF H, 0, N, AND C 
IN LIFE PROCESSES 
WATER: THE UNIQUE SOLVENT 
The Water Molecule 
Propert~es of Water 
Water as a Solvent 
PROPERTIES OF SOLUTIONS 
Concentration, Coll~gatlve Properties, 
and Activ~ty 
Ionizat~on of Water 
Ac~ds and Bases 
The B~olog~cal Importance of pH 
Henderson-Hasselbalch Equat~on 
Buffer Systems 
Electrlc Current ~n Aqueous Solutions 
SPOTLIGHT 3-1 ELECTRICAL TERMINOLOGY 
AND CONVENTIONS 
Blndlng of Ions to Macromolecules 
X CONTENTS 
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 
BIOLOGICAL MOLECULES 
Llplds 
Carbohydrates 
Protelns 
Nucle~c Ac~ds 
ENERGETICS OF LIVING CELLS 
Energy: Concepts and Defin~t~ons 
Transfer of Chem~cal Energy by Coupled React~ons 
ATP: Energy Carrier of the Cell 
Temperature and React~on Rates 
ENZYMES: GENERAL PROPERTIES 
Enzyme Speclficlty and Actlve S~tes 
Mechanism of Catalys~s by Enzymes 
Effect of Temperature and pH on Enzymat~c React~ons 
Cofactors 
Enzyme Klnetlcs 
Enzyme Inhlb~t~on 
REGULATION OF METABOLIC REACTIONS 
Control of Enzyme Synthes~s 
Control of Enzyme Actlvlty 
METABOLIC PRODUCTION OF ATP 
Oxldat~on, Phosphorylatlon, and Energy Transfer 
Glycolys~s 
Cltr~c Ac~d Cycle 
Effic~ency of Energy Metabol~sm 
Oxygen Debt 
Summary 
Rev~ew Quest~ons 
Suggested Readlngs 
CHAPTER 4 MEMBRANES, CHANNELS, 
AND TRANSPORT 
MEMBRANE STRUCTURE AND 
ORGANIZATION 
Membrane Composlt~on 
Flu~d Mosa~c Membranes 
SPOTLIGHT 4-1 THE CASE FOR A LIPID BILAYER 
MEMBRANE 
Var~at~on In Membrane Form 
CROSSING THE MEMBRANE: AN OVERVIEW 
D~ffus~on 
Membrane Flux 
Osmos~s 
Osmolarlty and Tonlclty 
Electr~cal Influences on Ion D~str~but~on 
Donnan Equll~brlurn 
OSMOTIC PROPERTIES OF CELLS 
Ion~c Steady State 
Cell Volume 
PASSIVE TRANSMEMBRANE MOVEMENTS 
Slmple D~ffus~on through the Llp~d Bllayer 
D~ffus~on through Membrane Channels 
SPOTLIGHT 4-2 ARTIFICIAL BILAYERS 
Facllltated Transport across Membranes 
ACTIVE TRANSPORT 
The Na+lK+ Pump as a Model of A a v e 
Transport 
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 
Ion Gradients as a Source of Cell Energy 
Coupled Transport 
MEMBRANE SELECTIVITY 
Selectlvlty for Electrolytes 
Selectlv~ty for Nonelectrolytes 
ENDOCYTOSIS AND EXOCYTOSIS 
Mechanlsms of Endocytosis 
Mechanlsms of Exocytosls 
JUNCTIONS BETWEEN CELLS 
Gap Junct~ons 
Tlght Junct~ons 
EPITHELIAL TRANSPORT 
Actlve Salt Transport across an Ep~thellum 
Transport of Water 
Summary 
Rev~ew Quest~ons 
Suggested Readlngs 
PART II PHYSIOLOGICAL 
PROCESSES 
CHAPTER 5 THE PHYSICAL BASIS 
OF NEURONAL FUNCTION 
OVERVIEW OF NEURONAL STRUCTURE, 
FUNCTION, AND ORGANIZATION 
Transmlss~on of S~gnals In a Slngle Neuron 
Transm~ss~on of Slgnals Between Neurons 
Organ~zat~on of the Nervous System 
MEMBRANE EXCITATION 
Measuring Membrane Potentials 
Dlstlngu~shlng Passlve and Actlve Membrane 
Electr~cal Propert~es 
SPOTLIGHT 5-1 THE DISCOVERY OF 
"ANIMAL ELECTRICITY" 
Role of Ion Channels 
PASSIVE ELECTRICAL PROPERTIES 
OF MEMBRANES 
Membrane Res~stance and Conductance 
Membrane Capac~tance 
ELECTROCHEMICAL POTENTIALS 
The Nernst Equat~on: Calculatlng the 
Equ~l~brlum Potent~al for Slngle Ions 
SPOTLIGHT 5-2 A QUANTITATIVE CONSIDERATION 
OF CHARGE SEPARATION ACROSS MEMBRANES 
The Goldman Equation: Calculatlng the 
Equll~brlum Potent~al for Mult~ple Ions 
THE RESTING POTENTIAL 
Role of Ion Grad~ents and Channels 
Role of Actlve Transport 
ACTION POTENTIALS 
General Propert~es of Act~on Potent~als 
Ion~c Baas of the Act~on Potent~al 
SPOTLIGHT 5-3 THE VOLTAGE-CLAMP METHOD 
Changes In Ion Concentrat~on durlng Exc~tat~on 
OTHER ELECTRICALLY EXCITED 
CHANNELS 
Summary 
Review Questions 
Suggested Readings 
CHAPTER 6 COMMUNICATION ALONG 
AND BETWEEN NEURONS 
TRANSMISSION OF SIGNALS IN THE 
NERVOUS SYSTEM: AN OVERVIEW 
TRANSMISSION OF INFORMATION WITHIN 
A SINGLE NEURON 
Pass~ve Spread of Electr~cal S~gnals 
Propagatlon of Actlon Potentials 
Speed of Propagat~on 
Rapid, Saltatory Conduction In Myelmated Axons 
SPOTLIGHT 6-1 EXTRACELLULAR SIGNS OF IMPULSE 
CONDUCTION 
SPOTLIGHT 6-2 AXON DIAMETER AND CONDUCTION 
VELOCITY 
TRANSMISSION OF INFORMATION 
BETWEEN NEURONS: SYNAPSES / 
Synapt~c Structure and Function: Electr~cal Synapses 
Synaptlc Structure and Function: Chermcal Synapses 
Fast Chemical Synapses 
SPOTLIGHT 6-3 PHARMACOLOGICAL AGENTS 
USEFUL IN SYNAPTIC STUDIES 
SPOTLIGHT 6-4 CALCULATION OF REVERSAL 
POTENTIAL 
PRESYNAFTIC RELEASE OF 
NEUROTRANSMITTERS 
Quanta1 Release of Neurotransm~tters 
Depolarizat~on-Release Coupllng 
Nonsplking Release 
THE CHEMICAL NATURE OF 
NEUROTRANSMITTERS 
Fast, D~rect Neurotransm~ss~on 
Slow, Indirect Neurotransmission 
POSTSYNAPTIC MECHANISMS 
Receptors and Channels in Fast, Direct Neurotransmission 
Receptors m Slow, Ind~rect Neurotransm~ss~on 
Neuromodulat~on 
INTEGRATION AT SYNAPSES 
SYNAPTIC PLASTICITY 
Homosynaptlc Modulation: Facil~tatlon 
Homosynapt~c Modulat~on: Posttetan~c Potentlation 
Heterosynapt~c Modulation 
" Long Term Potent~at~on 
Summary 
Rev~ew Questions 
Suggested Read~ngs 
CHAPTER 7 SENSING THE 
ENVIRONMENT 
GENERAL PROPERTIES OF SENSORY 
RECEPTION 
Properties of Receptor Cells 
Common Mechanisms and Molecules of Sensory 
Transduction 
From Transduction to Neuronal Output 
C O N T E N T S xi 
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 
Encod~ng Stlmulus Intensities 224 
Input-Output Relations 225 
Range Fractionation 226 
Control of Sensory Senslt~v~ty 226 
THE CHEMICAL SENSES: TASTE 
AND SMELL 231 
Mechan~sms of Taste Recept~on 232 
Mechanisms of Olfactory Reception 235 
MECHANORECEPTION 238 
H a ~ r Cells 238 
Organs of Equ~librlum 241 
The Vertebrate Ear 242 
An Insect Ear 248 
ELECTRORECEPTION 248 
THERMORECEPTION 250 
VISION 251 
Optlc
Mechan~sms: Evolut~on and Funct~on 252 
Compound Eyes 253 
SPOTLIGHT 7-1 SUBJECTIVE CORRELATES OF PRIMARY 
PHOTORESPONSES 256 
The Vertebrate Eye 257 
Photoreception: Convert~ng Photons into Neuronal Signals 261 
SPOTLIGHT 7-2 THE ELECTRORETINOGRAM 263 
SPOTLIGHT 7-3 LIGHT, PAINT, AND COLOR VISION 268 
LIMITATIONS O N SENSORY RECEPTION 269 
Summary 270 
Revlew Quest~ons 271 
Suggested Read~ngs 271 
CHAPTER 8 GLANDS: MECHANISMS 
AND COSTS OF SECRETION 
CELLULAR SECRETION 
Types and Funct~ons of Secretions 
Surface Secretions: The Cell Coat and Mucus 
Packag~ng and Transport of Secreted Material 
SPOTLIGHT 8-1 SUBSTANCES WITH SIMILAR 
STRUCTURES AND FUNCTIONS SECRETED BY 
DIFFERENT ORGANISMS 
Storage of Secreted Substances 
Secretory Mechanlsms 
GLANDULAR SECRETIONS 
Types and General Properties of Glands 
Endocrine Glands 
Exocr~ne Glands 
ENERGY COST OF GLANDULAR ACTIVITY 
Summary 
Rev~ew Questions 
Suggested Read~ngs 
CHAPTER 9 HORMONES: REGULATION 
AND ACTION 301 
ENDOCRINE SYSTEMS: OVERVIEW 302 
Chem~cal Types and General Functions of Hormones 3 02 
Regulat~on of Hormone Secretion 303 
NEUROENDOCRINE SYSTEMS 303 
Hypothalmlc Control of the Anterlor Pitu~tary 
Gland 304 
xii CONTENTS 
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 
Glandular Hormones Released from the Anterlor 
P~tuitary Gland 305 
Neurohormones Released from the Posterlor 
P~tu~tary Gland 308 
SPOTLIGHT 9-1 PEPTIDE HORMONES 310 
CELLULAR MECHANISMS O F HORMONE 
ACTION 311 
Lipld-Soluble Hormones and Cytoplasmic Receptors 311 
Lipld-Insoluble Hormones and Intracellular Slgnal~ng 312 
SPOTLIGHT 9-2 AMPLIFICATION BY ENZYME CASCADES 322 
PHYSIOLOGICAL EFFECTS O F HORMONES 328 
Metabolic and Developmental Hormones 328 
Hormones That Regulate Water and Electrolyte Balance 336 
Reproduct~ve Hormones 338 
Prostagland~ns 342 
HORMONAL ACTION IN INVERTEBRATES 343 
Summary 346 
Revlew Quest~ons 348 
Suggested Readings 349 
CHAPTER 10 MUSCLES AND ANIMAL 
MOVEMENT 
STRUCTURAL BASIS O F MUSCLE CONTRACTION 
Myofilament Substructure 
Contraction of Sarcomeres: The Slldlng Fllament Theory 
Cross-Bridges and the Productlon of Force 
SPOTLIGHT 10-1 PARALLEL AND SERIES 
ARRANGEMENTS: THE GEOMETRY OF MUSCLE 
MECHANICS OF MUSCLE CONTRACTION 
Relatlon Between Force and Shortening Veloclty 
SPOTLIGHT 10-2 SKINNED MUSCLE FIBERS 
Effect of Cross-Brldges on Force-Veloclty Relatlon 
REGULATION OE CONTRACTION 
Role of Calclum In Cross-Brldge Attachment 
Excltatlon-Contraction Coupllng 
Contraction-Relaxat~on Cycle 
THE TRANSICNT PRODUCTION O F FORCE 
Serles Elast~c Component 
The Act~ve State 
Twitches and Tetanus 
ENERGETICS O F MUSCLE CONTRACTION 
ATP Usage by Myosln ATPase and Calclum Pumps 
Regeneration of ATP durlng Muscle Actlvlty 
FIBER TYPES IN VERTEBRATE SKELETAL 
MUSCLE 
Class~ficat~on of Flber Types 
Functional Rat~onale for Different Flber Types 
ADAPTATION OE MUSCLES FOR VARIOUS 
ACTIVITIES 
Adaptatlon for Power: Jumplng Frogs 
Diversity of Function: Swlmmmg Fish 
Adaptatlon for Speed: Sound Productlon 
Hlgh-Power, Hlgh Frequency Muscles:Asynchronous 
Fllght Muscles 
NEURONAL CONTROL OF MUSCLE 
CONTRACTION 
Motor Control in Vertebrates 
Motor Control ln Arthropods 
CARDIAC MUSCLE 
SMOOTH MUSCLE 
Summary 
Revlew Questions 
Suggested Readings 
CHAPTER 11 BEHAVIOR: INITIATION, 
PATERNS, AND CONTROL 405 
SPOTLIGHT 1 1-1 BEHAVIOR IN ANIMALS THAT LACK A 
NERVOUS SYSTEM 403 
EVOLUTION O F NERVOUS SYSTEMS 408 
ORGANIZATION O F THE VERTEBRATE 
NERVOUS SYSTEM 412 
Major Dlv~slons of the Central Nervous System 413 
The Automatic Nervous System 420 
ANIMAL BEHAVIOR 423 
Bas~c Behavioral Concepts 423 
Examples of Behavlor 426 
PROPERTIES OF NEURONAL CIRCUITS 432 
Pieces of the Neuronal Puzzle 433 
Sensory Networks 434 
$SPOTLIGHT 11-2 TUNING CURVES: THE RESPONSE OF A 
NEURON PLOllED AGAINST THE PARAMETERS OF A 
STIMULUS 436 
SPOTLIGHT 11-3 SPECIFICITY OF NEURONAL 
CONNECTIONS AND INTERACTIONS 447 
Motor Networks 453 
Summary 461 
Revlew Questions 462 
Suggested Reahngs 462 
PART Ill INTEGRATION OF 
PHYSIOLOGICAL SYSTEMS 
CHAPTER 12 CIRCULATION 
GENERAL PLAN O F THE CIRCULATORY 
SYSTEM 
Open C~rculat~ons 
Closed C~rculatlons 
THE HEART 
Electrical Act~v~ty of the Heart 
Coronary Clrculat~on 
Mechanical Properties of the Heart 
SPOTLIGHT 12-1 THE FRANK-STARLING 
MECHANISM 
The Pericardlum 
Vertebrate Hearts: Comparative Functional Morphology 
HEMODYNAMICS 
Laminar and Turbulent Flow 
Relat~onship between Pressure and Flow 
THE PERIPHERAL CIRCULATION 
Arterlal System 
Venous System 
Capillaries and the M~croc~rculat~on 
THE LYMPHATIC SYSTEM 
. . . 
CONTENTS xi11 
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 
CIRCULATION AND THE IMMUNE RESPONSE 
REGULATION OF CIRCULATION 
Control of the Central Cardiovascular System 
Control of the M~croc~rculat~on 
CARDIOVASCULAR RESPONSE T O 
EXTREME CONDITIONS 
Exerc~se 
D~vmg 
Hemorrhage 
Summary 
Rev~ew Questions 
Suggested Readings 
CHAPTER 13 GAS EXCHANGE 
AND ACID-BASE BALANCE 
GENERAL CONSIDERATIONS 
SPOTLIGHT 13-1 EARLY EXPERIMENTS ON GAS 
EXCHANGE IN ANIMALS 
OXYGEN AND CARBON DIOXIDE 
IN BLOOD 
Resp~ratory P~gments 
SPOTLIGHT 13-2 THE GAS LAWS 
Oxygen Transport In Blood 
Carbon D~ox~de Transport m Blood 
Transfer of Gases to and from the Blood 
REGULATION OF BODY pH 
Hydrogen Ion Production and Excret~on 
Hydrogen Ion D~str~but~on between Compartments 
Factors Influenc~ng Intracellular pH 
Factors Influenc~ng Body pH 
GAS TRANSFER IN AIR: LUNGS AND 
OTHER SYSTEMS 
Funct~onal Anatomy of the Lung 
Pulmonary C~rculat~on 
SPOTLIGHT 13-3 LUNG VOLUMES 
Vent~lation of the Lung 
Pulmonary Surfactants 
Heat and Water Loss across the Lung 
Gas Transfer In B~rd Eggs 
Insect Tracheal Systems 
GAS TRANSFER IN WATER: GILLS 
Flow and Gas Exchange across G~lls 
Funct~onal Anatomy of the G~ll 
REGULATION OF GAS TRANSFER AND 
RESPIRATION 
Ventdat~on-to-Perfus~on Rat~os 
Neural Regulat~on of Breath~ng 
RESPIRATORY RESPONSES T O EXTREME 
CONDITIONS 
Reduced Oxygen Levels (Hypox~a) 
Increased Carbon D~ox~de Levels (Hypercapma) 
D ~ v ~ n g by Air-Breathmg An~mals 
Exerc~se 
SWIMBLADDERS: OXYGEN ACCUMULATION 
AGAINST LARGE GRADIENTS 
The Rete M~rab~le 
Oxygen Secret~on 
Summary 
Review Questions 
Suggested Readings 
CHAPTER 14 IONIC AND OSMOTIC 
BALANCE 571 
PROBLEMS OF OSMOREGULATION 571 
OBLIGATORY EXCHANGE OF IONS AND WATER 574 
Grad~ents Between the An~mal and the Env~ronment 574 
Surface-to-Volume Ratlo 574 
Permeab~l~ty of the Integument 575 
Feedmg, Metabol~c Factors, and Excret~on 577 
Temperature, Exerc~se, and Resp~rat~on 5 78 
OSMOREGULATORS AND OSMOCONFORMERS 580 
OSMOREGULATION IN AQUEOUS AN 
TERRESTRIAL ENVIRONMENTS 581 
Water-Breathmg An~mals 581 
hr-breathmg Animals 584 
OSMOREGULATORY ORGANS 587 
MAMMALIAN KIDNEY 587 
Anatomy of the Mammakn l d n e y 588 
Ur~ne Product~on 590 
SPOTLIGHT 14-1 RENAL CLEARANCE 595 
Regulat~on of pH by the K~dney 601 
Ur~ne-Concentratmg Mechan~sm 603 
SPOTLIGHT 14-2 COUNTERCURRENT SYSTEMS 604 
Control of Water Reabsorpt~on 606 
NONMAMMALIAN VERTEBRATE KIDNEYS 608 
EXTRARENAL OSMOREGULATORY ORGANS 
IN VERTEBRATES 608 
Salt Glands 608 
F~sh G~lls 613 
INVERTEBRATE OSMOREGULATORY ORGANS 616 
F~ltrat~on-Reabsorpt~on Systems 616 
Secretory-Reabsorpt~on Systems 617 
EXCRETION OF NITROGENOUS WASTES 620 
Ammon~a-Excretmg (Ammonotel~c) An~mals 621 
Urea-Excret~ng (Ureotehc) An~mals 623 
Ur~c Ac~d-Excretmng (Ur~cotel~c) An~mals 624 
Summary 624 
Rev~ew Quest~ons 625 
Suggested Read~ngs 625 
CHAPTER 15 ACQUIRING ENERGY: 
FEEDING, DIGESTION, 
AND METABOLISM 
FEEDING METHODS 
Food Absorpt~on through
Extenor Body Surfaces 
Endocytos~s 
Filter Feeding 
Fluid Feed~ng 
Se~z~ng of Prey 
Herb~vory and Graz~ng To Collect Food 
OVERVIEW OF ALIMENTARY SYSTEMS 
Headgut: Food Recept~on 
Foregut: Food Conduct~on, Storage, and Digestion 
M~dgut: Chemical D~gest~on and Absorpt~on 
xiv C O N T E N T S 
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 
Hindgut: Water and Ion Absorption and Defecation 643 
Dynamics of Gut Structure-Influence of Diet 644 
MOTILITY OF THE ALIMENTARY CANAL 644 
Muscular and Ciliary Mot~lity 645 
Peristalsis 645 
Control of Motility 646 
GASTROINTESTINAL SECRETIONS 649 
Exocr~ne Secret~ons of the Ahrnentary Canal 650 
Control of Dlgestlve Secret~ons 653 
SPOTLIGHT 15-1 BEHAVIORAL CONDITIONING IN 
FEEDING AND DIGESTION 654 
ABSORPTION 657 
Nutnent Uptake in the Intestme 657 
Blood Transport of Nutr~ents 658 
Water and Electrolyte Balance tn the Gut 659 
NUTRITIONAL REQUIREMENTS 66 1 
Energy Balance 66 1 
Nutr~ent Molecules 66 1 
Summary 663 
Review Questions 664 
Suggested Readlngs 664 
CHAPTER 16 USING ENERGY MEETING 
ENVIRONMENTAL CHALLENGES 665 
THE CONCEPT OF ENERGY METABOLISM 665 
MEASURING METABOLIC RATE 666 
Basal and Standard Metabol~c Rate 666 
Metabol~c Scope 667 
D~rect Calor~rnetry 668 
SPOTLIGHT 16-1 ENERGY UNITS (OR WHEN IS A 
CALORIE NOTA CALORIE?) 668 
Indlrect Calorimetry-Measurement from Food 
Intake and Waste Excretion 668 
Ind~rect Measures of Metabol~c Rate 669 
Resp~ratory Quohent 670 
Energy Storage 671 
Spec~fic Dynam~c Act~on 672 
BODY SIZE AND METABOLIC RATE 672 
SPOTLIGHT 16-2 THE REYNOLDS NUMBER: 
IMPLICATIONS FOR BIG AND SMALL ANIMALS 676 
TEMPERATURE AND ANIMAL ENERGETICS 677 
Temperature Dependence of Metabohc Rate 677 
Determ~nants of Body Heat and Temperature 680 
Temperature Classlficat~ons of Anlmals 682 
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 
TEMPERATURE RELATIONS 
OF ECTOTHERMS 
Ectotherms In Freezlng and Cold Env~ronments , 
Ectotherms ln Water and Hot Envlronments 
Costs and Benefits of Ectothermy: A Compar~son 
w ~ t h Endothermy 
TEMPERATURE RELATIONS 
OF HETEROTHERMS 
TEMPERATURE RELATIONS 
OF ENDOTHERMS 
Mechan~sms for Body Temperature Regulation 
Thermostatic Regulat~on of Body Temperature 
Fever 
DORMANCY: SPECIALIZED METABOLIC 
STATES 
Sleep 
Torpor 
H~bernat~on and Wlnter Sleep 
Estlvat~on 
ENERGETICS OF LOCOMOTION 
An~mal Sue, Velocity, and Cost of Locomohon 
Physlcal Factors Affecting Locomotlon 
Aquat~c, Aer~al, and Terrestrial Locomotlon 
BODY RHYTHMS AND ENERGETICS 
C~rcadlan Rhythms 
Nonclrcad~an Endogenous Rhythms 
Temperature Regulat~on, Metabohsm, and B~olog~cal 
Rhythms 
ENERGETICS OF REPRODUCTION 
Patterns of Energet~c Investment In Reproduct~on 
The "Cost" of Gamete Product~on 
Parental Care as an Energy Cost of Reproduct~on 
ENERGY, ENVIRONMENT, AND EVOLUTION 
Summary 
Rev~ew Quest~ons 
Suggested Readings 
Appendzx 1: SI Units 
Appendlx 2: Logs and Exponentials 
Appendrx 3: Conversions, Formulas, Physzcal and 
Chemrcal Constants, Definitions 
References Czted 
Glossary 
Index 
I t is nearly ten years since the third edition of Animal Physiology first appeared, written by Roger Eckert with 
the help of David Randall. Roger died in 1986 while revis- 
ing the third edition, which was completed by George Au- 
gustine and David Randall. That book formed the basis for 
the fourth edition, which is fittingly referred to as Eckert An- 
imal Physiology. Although this new edition has been exten- 
sively revised and redesigned, the approach that so success- 
fully characterized earlier editions has been maintained: the 
use of comparative examples to illustrate general principles, 
often supported by experimental data. In addition, we have 
emphasized the principle of homeostasis, and we have up- 
dated the molecular and cellular coverage. Retained in this 
edition is the comprehensive coverage of tissues, organs, and 
organ systems. Cellular and molecular topics are integrated 
early in the book so that common threads are developed to 
explain and compare the interactions between regulated 
physiological systems that produce coordinated responses 
to environmental change in a wide variety of animal groups. 
The basic principles and mechanisms of animal physiology 
and the adaptations of animals that enable them to exist in 
so many different environments form the central theme of 
this book. 
The diversity and adaptations of the several million 
species that make up the animal kingdom provide endless 
fascination and delight to those who love nature. Not the 
least of this pleasure derives from a consideration of how the 
bodies of animals function. At first it might appear that with 
so many kinds of animals adapted to such a variety of life- 
styles and environments, the task of understanding and ap- 
preciating the physiology of animals would be overwhelm- 
ing. Fortunately (for scientist and student alike), the 
concepts and principles that provide a basis for under- 
standing animal function are relatively few, for evolution 
has been conservative as well as inventive. 
A beginning course in physiology is a challenge for both 
teacher and student because of the interdisciplinary nature 
of the subject, which integrates chemistry, physics, and 
biology. Most students are eager to come to grips with the 
subject and get on with the more exciting levels of modern 
scientific insight. For this reason, Eckert Animal Physiology 
has been organized to present the essential background ma- 
terial in a way that allows students to review it on their own 
and go on quickly to consider animal function and to un- 
derstand its experimental elucidation. 
Eckert Animal Physiology develops the major concepts 
in a simple and direct manner, stressing principles and mech- 
anisms over the compilation of information and illustrating 
the functional strategies of animals that have evolved within 
the bounds of chemical and physical possibility. Common 
principles and patterns, rather than exceptions, are empha- 
sized. Examples are selected from the broad spectrum of an- 
imal life, consciously illustrating similarities between or- 
ganisms; for example, similar compounds are associated 
with reproduction in both humans and yeast. Thus, the 
more esoteric and peripheral details receives only passing at- 
tention, or none at all, so as not to distract from central 
ideas. We use the device of a narrative, describing experi- 
ments, to provide a feeling for methods of investigation 
while presenting information. 
ORGANIZATION OF THE BOOK 
For the first time, the chapters are organized into three 
parts, which we feel will promote an understanding of an- 
imals as integrated systems at every level of organization. 
Each part is introduced by an opening statement that gives 
students an overview of the material to follow. Part I con- 
tains four chapters and is concerned with the central prin- 
ciples of physiology. Part I1 (Chapters 5 - 11) deals with 
physiological processes, while Part I11 (Chapters 12-16) 
discusses how these basic processes are integrated in ani- 
mals living in a variety of environments. All 16 chapters 
have been extensively reworked and reorganized to stay 
abreast of new scienthc developments. 
xvi P R E F A C E 
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 
NEW TO THIS EDITION 
A new chapter on methodology (Chapter 2) in Part I, in 
which some of the latest molecular techniques are dis- 
cussed and illustrated, along with traditional methods. 
This emphasis on molecular coverage continues 
throughout the book; Chapters 5,6, and 7, for exam- 
ple, are updated with recent insights into the cellular 
and molecular underpinnings of membrane excitation, 
synaptic
transmission, and sensory transduction. 
Part I1 features a new chapter (Chapter 8, Glands: 
Mechanisms and Costs of Secretion), which brings to- 
gether information on an important, but frequently ne- 
glected, effector system. 
In Part 11, Chapter 11 (Behavior: Initiation, Patterns, 
and Control) preserves and expands the descriptions of 
vertebrate and invertebrate nervous systems found in 
previous editions, presenting an up-to-date view of sys- 
tems neurobiology, one of the fastest-growing areas of 
neurobiology. Several concepts from neuroethology, 
which bridges the gap between the pure study of be- 
havior and the study of cellular function in the nervous 
system, are introduced, along with examples of impor- 
tant recent neuroethological studies. 
The role of the nervous system in maintaining home- 
ostasis through the modulation of all systems has been 
incorporated into Part 111, which further advances the 
integrated approach of the book. 
There is an increased emphasis throughout the book on 
environmental adaptations, and specific examples of 
environmental adaptation (such as water balance in ele- 
phant seals in Chapter 14) illustrate the general princi- 
ples of comparative physiology. 
Some of the new topics introduced in the fourth edition 
include a section on the immune response in Chapter 
12 (Circulation), and a section on biorhythms in Chap- 
ter 13 (Using Energy: Meeting Environmental 
Challenges). 
PEDAGOGY 
The ideas developed in the text are illuminated and 
.augmented by liberal use of illustrations and figure 
legends. For the first time, full color drawings have been 
added, creating a high quality visual program to further 
motivate students. 
Spotlights provide in-depth information about the ex- 
periments and individuals associated with important 
advances in the subject matter, the derivation of some 
equations, or simply historical background on a topic 
under discussion. 
Thought questions within chapter text (look for 
the a) encourage problem-based learning and stim- 
ulate discussion on various aspects of the material 
presented. 
The text narrative includes effective, integrated exam- 
ples to support principles; while presenting information, it 
provides consistent thematic coverage and a feeling for 
methods of investigation. References to the literature 
within the body of the text and in figure legends are made 
unobtrusively, but with sufficient frequency that students 
can become aware of the role of scientists and their litera- 
ture as a subject is developed. Further pedagogical aids in- 
clude key terms that are explained and appear in boldface 
type at their first mention in the text, and that are formally 
defined in a useful, comprehensive glossary. End of chapter 
materials include a summary, which provides the student 
with a quick review of important points covered in the 
chapter, review questions, and an annotated list of sug- 
gested further readings. Students will find the following re- 
sources at the back of the book: appendixes that provide in- 
formation on units, equations, and formulas; the glossary; 
and a bibliography that includes the full citations of all ref- 
erences cited in the chapters. Our goal has been to produce 
a balanced, up-to-date treatment of animal function that is 
characterized by its clarity of exposition. We hope that 
readers will find Eckert Animal Physiology valuable, and 
we welcome your constructive criticism and suggestions. 
September 1996 DAVID RANDALL 
WARREN BURGGREN 
KATHLEEN FRENCH 
E ckert Animal Physiology has benefitted greatly from the contributions of several people whose efforts 
we gratefully acknowledge. Russell Fernald, Stanford Uni- 
versity, participated in the planning and reorganization 
of the book and the initial revision of many of the chap- 
ters. Lawrence C. Rome, University of Pennsylvania, 
wrote the initial draft of Chapter 10 (Muscles and 
Animal Movement), which contained most of the updated 
material except for the sections on cardiac and smooth 
muscle and on neuronal control. Harold Atwood, 
University of Toronto, was involved in some early 
discusions of the revision. Further, we are grateful for 
the informal comments of associates around the world, 
and for the formal reviews of manuscript, which were 
provided by the follow~ng colleagues from across the 
country: 
Joseph Bastian, University of Oklahoma 
Robert B. Barlow, Institute for Sensory Research, 
Syracuse University 
Francisco Bezanilla, UCLA School of Medicine- 
Center for the Health Sciences 
Phillip Brownell, Oregon State University 
Richard Bruch, Louisiana State University 
Wayne W. Carley, National Association of Biology, 
Teachers 
Ingrith Deyrup-Olsen, University of Washington 
' Dale Erskine, Lebanon Valley College 
A. Verdi Farmanfarmaian, Rutgers University 
Robert Full, University of California at Berkeley 
Carl Gans, University of Michigan 
Edwin R. Griff, University of Cincinnati 
Kimberly Hammond, University of California at 
Riverside 
David F. Hanes, Sonoma State University 
Michael Hedrick, California State University- Hayward 
James W. Hicks, University of California at Irvine 
Sara M . Hiebert, Swarthmore College 
William H. Karasov, University of Wisconsin at Madison 
Mark Konishi, California Institute of Technology 
Bill Kristan, University of California at Sun Diego 
Paul Lennard, Emory University 
Jon E. Levine, Northwestern University 
Harvey B. Lillywhite, University of Florida, Gainesville 
Duane R. McPherson, SUNY at Geneseo 
Duncan S. MacKenzie, Texas A&M University 
Eric Mundall, late, University of California at 
Los Angeles 
Kenneth Nagy, University of California at Los Angeles 
Richard A. Nyhoff, Calvin College 
Richard W. Olsen, UCLA School of Medicine 
C. Leo Ortiz, University of California at Santa Cruz 
Harry Peery, University of Toronto 
J. Larry Renfro, University of Connecticut 
Marc M. Roy, Beloit College 
Roland Roy, Brain Research Institute, UCLA School of 
Medicine 
Jonathon Scholey, University of California at Davis 
C. Eugene Settle, University of Arizona 
Michael P. Sheetz, Washington University School o f 
Medicine 
Gregory Snyder, University of Colorado at Boulder 
Joe Henry Steinbach, Washington University School o f 
Medicine 
Curt Swanson, Wayne State University 
Malcolm H . Taylor, University of Delaware-School o f 
Life and Health Sciences 
Ulrich A. Walker, Columbia University-College o f 
Physicians 
Eric P. Widmaier, Boston University 
Andrea H. Worthington, Siena College 
Ernest M . Wright, UCLA School of Medicine-Center 
for the Health Sciences 
Finally, the good sense and kind words of Kay Ueno and 
Kate Ahr, our editors, have much improved the book and 
ensured its publication. 
DAVID RANDALL 
WARREN BURGGREN 
KATHLEEN FRENCH 
xvii 
0 ur knowledge of animal physiology is based on infor- mation (data) derived from experimentation. Since 
the ultimate goal of animal physiology is to understand 
how a process operates within an organism, experiments 
must be designed to allow the measurement of key vari- 
ables (e.g., metabolic rate, blood flow, urine production, 
muscle contraction) in the animal (or its cells or tissues) 
while it is in a known state such as resting, exercising, di- 
gesting, or sleeping. This kind of experimentation is partic- 
ularly challenging and requires the use of a variety of tech- 
niques and methods. Many of the experimental techniques 
and measuring devices common in animal physiology are 
"time-honored." These include pressure transducers to 
measure pressure, catheter implantation to draw blood or 
inject samples, respirometers for determining metabolic 
rates, and numerous others. A description of each of these 
is beyond the scope of this
chapter, especially since such 
fundamental techniques are well described in texts such as 
J. N. Cameron's Principles of Physiological Measurement. 
In this chapter, we will focus on a few of the many molec- 
ular and cellular techniques that have recently been added 
to the physiologist's tool box, briefly describing them and 
illustrating their use in physiological research. First, how- 
ever, we consider the nature of hypotheses and the general 
principles that apply in testing them. 
By knowing why and how experiments in animal phys- 
iology are performed-whether they employ traditional or 
emerging methods-you will be much better able to eval- 
uate the strengths and limitations of the information you 
will learn in this book. 
FORMULATING AND TESTING 
HYPOTHESES 
Scientists use experimental data to create general laws of 
physiology-some literally centuries old, and some still 
emerging. These general laws, in turn, serve as the basis for 
formulating new hypotheses, which are specific predictions 
that can be tested by performing further experiments. An 
example of a general "law" supported by much existing 
data is that water-breathing animals regulate acid-base bal- 
ance by modifying the excretion of HCO,- in exhaled wa- 
ter, while air-breathing animals regulate acid-base balance 
by modifying the elimination of CO, gas in exhaled air. The 
following testable hypothesis could be derived from this 
general law: A transition from H C O , elimination to CO, 
elimination occurs when water-breathing tadpoles meta- 
morphose into air-breathing frogs. Although hypotheses 
are framed as statements rather than as questions, the goal 
of experimentation is to test the validity of hypotheses, and 
thus answer the implied questions. 
Physiological experiments should begin with a well- 
formed, specific hypothesis that focuses on a particular 
level of analysis and is amenable to a verifiable experimen- 
tal approach. Although a hypothesis such as killer whales 
have a very high cardiac output while in pursuit of seals 
may be interesting and in fact true, it is merely an intellec- 
tual exercise to suggest this hypothesis unless a feasible ex- 
perimental approach exists for gathering data necessary to 
accept (prove) or reject (disprove) it. However, the search 
for means to test novel hypotheses has been an important 
stimulus for development of new experimental techniques 
and measuring instruments. For example, telemetry devices 
currently available for gathering data on blood flow in 
small to medium-sized animals like ducks, fishes, and seals 
are being modified for use on even larger animals. 
The August Krogh Principle 
August Krogh was a Danish animal physiologist with ex- 
tremely broad interests in comparative physiology. Dozens \ 
of key research articles bearing his name have served as the 
basis for whole areas of further experimentation in the area 
of respiration and gas exchange. Indeed, Krogh's work in 
the late 1800s and early 1900s eventually led to his winning 
the Nobel Prize for physiology. One of the reasons for 
Krogh's extraordinary success as a physiologist was his un- 
canny ability to choose just the right experimental animal 
with which to test his hypotheses. His view was that for 
every defined physiological problem, there was an optimally 
suited animal that would most efficiently yield an answer. 
16 P R I N C I P L E S OF P H Y S I O L O G Y 
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 
The design of experiments based on the unusual char- 
acteristics of an animal has come to be known as the Au- 
gust Krogh principle (Krebs, 1975). Illustrations of this 
principle abound in this book and throughout modern an- 
imal physiology. For example, in the 1970s a group of an- 
imal physiologists, interested in the evolution of air breath- 
ing in crustaceans, were studying relatively tiny intertidal 
crabs, but they were frustrated because the small size of 
these animals kept them from "giving up" their physiolog- 
ical secrets. Evoking the August Krogh principle, which 
suggested that there was an ideal animal with which to 
carry out their studies, these physiologists organized an ex- 
pedition to the Palau Islands in the South Pacific. These is- 
lands are home to the "coconut," or "robber," crab, a ter- 
restrial hermit crab weighing up to 3 kg. The monstrous 
size of these animals (for a terrestrial crab) allowed numer- 
ous experiments yielding important new data during the 
one-month expedition. 
As another example, animal physiologists interested in 
cardiac performance in fishes often have a difficult time 
measuring pressure and flow and sampling blood from the 
heart because of its typical location in bony fish (i.e., 
teleosts). Yet, the sea robm, a deep-water (benthic) marine 
teleost that is quite unremarkable in most respects (al- 
though it is down-right ugly!), has an unusually large heart, 
which is much easier to access than in other fishes. By fol- 
lowing the August Krogh principle and using the sea robin 
as the basis for their experiments, comparative cardiovas- 
cular physiologists now know more about heart function 
in fishes than they would if they had continued to struggle 
with the relatively unforgiving anatomy of the trout, 
salmon, or catfish. 
Experimental Design and Physiological Level 
In designing an experiment, the first and most important 
decision a physiologist must make is about the level at 
which the physiological problem will be analyzed. This 
choice of level determines the methodology (and choice of 
animal) appropriate for measuring the experimental vari- 
ables of interest. 
Historically, techniques for exploring physiological 
problems at the level of the whole animal were developed 
first; subsequently, and with increasing rapidity in recent 
decades, have come new techniques for experimenting at 
the cellular and now at the molecular level. Conceptually, 
however, we generally operate in the reverse order: starting 
at the molecular level, then moving successively to the cel- 
lular, tissue, organ, and finally whole-animal levels, much 
as outlined in Figure 1-1. Consequently, in the following 
sections, we describe some representative experimental 
methods for studying physiological processes, beginning at 
the molecular level. Much of the information presented in 
other chapters of this book is based on experimental results 
obtained with these various techniques. Only by learning 
how and why these methods work, as well as some of their 
limitations, can you adequately assess the information 
presented. 
Note that no level of analysis is intrinsically more valu- 
able or important than any other. Indeed, the best under- 
standing of animal physiology comes from integrating 
knowledge about the contributing components from the 
molecular through organ-system level. Having said this, we 
recognize the strong trend in animal physiology (as in all of 
biology) during the last decade towards "reductionism," 
the study of cellular and molecular mechanisms in an at- 
tempt to explain more complex processes at higher organi- 
zational levels. Ultimately, some of the most valuable ex- 
periments are those at a level of analysis that allows insights 
about processes at adjacent organizational levels. 
Although researchers and students often are fascinated 
by new and frequently expensive methodologies, incisive 
results can be obtained with well-designed experiments us- 
ing relatively simple instruments and techniques. In other 
words, a well-conceived experimental design often can 
compensate for the lack of the latest, cutting-edge equip- 
ment and techniques. 
MOLECULAR TECHNIQUES 
The past few decades have seen a veritable explosion in the 
number and sophistication of available techniques for 
probing molecular events,
with new methods and refine- 
ments constantly emerging. The variety of molecular tech- 
niques available have had major implications for biological 
research in general, and animal physiology has certainly 
benefited from molecular approaches. In this section we de- 
scribe just a few of the powerful molecular techniques that 
have been used to answer questions in animal physiology. 
More detailed discussion of these and related techniques 
are presented in textbooks such as Molecular Cell Biology 
by H. D. Lodish et al. 
Tracing Molecules with Radioisotopes 
Greater understanding of physiological processes can often 
be achieved by knowing the movements of molecules 
within and between cells. For example, we can more eas- 
ily understand the role of a particular neurohormone in 
regulating physiological processes if its movements can be 
traced from its site of synthesis to its site of release and on 
to its site of action. Many types of experiments that follow 
the movement of physiologically important molecules em- 
ploy radioisotopes, the relatively unstable, disintegrating 
radioactive isotopes of the chemical elements. The natural 
disintegration of radioisotopes is accompanied by release 
of high-energy particles, which can be detected by appro- 
priate instruments. With the exception of 12jI, which emits 
y particles, the isotopes commonly used in biological re- 
search emit p particles. 
Although radioisotopes occur naturally, those normally 
used in experimental studies are produced in nuclear reac- 
tors. The most commonly used isotopes in biological re- 
search are 32P, 12jI, 35S, 14C, 4SCa, and 3H. A radioisotope of 
an element normally present in the molecule of interesttan 
be incorporated in vitro or in vivo either directly into the 
molecule or into a precursor molecule that will eventually Caudate-putamen 
be converted into the molecule of interest. The resulting ra- 
diolabeled molecule has the same chemical and biochemi- 
cal properties as the unlabeled molecule. An amazing array 
of so-called radiolabeled biologically active molecules (e.g., 
amino acids, sugars, hormones, proteins) are now readily 
available (at a substantial price) from companies that spe- 
cialize in their production. Once a molecule has been radi- 
olabeled, the particles emitted from the radioisotope can be 
used to detect the presence of the molecule, even at very 
low concentrations. 
In one type of tracing experiment, the radiolabeled 
molecule of interest or its precursor is administered to an 
animal, isolated organ, or cells growing in uitro culture, 
and then samples are removed periodically for measure- 
ment of particle emission. Two types of instruments are 
used to detect emitted particles. A Geiger counter detects 
ionization produced in a gas by emitted energy. A scintil- 
lation counter detects and counts tiny flashes of light that 
these particles create as they pass through a specialized 
"scintillation fluid." The amount of radiation detected by 
either instrument is related directly to the amount of the ra- 
diolabeled molecule present in the sample. 
In another type of experiment, the location of radio- 
labeled molecules within a tissue section is pinpointed by 
autoradiography. In this technique, which literally "takes a 
picture" of the radioisotopes in tissues, a thin tissue slice 
containing a radioisotope is laid on a photographic emul- 
sion. Over the course of days or weeks, particles emitted 
from the radioisotope expose the photographic emulsion, 
producing black grains that correspond to the location of 
the labeled molecules in the tissue (Figure 2-1). This quali- 
tative record can be quantitated by measuring the amount 
of exposure of the emulsion in a densitometer and com- 
paring it with exposures caused by standards of known 
concentration; in this way, the actual concentration of a 
radiolabeled molecule in the tissue or portions of it can be 
determined. Autoradiography has been particularly useful 
in neurobiology, endocrinology, immunology, and other ar- 
eas of physiology involving cell-to-cell communication. 
Tracing Molecules with Monoclonal Antibodies 
Examination of a biological structure in a fixed tissue slice 
on a microscope slide can be daunting. Even when the tis- 
sue has been stained so that the cell nuclei are dark purple 
and the cell membranes a somewhat lighter shade, for ex- 
ample, it remains difficult to discern much about the details 
of the tissue. Much better visualization of the structural de- 
tails of cells is possible with antibody staining. This re- 
markable technique permits localization of molecules pres- 
ent in such extremely low concentrations that they are 
difficult to study by other techniques. 
Antibody staining generally involves covalently linking 
a flourescent dye to an antibody that recognizes a specific de- 
terminant on an antigen molecule. (Although we often think 
of antigens as disease-causing microbes or invading foreign 
materials like pollen, normal, biologically active molecules, 
Figure 2-1 Autoradiograms can reveal biochemical and structural details 
that cannot be seen with traditional techniques for tissue fixation and 
staining. This autoradiograph shows a frontal section through the rat 
brain after cannabinoid receptors have been bound by a radiolabelled 
synthetic cannabinoid (closely resembling the active ingredient of mari- 
juana). The most radioactive areas (that is, the areas with the most 
cannabinoid receptors) have most heavily exposed the photograph film 
on which the brain slice was laid, and show up primarily as the dark ar- 
eas in the striaturn (caudate-putamen), which mediate motor functions. 
[Courtesy of Miles Herkenham, NIMH.] 
such as neurotransmitters and cell growth regulators, can 
act as antigens and induce production of specific antibod- 
ies when injected into an appropriate animal.) Identical an- 
tibodies produced in response to an antigen are called mon- 
oclonal antibodies; however, most natural antigens have 
multiple, rather than single determinants, thus the produc- 
tion of several different antibodies is likely. A mixture of an- 
tibodies that recognize different determinants on the same 
antigen is called polyclonal. Once antibodies that recognize 
discrete sites on a molecule of interest have been produced 
and linked to a flourescent dye, thay can be injected into the 
cells or tissues under study. Over the past decade, re- 
searchers increasingly have used a combination of mono- 
clonal and polyclonal antibodies for antibody staining, par- 
ticularly in irnmunofluorescent microscopy (Figure 2-2). 
Alternatively, radiolabeled monoclonal antibodies can 
be used and the location of any antigen-antibody com- 
plexes that form in a sample detected by autoradiography. 
This approach has been used to localize the hormones epi- 
nephrine and norepinephrine within certain cells of the 
adrenal medulla, as described in Chapter 8 on glands. 
Monoclonal antibodies can be used not only to track down 
specific molecules within cells but also to purify them, as 
described in a later section. Such purified molecules are suit- 
able for detailed studies on their structure and function. 
The crucial advance that made antibody staining fea- 
sible was development of a method for producing large 
amounts of monoclonal antibody. Isolation and purifica- 
tion of a single type of monoclonal antibody from anti- 
serum taken from animals exposed to the corresponding 
antigen is not practical, because each type of antibody is 
present only in very small amounts. Moreover, the B 
Figure 2-2 Both monoclonal and polyclonal antibodies are frequently 
used in antibody staining. In this irnrnunofluorescent micrograph of rat 
spinal cord cultured 10 days, a mouse monoclonal antibody (green) and 
a rabbit polyclonal antibody (red) that is
specific for a single protein, along 
with a blue fluorescent dye that binds DNA directly, are used. Here we 
see neurons (red), astrocytes (green), and DNA (blue). [Courtesy of Nancy 
L. Kedersha/lrnrnuno Gen.] 
lymphocytes (or B cells) that produce antibodies normally 
die within a few days, and thus cannot be grown for ex- 
tended periods in culture. In the mid-1970s, G. Kohler and 
C. Milstein discovered that normal B cells could be fused 
with cancerous lymphocytes, called myeloma cells, which 
grow indefinitely in culture (i.e., they form an "immortal" 
cell line). The resulting hybrid cells, termed hybridomas, 
are spread out on a solid growth medium in a culture dish. 
Each cell grows into a clone of identical cells, with each 
clone secreting a single monoclonal antibody. Clones are 
then screened to identify those that secrete the desired an- 
tibody; these self-perpetuating cell lines can be maintained 
in culture and used to obtain large quantities of homoge- 
neous monoclonal antibody (Figure 2-3). Although indi- 
vidual investigators can make and maintain their own hy- 
bridoma cell lines, many now choose to have specific 
monoclonal antibodies prepared by companies specializing 
in their production. (The next time you are in your univer- 
sity or college library, find the journal Science and take a 
look at the classified ads in the back.) The development of 
monoclonal antibody technology by Kohler and Milstein 
so revolutionized molecular studies that they received the 
Nobel Prize for their research. 
Genetic Engineering 
Genetic engineering encompasses various techniques for 
manipulating the genetic material of an organism. This ap- 
proach is increasingly used in both agriculture and medi- 
cine, and it offers considerable promise for investigators in 
animal physiology. These techniques make it possible to 
produce large quantities of biologically important mole- 
cules (e.g., hormones) normally present at very low con- 
centrations, animals with mutations that affect specific 
physiological processes, and animals that synthesize above- 
or below-normal amounts of specific gene products. 
Genetic engineering begins with identification of the 
structural gene that codes for a specific protein within the 
DNA isolated from an organism of interest. For example, 
the gene that encodes human insulin can be identified in 
DNA isolated from human cells. The section of DNA con- 
taining the insulin gene of interest can be "clipped out" of 
the original very long human DNA strands and then in- 
serted into a cloning vector, which is a DNA element that 
can replicate within appropriate host cells independently of 
the host cells' DNA. Insertion of a fragment of foreign 
DNA (e.g., the human insulin gene) into a cloning vector 
yields a recombinant DNA, which is any DNA molecule 
containing DNA from two or more different sources. 
Bacterial plasmids are a common type of cloning vec- 
tor. These are extrachromosomal circular DNA molecules 
that replicate themselves within bacterial cells. Under cer- 
tain conditions a recombinant plasmid containing a gene of 
interest is taken up by the common bacterium Escherichia 
coli, a process called transformation (Figure 2-4). Nor- 
mally, only a single plasmid molecule is taken up by any 
one bacterial cell. Within a transformed cell, the incorpo- 
rated plasmid can replicate, and as the cell divides a group 
of identical cells, or clone, develops. Each cell in a clone 
contains at least one plasmid with the gene of interest. This 
general genetic engineering procedure, called DNA or gene 
cloning, can be used to obtain a DNA "library" consisting 
of multiple bacterial clones, each of which contains a spe- 
cific gene from humans or other species. Several variations 
of DNA cloning are used depending on the size and num- 
ber of the genes in the organism being studied. 
Clonal populations for medicine and research 
Under appropriate environmental conditions, the recombi- 
nant DNA in an "engineered" E. coli clone is transcribed 
into messenger RNA, which is used to direct synthesis of 
the encoded protein. Commercial companies, for example, 
grow E. coli cells carrying recombinant DNA containing 
the gene for human insulin or other hormones in huge vats; 
after the bacterial cells are harvested, large quantities of the 
human hormone can be isolated relatively easily. In the 
past, hormones needed for treating humans with endocrine 
disorders were extracted from the tissues of other mammals 
such as cows and pigs. Because hormones are present in 
quite low concentrations, this is a time-consuming and ex- 
pensive process. Producing these hormones with genetically 
engineered bacteria has proven to be far less expensive and 
yields a purer product. Moreover, hormones isolated from 
other mammalian species often induce an immune response 
in humans, a complication not encountered with human 
hormones obtained from engineered bacteria. 
Recombinant DNA technology is also a powerful tool in 
basic research on human genetic disorders. By isolating and 
studying genes associated with hereditary diseases, scientists 
can determine the molecular basis of these diseases. This will 
W Figure 2-3 Hybrldoma cell llnes secrete "pure" (homogeneous) monoclonal antibod- 
les To prepare monoclonal antlbodles, antl- 
body-producing spleen cells flrst are fused 
w~th myeloma cells orlglnally derlved from B Cell-culture lymphocytes The hybr~d cells, or hybrldomas, 
that secrete antlbody speclflc for the proteln 
of Interest are separated out They can be 
Fuse In malntalned In cell culture, where they secrete 
polyethylene glycol large quantites of the speclflc antibody, or in 
n \ n jected into a host mouse, where they induce 
0 0 - @ Q @ 
000 @ @ 
Spleen cells Myeloma cells 
I Transfer cells to selective medium 
1 Select hybrid cells 
1 Select cells that produce a desired antibody 
Grow in 
mass culture 
Antibody Antibody 
the production of the antlbody. 
certainly lead to better methods of controlling or even cur- normal gene product helped alleviate most of the symp- 
ing them. Over the last several years numerous laboratories toms of cystic fibrosis in the treated patients. 
worldwide have been engaged in a massive project to "map" 
the locations of all human genes on the long strands of DNA 
in human chromosomes and determine their nucleotide se- 
quences. This Human Genome Project is providing invalu- 
able data for researchers studying genetic diseases. 
DNA cloning and recombinant DNA technology also 
form the basis for gene therapy. In this approach to treat- 
ing those with genetic disorders, the normal form of the 
gene that is missing or defective is introduced into patients. 
For example, persons with cystic fibrosis have a defective 
CFTR gene and thus cannot produce the normal protein 
encoded by this gene. One result of this defect is production 
of a very thick mucus in the lungs' airways, which leads to 
potentially lethal breathing problems. Molecular biologists 
have engineered common cold viruses with the normal 
CFTR gene. When some cystic fibrosis patients were in- 
fected with an engineered cold virus, the viral particles car- 
ried the normal human gene into the patients' lung cells, 
where it became established. Subsequent synthesis of the 
20 PRINCIPLES OF PHYSIOLOGY 
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 
Plasmid vector mutations that cause a heart with abnormallv thin ven- 
tricular walls and another with a constriction of the arte- 
DNA fragment rial outflow tract of the heart. Both of these conditions 
mimic human disease states. 
Mutations often produce abnormal effects only in the 
homozygous state (i.e., when an individual
receives a mu- 
tated form of a gene from each parent). Even when a mu- 
tation causes a lethal condition incompatible with long- 
term survival, it can be "preserved" in the parents, who are 
heterozygous for the mutation, carrying one normal and 
one mutated form of the gene. Each time these parents 
breed, some of the offspring will be homozygous and show 
the abnormal effects. Thus, the heterozygous parents are 
a "living gene library" of these mutations. 
Bacterial chromosome 
Figure 2-4 DNA cloning is a way to isolate and maintain individual 
genes. In the cloning procedure illustrated, a specific DNA fragment to 
be cloned is inserted Into a plasmid vector, whichalso contains a gene 
conferring resistance to the antibiotic ampicill~n. When the resulting re- 
combinant plasm~ds are mixed with E. colicells, a few cells take up a plas- 
mid, which can replicate within the cells. If the cells are placed in media 
containing ampicillin, only those that have taken up the vectorwill grow. 
As each selected cell multiplies, it eventually forms a colony of cells 
(clone) all containing the same recombinant plasmid. 
"Made-to-order" mutants 
As mentioned in Chapter 1, mutations are permanent 
changes in the nucleotide sequence of DNA. Mutations, 
which can occur spontaneously or be induced experimen- 
tally, are duplicated and passed on to daughter cells at the 
time of cell division. Mutant genes can tell us a great deal 
about how physiological processes work. The specific dis- 
ruption in a physiological process resulting from a single 
mutant gene can pinpolnt the functions controlled by par- 
ticular genes, information that may not be revealed by con- 
ventional physiological techniques. 
For example, cardlovascular physiologists are produc- 
ing and analyzing the effects of mutations in zebrafish to 
understand heart development. In research described by 
J-N. Chen and M. Fishman (1997), dozens of specific car- 
diovascular mutations have been produced in zebrafish. 
The process starts when adult zebrafish are exposed to 
powerful mutagens-compounds that produce perma- 
nent mutations in the germ cell line. Subsequent matings 
of the F, and F2 generations lead to embryos with large 
numbers of mutations. Very rarely, an embryo will appear 
with just one specific mutation in a structure or process of 
interest. The Fishman group, for instance, has identified 
Transgenic animals 
Transgenic animals are another type of genetically engi- 
neered organism with the potential for making great con- 
tributions to physiology. A transgenic animal is one whose 
genetic constitution has been experimentally altered by the 
addition or substitution of genes from other animals of 
the same or other species. Transgenic animals (especially, 
mice) are at the forefront of the menagerie of animal mod- 
els that are helping researchers understand basic physio- 
logical processes and the disease states that results from 
their dysfunction. 
Numerous techniques have been employed to produce 
transgenic animals. In one method, "foreign" DNA con- 
taining a gene of interest, called a transgene, is injected into 
a pronucleus of fertilized eggs (commonly from mice), 
which then are implanted into pseudopregnant females. At 
a relatively low frequency, the transgene is incorporated 
into the chromosomal DNA of the developing embryos, 
leading to offspring that carry the transgene in all their 
germ-line cells and somatic cells (Figure 2-5). Mice ex- 
pressing the transgene then are mated to produce a trans- 
genic line. This approach is used to add functional genes, 
either extra copies of a gene already present in the animal 
or a gene not normally present, leading to overexpression 
of the gene product. Subsequent analysis of the morphol- 
ogy and physiology of the transgenic animals can provide 
considerable insight into physiological processes that can- 
not easily be investigated in other ways. 
Transgenic animals characterized by underexpression 
or complete lack of expression of a particular gene can be 
equally informative. M. R. Capecchi (1994) has reviewed 
a procedure for replacing a functional gene with a defective 
one, thereby producing so-called knockout mice. These 
mice cannot express the protein originally coded for by the 
replaced gene and thus lack the functions mediated by the 
missing protein. The molecular and genetic basis of physi- 
ological processes can be determined by examining the ef- 
fect of such functional ablation of genes. Knockout mice 
are used extensively to unravel human physiological 
processes, because human and mouse genes are greater 
than 98% identical. To cite a couple of examples, re- 
searchers are investigating the normal genes that regulate 
EXPERIMENTAL METHODS FOR EXPLORING PHYSIOLOGY 21 
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 
I Fertilized eggs collected from female 
I Injected eggs implanted into oviduct of new female 
individual cells, are used to measure various properties of 
cells or inject materials into them. Although cellular phys- 
iologists employ these devices in a variety of ways, the 
technology used to make them is decades old. Essentially, 
a region in the middle of a glass capillary tube is heated to 
the point of melting. The ends of the tube are then pulled 
apart, which draws the soft spot in the glass down to an 
invisibly small diameter before it breaks and separates. 
Two micropipettes, each with a drawn-down tip as small 
as just a micron in diameter, are produced as a result. 
When a micropipette is filled with an appropriate solu- 
tion, it can function as a microelectrode. Typically, a mi- 
cropipette (or microelectrode) is mounted in a microma- 
nipulator, a mechanical device that holds the pipette 
steady and allows its tip to be moved incrementally in 
three different planes. 
Measuring electrzcal properties 
Since neurons communicate via electrical signals, micro- 
electrodes can be used to "eavesdrop" on their communi- 
cation by measuring the electrical signals across the cell 
membrane and changes In these signals under different con- 
ditions. The microelectrodes used to measure the electric 
10-30% of offspring contain transgene potential (voltage) across the cell membrane cause virtually 
1 no flow of current from the cell into the electrode. Thus lit- tle or no disruption of the nerve cell occurs even as its com- axQ munication A microelectrode with neighboring for recording cells is electrical being detected. signals from neurons or muscle cells is made by filling a micropipette 
Breed transgenics to ma~ntain DNA in germ line with an ionlc conducting solution (typically KCl) and con- 
Figure 2-5 A transgenic animal is produced by adding or substituting 
genes from another animal of the same or different species. To introduce 
a transgene into mice, cloned "foreign" DNA is injected into fertilized 
eggs, which then are implanted into a female. A proportion of the viable 
offspring will retain the transgene, which can be maintained in the germ 
line by selective breeding. 
early heart development in the embryo and the oncogenes 
responsible for some typ~s of cancer in studies with knock- 
out mice. 
CELLULAR TECHNIQUES 
Understanding cells and cellular behavior is a goal of many 
experiments in physiology. With a knowledge of cellular 
behavior and communication, we can begin to understand 
how communities of cells function as tissues, and tissues as 
organs. Physiological analysis at the cellular level has been 
pursued most vigorously using several now-standard tech- 
niques. In this section we discuss three very common and 
productive cellular techniques: recording with microelec- 
trodes, microscopy, and cell culture. 
Uses of Microelectrodes
and Micropipettes 
Many experiments in cellular physiology make use of mi- 
cropipettes or various types of microelectrodes. These tiny 
glass "needles," which can be inserted into tissues or even 
necting it to an appropriate amplifier. A second electrode 
connected to the amplifier is placed in the fluid or organism 
in the vicinity of the first electrode. When the tip of the first 
electrode is pushed through the cell membrane into the cy- 
toplasm, it completes an electric circuit whose properties 
(voltage, current flow) can be measured. Since microelec- 
trode recording techniques were introduced in the 1950s, 
our understanding about the electrical activities within a 
cell have increased dramatically. 
One of the most revolutionary advances in microelec- 
trode recording methodology is patch clamping. With this 
technique, the behavior of a single protein molecule con- 
stituting an ion channel can be recorded in situ (Latin for 
"in its normal place"), as illustrated in Figure 2-6. This 
method lies at the heart of the recent explosion of knowl- 
edge about membranes, including their channels and how 
they regulate the movement of materials across the mem- 
brane (see Chapters 4-6). 
Measuring ion and gas concentrations 
Specially constructed microelectrodes can be used to probe 
the intracellular concentration of common inorganic ions 
including H+, Na+, K+, C1- , Ca2+, and Mg2+. Because cells 
use movements of ions across cell membranes to commu- 
nicate and to do work, the magnitude, direction, and time 
course of ion movements provide important information 
about certain processes. ~croelectrodes that measure the 
22 P R I N C I P L E S O F P H Y S I O L O G Y 
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 
A 
Flu~d to conduct 
Figure 2-6 Patch-clamp recording permits determination of ion move- 
ment across a small patch of membrane containing transmembrane ion 
channels. (A) Diagram of patch clamp in place. When a fire-polished mi- 
croelectrode is placed against the cell surface, a very high resistance seal 
forms between the electrode tip and the membrane This t~ght seal 
allows direct measurement of the membrane features beneath the tip 
Typically, only a few transmembrane ion channels lie beneath the t~p, al- 
lowing the current flowthrough them to be measured directly (0) Photo- 
micrograph showing tip of a patch mlcropipette abutting the cell body 
of a nerve cell The tip has a diameter of about 0 5 p m [Part B from 
Sakmann, 1992.1 
partial pressure of gases (e.g., 0, and CO,) dissolved in a 
fluid also are now available. 
The tip of a microelectrode for measuring the concen- 
tration of a particular ion (e.g., Na+) is plugged with an 
ion-exchange resin that is permeable only to that ion. The 
remainder of the electrode (the "barrel") is filled with a 
known concentration of the same ion. The electrical po- 
tential measured by the microelectrode when no current 
flows reflects the ratio of the ion concentrations on the two 
sides of the ion-exchange barrier in the tip. Proton-selective 
microelectrodes are particularly useful for measuring the 
pH of blood and other body fluids. 
Measuring intracellular and blood pressure 
Microelectrodes are now being used to measure hydro- 
static pressures within individual cells and microscopic 
blood vessels-indeed, in any fluid-filled space into which 
the tip of a microelectrode can be inserted. To understand 
the principle of such micropressure systems, let's consider 
a small blood vessel. A microelectrode, filled with at least 
a 0.5 M NaCl solution and mounted in a micromanipula- 
tor, is inserted into the vessel of interest. The higher pres- 
sure inside the vessel causes the interface between the 
plasma and the solution filling the electrode to move into 
the electrode. This results in increased resistance across the 
electrode tip, because the resistance of plasma is higher 
than that of the NaCl solution. The change in resistance is 
measured and is proportional to the change in blood pres- 
sure. A motor-driven pump associated with the micro- 
pressure system produces a pressure in the microelectrode 
that just offsets the pressure in the vessel. This opposing 
pressure keeps the interface at a stationary position; there- 
fore, it is called a servo-null system. The required offset- 
ting pressure generated in the micropressure system is then 
monitored with a conventional pressure transducer such 
as would be used for measuring blood pressure in much 
larger vessels. 
Micropressure systems have greatly extended our 
knowledge of the development of cardiovascular function 
in developing embryos and larvae. These techniques have 
also allowed direct cardiovascular measurements in adults 
of very small animals like insects. 
Microinjecting materials into cells 
In addition to their use as microelectrodes, micropipettes 
also can be used to inject substances into individual cells. 
These substances may be active molecules that produce a 
measurable change in cell or tissue function. For instance, 
drugs that influence blood pressure and heart rate can be 
injected into very small blood vessels (e.g., those lining the 
shell of a bird egg) or into the microscopic heart of a frog 
embryo. 
Alternatively, the injected substance may be a dye used 
to mark injected cells, helping to reveal cell processes or to 
trace cells as they divide. A classic variation of this tech- 
nique involves horseradish peroxidase, an enzyme derived 
from the horseradish plant that forms a colored product 
from specific colorless substrates. When this enzyme is in- 
jected via a micropipette into the extensions (especially ax- 
o n ~ ) of neurons, it is taken up and transported back to the 
neuron's cell body; subsequent injection of the substrate 
generates a colored "trail" between the injection site and 
cell body. By this technique, peripheral nerves can be traced 
back to their origin in the central nervous system, a task 
that would defy even the most skilled neuroanatomist us- 
ing more traditional techniques. 
Structural Analysis of Cells their constituents, typically by cross-linking amino groups 
Cellular function is dependent on cellular structure, reaf- 
firming the central theme discussed in Chapter 1 that strong 
structure-function relationships govern animal physiology. 
Physiologists commonly use structural analyses at the cel- 
lular level to complement physiological measurements in 
order to discover how animals function. Such analyses de- 
pend on various types of microscopy, because animal cells 
are typically about 10-30 pm in diameter, which is well 
below the smallest particle visible to the human eye. 
Light microscopy 
Light microscopy, as its name implies, uses the photons of 
visible or near visible light to illuminate specially prepared 
cells. Under optimal conditions, the resolution, or resolving 
power, of light microscopes is a few microns; two objects 
that are located closer to each other than a microscope's 
resolution will appear as one. As the resolution of micro- 
scopes has been improved, our understanding of the struc- 
ture of cells and their components has increased. 
Since cells removed from a living animal rapidly die, tis- 
sue must be prepared quickly to prevent degradation of cel- 
lular constituents. Fixation is the addition of a specialized 
chemical (e.g., formalin) that kills the cells and immobilizes 
of proteins with covalent bonds. The fixed cells then are 
treated with dyes or other reagents that stain particular cel- 
lular features, allowing visualization of the cells, which oth- 
erwise are colorless and translucent. 
Fixation and staining of large blocks of tissue is im- 
practical and does not