Fish Physiology: Sensory Systems and Electric Organs

T Volume V Sensoy System and

Electric Organs

CONTRIBUTORS M. V. L. BENNETI'

0. LOWENSTEIN

J. DIAMOND

F. W. MUNZ

AKE FLOCK

R. W. MURRAY

TOSHIAKI J. HARA

WILLIAM N. TAVOLGA

DAVID INGLE

T. TOMITA

FISH PHYSIOLOGY Edited by W. S. HOAR DEPARTMENT OF ZOOLOGY UNIVERSITY O F BRITISH COLUMBIA VANCOUVER, CANADA

and

D . J. R A N D A L L DEPARTMENT OF ZOOLOGY UNIVERSITY OF BRITISH COLUMBIA VANCOWER, CANADA

Volume V

Sensory Systems and Electric Organs

(23

Academic Press New York and London

1971

COPYRIGHT @ 1971, BY ACADEMIC PRESS, INC. ALL RIGHTS RESERVED NO PART OF THIS BOOK MAY BE REPRODUCED IN ANY FORM, BY PHOTOSTAT, MICROFILM, RETRIEVAL SYSTEM, OR ANY OTHER MEANS, WITHOUT WRIlTEN PERMISSION FROM THE PUBLISHERS.

ACADEMIC PRESS, INC.

111 Fifth Avenue, New York, New York 10003

United Kingdom Edition published by ACADEMIC PRESS. INC. (LONDON) LTD. 24/28 Oval Road, London N W l ID’D

LIBRARY OF CONGRESS CATALOG CARDNUMBER: 76-84233

PRINTED IN THE UNITED STATES OF AMERICA

CONTENTS ix

LISTOF CONTRI~UTORS PREFACE

xi xiii

OF OTHERVOLUMES CONTENTS

1. Vision: Visual Pigments F. W. Munz 1

I. The Eye as an Optical System 11. Visual Pigments References

14 27

2. Vision: Electrophysiology of the Retina

T . Tomita I. 11. 111. IV. V. VI.

Introduction Electroretinogram Response of Single Ganglion Cells Response of Photoreceptors Responses in the Inner Nuclear Layer Retinal Mechanisms of Color Vision References

33 35 40 43 47 51 53

3. Vision: The Experimental Analysis of Visual Behavior

David Ingle I. 11. 111. IV. V. VI.

Introduction Relative Discrimination Weaknesses Configurational Properties of Shapes Perceptual Equivalence and Change in Spatial Position Selective Attention Toward a Unified Outlook on Visual Behavior References V

59 61 64 68 72 74 76

CONTENTS

vi

4. Chemoreception Toshiaki 1. Hara I. 11. 111. IV. V.

Introduction Anatomy of Chemical Sense Organs Behavioral Studies of Chemoreceptive Functions Electrophysiological Studies of Chemoreceptor Responses Biological Aspects of Chemoreception References

79 81 91 94 104 114

5. Temperature Receptors R. W. Murray I. Introduction 11. Thermal Sensitivity of Fishes 111. The Sense Organs Involved IV. Electrophysiology V. Thermal Responses of Other Sense Organs References

121 121 123 125 131 132

6. Sound Production and Detection William N . Tavolga I. 11. 111. IV. V.

Introduction Sound Production Sound Detection Acoustic Communication in Fish Problems and Prospects for the Future References

135 136 162 183 189 192

7. The Labyrinth 0. Lowenstein I. Structure 11. Function References

207 214 236

8. The Lateral Line Organ Mechanoreceptors Ake Flock I. 11. 111. IV. V.

Introduction Structure of the Sense Organ Sensory Excitation in the Hair Cell Transmission at the Sensory Synapse Initiation of Nerve Impulses

241 242 248 255 258

CONTENTS

VI. VII.

The Central Nervous System and Feedback Conclusion References

Vii

259 261 262

9. The Mauthner Cell 1. Diamond I. Introduction The Basic Anatomy of the Mauthner Neuron The Selective Activation of Mauthner Neurons The “Mauthner Reflex” The Spinal Circuitry The Anatomy of the Spinal Circuitry The Precision and Constancy of the Minimum Discrimination Time The Excitation of the Mauthner Cells The Functions of the Mauthner Cells References

11. 111. IV. V. VI. VII. VIII. IX.

265 267 271 278 284 297 310 315 331 344

10. Electric Organs

M . V. L. Bennett I. 11. 111. IV.

Introduction Electric Organs and Electrocytes Neural Control of Electric Organs Conclusions and Prospects References

347 355 460 483 484

11. Electroreception M . V. L. Bennett I. 11. 111. IV. V. VI. VII.

Introduction Distribution of Electroreceptors Tonic Electroreceptors Phasic Electroreceptors Receptor Function in Electroreception Evolution of Electrosensory Systems and Electric Organs Implications for Receptor Function in General References

493 496 503 520 544 561 564 568

AUTHORINDEX

575

SYSTEMATIC INDEX

587

SUBJECTINDEX

594

LIST OF CONTRIBUTORS Numbers in parentheses indicate the pages on which the authors’ contributions begin.

M. V. L. BENNETT(347, 493), Department of Anatomy, Albert Einstein College of Medicine, Yeshiva University, Bronx, N e w York

J . DIAMOND*(265), Department

of Physiology, University College

London, London, England Am FLOCK (241), King Gustaf V Research Institute, and Department of Otolaryngology, Karolinska Sjukhuset, Stockholm, Sweden TOSHIAKI J. H A R A(79), ~ Department of Physiology, Kumumoto University Medical School, Kumumoto, Japan

DAVIDINGLE (59), The Neurophysiological Laboratory, McLean Hospital, Belmont, Massachusetts 0. LOWENSTEIN (207), Department of Zoology and Comparative Physiology, University of Birmingham, Birmingham, England F. W. MUNZ (l), Department of Biology, Uniuersity of Oregon, Eugene, Oregon R. W . MURRAY(121), Department of Zoology and Comparative Physiology, University of Birminghum, Birmingham, England WILLIAMN . TAvoLGh ( 1351, Department of Biology, T h e City College of the City University of New York, New York, New York

T. TOMITA(33), Department of Physiology, Keio University, School of Medicine, Shinjuku-ku, Tokyo, Japan

* Present address: Department of Neurosciences, McMaster University, Hamilton, Ontario, Canada. f Present address: Fisheries Research Board of Canada, Freshwater Institute, Winnipeg, Manitoba, Canada. ix

This Page Intentionally Left Blank

PREFACE Volume V of this treatise is concerned with sensory systems and some aspects of function of the central nervous system. Sensory systems have been extensively studied in fish not only because of a wide general interest in the behavioral and sensory physiology of this group but also because, in many instances, fish are technically suitable for general studies of sensory systems and have certain receptors not present in other groups. Electroreceptors fall into this category; these receptors are unique to fishes, and studies of this system have application to receptor function in general. Electric organs, an effector rather than receptor system, are discussed in this volume because of the functional relationships between electroreception and electric organ discharge. The Mauthner neuron which is another system studied both to increase understanding of neuronal organization in fish and because the Mauthner cell constitutes a useful preparation for studying synaptic function and the integration of activity in neuronal networks in general is discussed in another chapter. Neurophysiology, particularly sensory physiology, is a very active area of biology. The chapters in this volume, perhaps more than in other volumes, can only present a summary of the present state of science in this rapidly expanding and developing field. We hope that this volume reflects some of the excitement and activity in sensory physiology and will be a useful introduction to students in this area of biology. W. S. HOAR D. J. RANDALL

xi

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CONTENTS OF OTHER VOLUMES Volume I The Body Compartments and the Distribution of Electrolytes W. N . Holmes and Edward M . Donaldson The Kidney Cbveland P. Hickman, Jr., and Benjamin F . Trump Salt Secretion Frank P. Conte

The Effects of Salinity on the Eggs and Larvae of Teleosts F. G. T . Holliday Formation of Excretory Products Roy P. Forster and Leon Goldstein Intermediary Metabolism in Fishes P. W. Hochachka Nutrition, Digestion, and Energy Utilization Arthur M . Phillips, Jr. AUTHORINDEX-SYSTEMATIC

INDEX-SUB

JECT INDEX

Volume I1 The Pituitary Gland: Anatomy and Histophysiology J. N . Ball and Bridget 1. Baker The Neurohypophysis A. M . Perks Prolactin (Fish Prolactin or Paralactin) and Growth Hormone 1. N . Ball Thyroid Function and Its Control in Fishes Aubrey Gorbmun xiii

XiV

CONTENTS OF OTHER VOLUMES

The Endocrine Pancreas August E p p b The Adrenocortical Steroids, Adrenocorticotropin and the Corpuscles of Stannius 1. Chester Jones, D. K . 0 . Chan, I . W. Henderson, and J . N . Ball

The Ultimobranchial Glands and Calcium Regulation D. Harold Copp Urophysis and Caudal Neurosecretory System Howard A. Bern AUTHORINDEX-SYSTEMATICINDEX-SUBJECT INDEX Volume I11 Reproduction Willium S. Hoar Hormones and Reproductive Behavior in Fishes N . R. Liley Sex Differentiation Toki-o Yamumoto Development: Eggs and Larvae J . H . S . Blaxber Fish Cell and Tissue Culture Ken Wolf and M . C . Quimby Chromatophores and Pigments Ryozo Fujii Bioluminescence J. A. C . Nicol Poisons and Venoms Findlay E . Russell AUTHORINDEX-SYSTEMATIC INDEX-SUBJECT INDEX Volume IV Anatomy and Physiology of the Central Nervous System Jerald 1. Bernstein

CONTENTS OF OTHER VOLUMES

xv

The Pineal Organ James Clurke Fenwick Autonomic Nervous Systems Graeme Campbell The Circulatory System D. 1. Randall Acid-Base Balance C. Albers Properties of Fish Hemoglobins Austen Riggs Gas Exchange in Fish

D. J. Randall The Regulation of Breathing G . Shelton Air Breathing in Fishes Kiell Johansen

The Swim Bladder as a Hydrostatic Organ Johan B. Steen Hydrostatic Pressure Malcolm S. Gordon Immunology of Fish John E . Cushing AUTHOR INDEX-SYSTEMATIC INDEX-SUBJECT INDEX Volume VI The Effect of Environmental Factors on the Physiology of Fish: An Examination of the Different Categories of Physiological Adaptation F . E. J. Fry Action of the Environment on Biochemical Systems P. W . Hochachka and G. N . Somero Freezing Resistance in Fishes Arthur L. DeVries

xvi

CONTENTS OF OTHER VOLUMES

Learning and Memory Paul Rozin and Henry Gleitman The Ethological Analysis of Fish Behavior G . P . Baerends Biological Rhythms H . 0 . Schwasmnn Orientation and Fish Migration A. D . H a s h Special Techniques D. J . Randall and W. S. Hoar

AUTHORINDEX-SYSTEMATICINDEX-SUBJECT INDEX

1 VISION: VISUAL PIGMENTS F . W.M U N Z

. . . . . . I. The Eye as an Optical System A. Structure of the Eye . . . . . . . B. Image Formation and Accommodation . . . . C. Light and Dark Adaptation . . . . . . D. Specializations for Deep-sea Life . . . . . 11. Visual Pigments . . . . . . . . . A. Photochemistry . . . . . . . . . B. Methods of Study . . . . . . . . C. A Choice of Retinenes: Rhodopsin and Porphyropsin D. A Multiplicity of Opsins . . . . . . E. Pigments of Color Vision . . . . . . References . . . . . . . . . . .

.

. . . . .

.

. . .

. .

. .

. . . .

. . . .

. .

1 1 5 8 12 14 14 15 19 23 25

27

I. THE EYE AS AN OPTICAL SYSTEM

The fascinating and detailed study by Walls (1942) remains a primary source of information on functional anatomy of the eye, but the less well known works of Rochon-Duvigneaud (1943, 1958) should also be consulted. Valuable reviews of the structure and function of fish eyes have been prepared by Brett (1957) and Nicol (1963). A treatment by Prince (1956) contains considerable information on fishes, but its usefulness is reduced by incomplete documentation. The present brief account is intended to give necessary background, together with the results of recent research; wherever specific reference is omitted, please refer to Walls (1942). A. Structure of the Eye The eyes of fishes are constructed along the general vertebrate plan (Fig, 1 ) , They are more or less flattened and have the normal complement 1

2

F. W. MUNZ

&epchoOriM

VFd

lymph space

susmnsorv I I I \

Fig. 1. Diagrammatic vertical section of a typical teleost eye. Not all structures shown are present in every teleost eye; e.g., hyaloid vessels are not present in conjunction with a falciform process. From Walls ( 1942).

of s i x oculomotor muscles. Except in cyclostomes, in which it is fibrous, the sclera is usually reinforced with cartilage. This is often calcified in elasmobranchs; teleosts frequently have one or two scleral ossicles in addition to cartilaginous support. Little refraction occurs at the corneal surface, for its refractive index approximately equals that of water. In air, however, the fish eye is myopic because of the added refraction. Baylor (1967b) has shown that in flying fish the corneal surface is pyramidal, with the lower third covered by a flat face. When the flying fish is airborne, its downward vision should be relatively undistorted. Many fastswimming teleosts have additional streamlining and protective structures, the transparent “adipose eyelids.” These vertical folds, which are really adipose in Mugil, but not in clupeoids or scombroids, overlie the cornea in various configurations. Many sharks have mobile eyelids; in some species there is an active nictitating membrane.

1.

VISUAL PIGMENTS

3

The lens is usually spherical and protrudes through the pupil. In lampreys it is held against the cornea by the vitreous humor; no suspensory ligaments or muscles are attached to the lens. In most teleosts the pupil is immobile, but in many elasmobranchs the iris is capable of extensive contraction. In the absence of corneal refraction, protrusion of the lens through the pupil assures a wide field of view. The eye accommodates by small movements of the lens. In teleosts, it is pulled backward by a retractor muscle; in elasmobranchs, it is pulled forward by a protractor muscle. The lens or cornea of many fishes contains pigments that filter out violet or ultraviolet radiation, probably improving visual acuity (Kennedy and Milkman, 1956; Denton, 1957; Motais, 1957). Little is known about the aqueous or vitreous humors; among different species, the vitreous ranges in consistency from a liquid to a firm gel. Continuous with the peripheral border of the iris is the complex tissue named “choroid” ( alternatively spelled chorioid) . The choroid combines the functions of nourishing the retina and of absorbing stray light or reflecting it by a tapetum lucidum back through the retina (Section I, C ) . In common with other nervous tissue, the retina has a high oxygen consumption (Lindeman, 1943). The innermost part of the choroid, lying just behind the retina, is modified into a choriocapillary structure. In most teleosts, but not in elasmobranchs, the choroid projects through the optic cleft into the posterior chamber of the eye, as the richly vascular, pigmented falciform process (Fig. 1; see Hanyu, 1959). The implication of a nutritive function for this process is strengthened by the occurrence of vitreal blood vessels closely applied to the retinal surface (Fig. 1) only in those species lacking the falciform process (e.g., eels, puffers, and anglerfishes). Most teleosts ( and Amia) have a peculiar, specialized “choroid gland,” which is actually a rete mirabile, located behind the retina. Its structural similarity to the gas gland of teleost swim bladders led Wittenberg and Wittenberg (1962) to measure the oxygen pressure in the eyes of living marine fishes. In the vitreous, immediately in front of the retina, the partial pressure of oxygen was highest (average values 250420 mm Hg) in teleosts with a prominent rete. Teleosts with smaller retia had lower oxygen pressures (20-210 mm); elasmobranchs and teleosts which lack a choroid gland had still lower pressures ( 10-20 mm) . Clearly, active secretion of oxygen (the second case known in animals) is associated with the choroid gland. Those fishes that have lost the pseudobranch invariably lack a choroid gland also. Only a general description of the retina is given here; additional information may be found in the section on visual electrophysiology. Fish retinae are organized according to the ordinary vertebrate plan. Innermost are the various neuronal and glial elements, which are relatively transparent. Light passes through these to the photoreceptor (“visual”) cells.

4

F. W. MUNZ

Outermost, adjacent to the choroid, is the pigment epithelium (Section I, C). The visual cells of lampreys are usually of two morphologically distinct types, but their affinities with rods and cones of other vertebrates are not certain (Walls, 1942). Elasmobranchs and teleosts each depart from the familiar duplex pattern of vertebrates, but in different ways. Most elasmobranchs are thought to have pure-rod retinae, but some sharks ( Mustelus, Lamna, Squutina, Negaprwn, Carcharinus, Sphyrna, and Ginglymostm) and rays (Myliobatis and Dasyatis) are reported to have cones as well as rods (Walls, 1942; Rochon-Duvigneaud, 1943; Gruber et al., 1963; Hamasaki and Gruber, 1965; Tamura and Niwa, 1967). In contrast, teleosts typically have both cones and rods. In addition to the ordinary, single cones, they also have peculiar visual cells called “twin cones.” These differ from the double cones found in most vertebrate groups, in that the “twins” in each cell pair are morphologically similar and fused longitudinally. Certain teleosts, in which a tapetum is well developed, lack single cones (e.g., anchovies; see Tamura, 1957; OConnell, 1963). In many deep-sea fishes, cones are entirely absent (Section I, D). It is probably typical of teleosts that the visual cells are not distributed uniformly over the retina. Commonly there is a specialized temporal “area” in which cones are more numerous. The area may represent a concentration of twin cones alone (as in anchovies, which lack single cones) or of both twin and single cones ( OConnell, 1963). A cone-rich area also occurs in the shark, Mustelus. The fovea is a further retinal specialization for increased visual acuity and consists of a depression or pit in the retina, overlying the area of more numerous (and often smaller) visual cells. Blood vessels are typically absent from the fovea. The occurrence of a fovea is accompanied by the ability to fixate the image of a moving object on this special region by voluntary eye movements. Fixation is normally associated with binocular vision. In the laterally placed eyes of fishes, it is not surprising, therefore, that foveae are usually located near the posterior (temporal) border of the retina. Rods and twin cones are nearly or entirely absent in the fovea, which has concentrations of single cones. The pupil is usually larger than the lens in these eyes; in other words, there is an aphakic space anterior to the lens (Walls, 1942). This reduction of the iris allows light from objects directly ahead of the fish to be focused on the fovea. Retinal images of objects viewed to the side must be degraded, of course, by light passing through the aphakic space. Actually, foveae are rare in teleosts; they have been described in about 20 littoral marine species (Kahmann, 1936; Baron and Verrier, 1951). Foveae have also been reported in freshwater species of Fundulus and Umbridw (Prince, 1956). Temporal foveae occur in the

1.

VISUAL PIGMENTS

5

pure-rod retinae of several genera of deep-sea fishes: Platytroctes and Bathytroctes (Brauer, 1908; the latter may be Sear&, according to Munk, 1966), Bathylugus (Vilter, 1954a,b; Munk, 1966), Scopelosaurus and Searsia ( Marshall, 1966), and Platytroctegen ( Munk, 1966). Because several families are represented, it seems probable that pure-rod foveae have evolved more than once. As in shallow-water teleosts, a prominent aphakic space is correlated with these temporal foveae (Marshall, 1966). One other pure-rod fovea has been reported in the rhynchocephalian, Sphenodon (Walls, 1942), but Vilter (1951) has shown that its visual cells are cones. Therefore, the pure-rod foveae of deep-sea fishes are unique.

B. Image Formation and Accommodation In all groups of fishes the corneal index of refraction is about the same as that of water (1.33)and the ocular humors. Refraction and image formation, therefore, depend almost entirely upon the lens. The lens is spherical, with a very high effective index of refraction (about 1.67). Since the maximum index for any transparent material of biological origin is about 1.53 (Pumphrey, 1961), the fish lens cannot be homogeneous. That its refractive index is highest at the center (1.53) and gradually decreases (to 1.33) toward the outside was confirmed by Pumphrey ( 1961), who showed that the lens has no spherical aberration. This permits unaberrated image formation without stopping down the lens ( f / 0 . 8 in teleosts). Stopping down would be disadvantageous because the lack of corneal refraction means that the lens must protrude through the iris to achieve a wide visual field. This same gradation of refractive index from 1.53 to 1.33occurs in the small lenses of young fish and large lenses of older fish. The lens substance shows concentric discontinuities, suggestive of growth increments; but the way in which the index of refraction of the inner and outer parts could be altered continuously and differentially during growth is not understood. The lens lacks any appreciable chromatic aberration, but the means by which this is achieved is also unknown (Pumphrey, 1961). An almost constant feature of fish eyes is the distance from the center of the lens to the retina divided by the radius of the lens. This ratio is about 2.55 ( Matthiessen’s ratio). Accommodation results from changing the distance between the lens and the retina rather than altering lenticular shape. Lampreys have a unique corneal muscle that inserts on the spectacle covering the eye. When it contracts, the cornea is flattened, pushing the lens closer to the

6

F. W. MUNZ

retina and bringing more distant objects into focus. In elasmobranchs there are smooth muscle fibers in the ciliary body that pull the lens closer to the cornea. (The ciliary body is a continuation of the choroid that separates the anterior and posterior chambers and from which the iris projects. ) Thus, accommodation would bring near objects into focus. This mechanism has not been demonstrated unequivocally in elasmobranchs ( Nicol, 1963). Pumphrey (1961) has stated very clearly what he believes is the method of accommodation in trout and, probably, in other teleosts. When a distant object to the side of the animal is viewed by the unaccommodated eye, its image is focused on the retina (Fig. 2A). Because of the short focal length of the lens, closer objects are imaged only a few microns farther from the lens. Therefore, even quite nearby objects should also be in good focus. During accommodation, the retractor lentis muscle (of ectodermal origin) moves the lens posteriorly, rather than toward the fundus (back) of the eye (Fig. 2A). Laterally placed objects should still be imaged on the retina fairly sharply. The view of objects directly in front of the trout is very different. The retina is not a hemisphere concentric with the lens, but it is somewhat ellipsoidal. For such objects viewed by the unaccommodated eye, the distance from lens to retina is slightly greater than for laterally placed objects. To the front, therefore, the trout is near-sighted (myopic); Pumphrey stated that objects between 10 and 20 cm away would be seen clearly (Fig. 2B). At rest, the eyes are well adjusted for binocular viewing of any prey at close range. The effect of accommodation is to decrease the distance from lens to retina, bringing more distant objects into focus. In this connection, recall the temporal location of fish foveae and specialized retinal areas. Certain data recently obtained by Baylor and Shaw (1962) are relevant to accommodation and Pumphrey’s interpretation. In the alewife, Alosa, the retina is also ellipsoidal rather than spherical. Retinoscopic measurements were made of refractive error in the eyes of living, immersed, marine fishes ( 5 elasmobranch and 17 teleost species). The eyes were all far-sighted (hypermetropic). In the alewife and silversides, Menidia, measurements were made from positions lateral and anterior to the fish. From the anterior position, the average refractive error was 5 (alewife) or 8 (silversides) diopters less than from the lateral position. These differences are consistent with the ellipsoidal shape of the retina and are within the range of accommodation observed by Baylor and Shaw. The difficulty seems to be whether the unaccommodated eye is normal-sighted (emmetropic) as stated by Pumphrey ( 1961) or farsighted. A crucial question is what specific ocular structure is responsible for the retinoscopic reflection. Baylor and Shaw stated that reflection comes from behind the retina; Nicol (1963) has indicated that this point

1.

7

VISUAL PIGMENTS

anterior :

i posterior

(B) Fig. 2. ( A ) Diagrammatic horizontal section of the left eye in relation to the longitudinal axis of the fish‘s body, ts. Position of the lens at rest (solid line) and in full accommodation (broken line). ( B ) Diagrammatic representation of the horizontal visual field of a fish, eyes unaccommodated, showing the area in focus (diagonal lines). The triangular open area in front of the fish lies beyond the “far point”; the circular open area to the sides is within the locus of the “near point” (interrupted posteriorly by the shadow of the fish‘s body). Redrawn after Puniphrey ( 1961).

should be reexamined. At any rate, it now seems clear that fishes are not myopic, at least to the side, as some earlier authors maintained (Walls, 1942; Brett, 1957). Recently, Baylor ( 1967a) has examined nine species of teleost reef fishes. Their behavior suggested that they can accommodate for near vision; using the retinoscopic method, Baylor found that they tend to be emmetropic. When frightened, they often displayed an aggressive posture, in which they turned so as to view the human observer binocularly. In this situation, the eyes became hypermetropic; these observations would seem to fit Pumphrey’s treatment of image formation and accommodation (see Fig. 2 ) . In comparing his most recent results with the earlier findings of Baylor and Shaw (1962), Baylor ( 1967a) stated that “, . . it can be tentatively concluded . . . that reef fishes are

8

F. W. MUNL

better adapted for close-up vision than open-water fishes.” Some of the species examined in the earlier study, however, are benthic (e.g., Prionotus, Paralichthys, and Pseudopleuronectes ) and would presumably gain advantage from emmetropic rather than hypermetropic sight. An abstract of recent work by Bogatyrev (1966) indicates that fishes can focus their eyes for sharp vision over a wide range, from a near point of 5 cm or less out to infinity, in apparent agreement with the conclusions of Pumphrey. This problem merits further study. In addition to accommodation, several other devices can have somewhat the same result, but without requiring any active mechanism (Walls, 1942). (1) A pinhole pupil produces a fairly sharp image regardless of the distances from it to the object and to the retina. When light adapted, such elasmobranchs as Scyliorhinm and Raja have a pupil with a very small aperture. ( 2 ) A “ramp” retina, which is tilted away from the lens, could simultaneously have in focus images of objects located at different distances. In Raja, the upper portion of the retina is farther from the lens than the lower portion. Objects nearby on the ocean bottom could, therefore, be in focus at the same time as distant objects located above the animal. ( 3 ) The fact that the outer segments of the visual cells have considerable length means that objects at various distances would be equally in (or out of! ) focus; presumably this has more to do with increasing sensitivity than with accommodation. (4) Another structural modification is to have the eye permanently set for vision at two particularly useful distances; Walls suggested the analogy of bifocal spectacles. Subdivision of the retina into two parts in the tubular eyes of certain deep-sea fishes is one example (Section I, D ) . Better known is the “four-eyed” fish ( Anubbps; see Walls, 1942; Schwassman and Kruger, 1965). Anableps swims at the surface with its eyes partly out of water. The pupil of the light-adapted eye is divided horizontally by flaps of the iris. Objects in air are imaged on the ventral part of the retina, those in water on the dorsal part. The lens is egg-shaped; compensating for the lack of corneal refraction under water, there is greater curvature at the lens surfaces concerned with aquatic than with aerial vision. The two parts of the retina are specialized for their different functions ( Inouye and Noto, 1962; Schwassman and Kruger, 1965). C. Light and Dark Adaptation

Gnathostome fishes can change the effective light intensity at the receptor level by several means. Pupil movement and photomechanical

1. VISUAL

PIGMENTS

9

changes in the retina will be described, together with the tapetum lucidum. In some species, the tapetum can be occluded by a migration of black pigment, which is the reason for its mention here. The most familiar components of light and dark adaptation-changes in concentration of visual pigment and modifications in neuronal interaction-are described in Chapter 2. With the possible exception of deep-sea forms, elasmobranchs usually have a highly mobile pupil. According to Young (1933a), Nicol (1964), and Kuchnow and Gilbert ( 1967), the pupil closes quite rapidly (2-15 min) after the eye is exposed to light. Dilatation in darkness occurs more slowly, requiring 30 min or more. In elasmobranchs, Young (1933a) found that the irideal sphincter contracts in direct response to illumination and is not under nervous control. The dilatator muscle is innervated by the oculomotor nerve. Anguillu, one of very few teleosts with appreciable mobility of the pupil, has been studied by Seliger ( 1962). Light has a direct effect on excised pieces of iris, causing the sphincter muscle to contract. The action spectrum for this effect has a primary maximum near 500 nm, suggesting that the light may be absorbed by a rhodopsinlike pigment. In darkness the iris relaxes. The iris of Uranoscopus and Lophius has dual innervation, oculomotor to the dilatator and sympathetic to the sphincter muscles (Young, 1931, 1933b). Some flatfishes have a mobile pupillary operculum, but not all (Nicol, 1965a). In the great majority of teleosts, photomechanical (also called “retinomotor”) movements take the place of pupillary control of light intensity at the retinal level. The main processes were reviewed by Walls (1942), Brett ( 1957), and Nicol ( 1963) and need only be summarized. The outermost retinal cell layer is called the “pigment epithelium.” Long processes of the pigment cells extend toward the visual cells and interdigitate with their outer segments. In the dark-adapted eye, melanin granules within these pigment cells are drawn back, away from the visual cells (Fig. 3). Exposure to light is soon followed by migration of pigment granules into the processes. The visual cells have a contractile “myoid region proximal to the outer segment. When the rod-cell myoids change length, the outer segments move counter to the melanin in the pigment epithelium. Under conditions of dark adaptation, the rod outer segments are pulled proximally, toward the focal plane; and the pigment is retracted. In the lightadapted retina, the expanded pigment surrounds and shields the extended outer segments of the rods. The cone outer segments move also, in a manner opposite to the rods. They are never shielded by the pigment epithelium. Perhaps their extension in the dark-adapted retina merely clears the focal plane for the rod outer segments. In Ameiurus ( = Zctalurus), photomechanical movements follow a die1 cycle (Welsh and Osborn, 1937).

10

F. W. MUNZ

Light-adapted

Dark-adapted A

A

P. f

P.

i

V.

t

m. A m. 1

5

r

V

i J

I

0.n.i.

bl

J

Fig. 3. The retina of Clupea drawn semidiagrammatically in the dark- and lightadapted state. Key: bJ., bipolar layer; c., cone; e . , ellipsoid; e l m . , external limiting membrane; ni., length of cone inyoids; o.n.l., outer nuclear layer; o.s., outer segment; p., pigment epithelium; r., rod; and v., visual cell layer. From Blaxter and Jones (1967).

Additional information on photomechanical responses has recently been published by several authors. In young Oncorhynchus, light adaptation is complete in 20-25 min, but dark adaptation takes about an hour (Brett and Ali, 1958). As the salmon grow older, the time for light adap-

1. VISUAL

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11

tation shortens, but the time required for dark adaptation tends to increase (Ali, 1959). Light adaptation is more rapid and complete when Salmo is exposed to high intensities; after a preliminary exposure to bright light, dark adaptation takes longer ( Ali, 1962). Albino Salvelinus, which lack retinal melanin, undergo normal photomechanical migrations of rod and cone outer segments ( Ali, 1964b). Three flatfishes ( Microstomus, Pleuronectes, and Solea) that have immobile pupils have normal photomechanical movement ( Nicol, 1961b, 1965a); flatfishes with mobile pupils have not been investigated. The retina of Carassius shows a persistent circadian rhythm in its photomechanical changes. The cones of fish kept in constant darkness for 3 days continued to shift positions in synchrony with the die1 cycle (John et al., 1967). Goldfish were restrained and anesthetized by Ali (1964a), and then one eye was exposed to a bright light. The rods and cones of the dark-adapted eye did not move, but the retinal melanin expanded partially. Ali suggested that pigment migration may be influenced by hormones. Walls (1942) commented on the confusion surrounding the mechanisms controlling photomechanical movements; the situation is still far from clear. A mirror or tapetum lucidum at the back of the eye is a common device to increase the visual sensitivity of nocturnal animals. Although several morphologically distinct types were described by Walls ( 1942), only three are common in fishes. A retinal tapetum occurs in the pigment epithelium of many freshwater fishes ( cyprinids and percids ) . The epithelial cells contain particles or crystals of the reflective substance guanine. Melanin is present in the same cells and migrates normally, occluding the tapetum in bright light. A nonocclusible tapetum of the same type was said to occur in pelagic deep-sea teleosts (Walls, 1942). This statement was evidently based on the investigation of Brauer ( 1908), but Munk (1966) has not found retinal or choroidal tapeta in any deepsea teleost. Some surface-dwelling marine teleosts have a fibrous, choroidal tapetum which is shiny, like a tendon; this type is not occlusible. Almost all elasmobranchs have a choroidal tapetum lucidum just external to the choriocapillary layer; Myliobatis, a pelagic ray, seems to be the only exception (Denton and Nicol, 1964). The tapetum consists of flattened, often imbricate cells that contain guanine (Denton and Nicol, 1965; Best and Nicol, 1967). The reflecting plates are typically oriented perpendicularly to the light incident at each region of the retina. Reflection from these plates is highly directional (specular), which should minimize blurring of the image (Denton and Nicol, 1964, 1965). In benthic, neritic species (e.g., Scyliorhinus) the fundus has a black ventral area; a tapetum occurs elsewhere and is not occlusible (Nicol, 1961a,

12

F. W. MUNZ

Fig. 4. A sketch showing diagrammatically the geometric arrangements of reflecting cells and melanophore cell processes in the tapetum lucidum of Squalus. Illumination is normal to the surface of the plates. The pigment is shown in three stages of expansion. From Denton and Nicol (1964).

1964). The tapetum is also not occlusible in several deep-sea forms (squaloid sharks, Raja richardsonii, Hydrolagus afinis; Nicol, 1964). Active, pelagic sharks of the neritic zone (e.g., Squalus and Mustelus) have an occlusible tapetum over the entire fundus (Nicol, 1964). Reflectivity of the dark-adapted tapetum of Squulus is about 85%.When the fish is illuminated, choroidal melanophores send pigment out over the reflecting cells in order to conceal them (Fig. 4).The pigment retreats in darkness, this process requiring about 2 hr, in either direction. In Negaprion, the pigment migration is somewhat more rapid (Kuchnow and Gilbert, 1967). The pigment cells of the choroid seem to behave as independent effectors which are sensitive to light ( Nicol, 196%). Pigment migration is decreased by anoxia but is independent in each eye and is unaffected by nerve cutting, excision of endocrine glands, or administration of drugs. Nicol (1964) listed the elasmobranchs known to have occlusible, partly occlusible, and nonocclusible tapeta and suggested that the tapetum is concealed (except in deep-sea forms) either by pupil closure or by migration of pigment in order to avoid displaying eye-shine. The lack of retinal photomechanical movements in elasmobranchs is consistent with the typically pure rod retina. Absence of melanin in the retinal “pigment epithelium” is correlated with the development of a choroidal tapetum, and a mobile pupil allows this basically nocturnal visual system to cope also with higher light intensities.

D. Specializations for Deep-sea Life Among the most fascinating specializations for vision in a particular habitat are those of deep-sea animals. Ever since Brauer’s treatise (1908) revealed the startling diversity of ocular structures in deep-sea fishes,

1. VISUAL

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13

authors have speculated on their functional significance (see review by Marshall, 1954). Certain of these fragile animals can be caught at night at the ocean surface, but their vision has scarcely been studied. Anatomical investigations, however, have continued ( Munk, 1959, 1963, 1964a,b, 1965a,b, 1966; Pearcy et al., 1965). Many ocular features of deep-sea fishes must increase visual sensitivity. In deep-sea elasmobranchs and teleosts the retina typically contains only rods, which may be extremely numerous and have very long outer segments. The one well-established exception is Omosudis, which has an almost pure-cone retina (Munk, 1965b). Some teleosts have their rod outer segments arranged in several distinct layers (Vilter, 1954a; Munk, 1963, 1966; Pearcy et al., 1965). Presumably this arrangement increases sensitivity, perhaps without decreasing resolving power; other suggestions were also discussed by Munk (1963, 1966). Black pigment is often absent from the back of the eye, and a tapetum may be well developed in elasmobranchs (Denton and Nicol, 1964). Weale ( 1955) pointed out that binocular vision may approximately double the monocular sensitivity, the increase being as much or more than that from a tapetum. Binocular vision is well developed in a number of teleost groups, most extravagantly in those with tubular eyes (Brauer, 1908; Walls, 1942; Munk, 1966). The eye is elongate and the lens very large; the iris is absent and the mechanisms of accommodation are rudimentary or absent (Fig. 5A). The main retina is at the bottom of the tube (Matthiessen’s ratio still holds true, however); in effect, the tubular eye is equivalent to the axial part of a much larger conventionally shaped eye. Lightgathering power is increased at the expense of narrowing the visual field. This is usually compensated to some extent by an accessory retina located along the inner wall of the eye where it touches the lens. Presumably, it detects light and motion in a larger visual field. Tubular eyes are often directed upward (e.g., Argyropelecus and Opisthoproctus), but in some species are turned forward (Giganturu and Winteria). This orientation is probably related to the animal’s method of prey-capture or other behavior ( Clarke, 1963). Degeneration of the eyes in fishes from great depths has been examined by Munk (1964a, 1965a). The most interesting of these forms is Zpnops. This benthic animal has a spatulate snout, with transparent bony plates covering the orbital area. Beneath these bones are odd flattened organs that have been described either as photophores (Walls, 1942) or as modified eyes. Although they lack cornea and lens, Munk (1959) has shown unequivocally that they are eyes, with rod cells and optic nerve. Another very peculiar fish ( Bathylychnops) has eyes (Pearcy et al.,

14

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Fig. 5. Specialized eyes of deep-sea fishes. ( A ) Dorsally directed tubular eye of Scopelarchus. Thin arrow points at a piece of the accessory retina located below and medially to the optic nerve (from Munk, 1966, after Brauer, 1908). ( B ) Eye of Bathylychnops as seen in vertical section at right angles to equator of eyeball. Thin arrow points at choroid fissure (from Munk, 1966). Key: ar, accessory retina; c, cornea; i, iris; 1, lens; Id, laterad; lm, lens muscle; Ip, lens-pad; mr, main retina; on, optic nerve; r, retina; s, sclera; sg, secondary globe; sl, scleral lens; and w, window of the diverticulnm-retina. Thick arrows point to corneal borders.

1965; Munk, 1966) that put to shame the claim of AnabZeps to being called “four-eyed.” The primary globe of each eye is located on the flattened snout and directed dorsally, with a large binocular field. There is a secondary globe (Fig. 5B), which overhangs the jaw and is directed ventrally and slightly caudally. The two globes are continuous, but a flap keeps light from passing between them. The retina of the secondary globe is a diverticulum of the main retina. Although living individuals have been observed, the functions of this unique visual arrangement remain a matter for speculation. 11. VISUAL PIGMENTS

A. Photochemistry

The known visual pigments consist of a protein (called “opsin”), conjugated with a prosthetic group, the aldehyde of vitamin A (“retinene” or “retinal”). When light is absorbed, the retinene is isomerized, initiating a series of chemical rearrangements in the opsin. Excitatory events lead-

1. VISUAL

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15

ing to vision are triggered at some early stage in this sequence. The general processes of visual photochemistry are familiar and require no restatement (see Dartnall, 1957, 1962; Morton and Pitt, 1969; Wald, 1959, 1960). Abrahamson and Ostroy ( 1967) have recentIy reviewed the complex chain of events following illumination and have evaluated current theories about the structure of visual pigments and initiation of vision. These concepts have been derived from studies of the visual pigments of frogs and cattle and are not within the scope of the present review. Interest in the visual pigments of fishes derives primarily from their great diversity, which is discussed below. These differences are based on alterations of both parts of the molecule: a series of species-specific opsins has been described (e.g., Bridges, 1965a; Dartnall and Lythgoe, 1965) and two retinenes are known (Wald, 1959, 1960). Retinene,, the aldehyde of vitamin Al, has one double bond in its ring structure, whereas retinene, (derived from vitamin A,) has two. In principle, any opsin can combine with either retinene; in this way a pair of visual pigments can be formed. A number of these pairs have been described (Bridges, 1965a; Dartnall and Lythgoe, 1965; Munz and Schwanzara, 1967). The nomenclature of visual pigments has been a source of confusion (Dartnall, 1962, p. 389). It is convenient to attach names to the series of visual pigments based on each of the two retinenes. Visual pigments based on retinene, may be called “rhodopsins” and those based on retinene,, “porphyropsins,” without regard to their origin in rods or cones. This usage is operational, for the origin of extracted visual pigments is usually not known with certainty. Some authors would prefer to restrict these names to the visual pigments of rods (e.g., Wald, 1959, 1960)) but this limitation is unworkable at present (see Dartnall, 1962, p. 505). B. Methods of Study 1. PARTIALBLEACHING

The primary method for investigation and identification of visual pigments is known as partial bleaching. It has been developed chiefly by Dartnall, who has described the method in detail (Dartnall, 1962). Retinal extracts usually contain hemoglobin or other light-absorbing substances, in addition to visual pigments. Identification of any one component in the composite absorbance spectrum is made uncertain by the presence of the others ( Fig. 6A, curve 1 ) . Fortunately, visual pigments are photosensitive (i.e., bleached by light), but the usual impurities are not. (In Fig. 6A, note that as the absorbance of the visual pigment decreases in the visible region, a product of bleaching appears in the ultra-

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16

Wavelength (nrn)

Fig. 6. Partial bleaching experiment with a single retinenel pigment extracted from Scutophugus. ( A ) Curve 1, initial absorbance spectrum; curve 2, after 12 min exposure to k 660 nm light; curve 3, after 15 min further exposure to k 660 nm light; curve 4, after 6 min exposure to k 610 nm light. ( B ) Difference spectra; each bleaching operation shown as absorbance loss for the visual pigment and absorbance gain for the products of bleaching. Curves 1-2 and 2-3, results of k 660 nm irradiation; curve 3-4, result of k 610 nm irradiation. From Schwanzara ( 1967).

violet; this will be discussed later.) The absorbance changes that follow exposure to a bleaching light are called ''difference spectra" ( Fig. 6B). Under properly controlled conditions (temperature, pH, etc.), the difference spectrum closely approximates the absorbance spectrum of the pure visual pigment, over a broad wavelength region. It is important that the visual pigment be bleached in stages (Fig. 6) rather than all at once. If only a single visual pigment is present in the extract, the difference spectra are all the same, first to last, whatever the color of the bleaching light (Fig. 6 B ) . Suppose, on the other hand, that the unbleached extract contains a mixture of two visual pigments, both

1.

17

VISUAL PIGMENTS

photosensitive, in addition to stable, light-absorbing impurities ( Fig. 7A, curve 1 ) . If one of the visual pigments is more sensitive to the red light to which the extract is initially exposed it will be bleached preferentially, and the first difference spectrum will represent the absorbance spectrum of the red-sensitive component ( Fig. 7B, curve 1-2). Continued exposure to red or orange light will eventually destroy the remaining red-sensitive pigment, together with some of the second pigment, which is less sensitive to red light. Finally, this second component can also be bleached by an appropriate light and its difference spectrum plotted (Fig. 7B, curve 3-4). This description of the method is somewhat simplified; its successful application requires considerable skill. Mixtures have been

Wavelength (nm)

Fig. 7. Partial bleaching experiment with a retinal extract from Ctenobrycon. Both retinenel and retinenet components present in approximately equal amounts. ( A ) Curve 1, initial absorbance spectrum; curve 2, after 30 min exposure to h 661 nm light; curve 3, after 60 min exposure to h 641 nni light; curve 4, after 6 min exposure to h 610 nm light. ( B ) Difference spectra. Curve 1-2, result of ?, 661 nm irradiation; curve 2-3, result of h 641 nm irradiation; curve 3 4 , result of h 610 nm irradiation. From Schwanzara ( 1967 ).

F. W. MUNZ

500

600

Wavelength (nm)

Fig. 8. Identification of A,,,,, of the visual pigment pair extracted from Ctenohrycon. Curve 1, difference spectrum (curve 3-4 from Fig. 7 B ) ; curve 2, constructed from Dartnall's nomogram assuming A,,:, equal to 503 nm. Curve 3, difference spectrum (curve 1-2 from Fig. 7 B ) ; curve 4, constructed from the nomogram of Munz and Schwanzara, assuming A,,,3x equal to 527 nm. Each curve scaled to 100%at its maximum. From Schwanzara ( 1967 ).

found so often that Dartnall and Lythgoe (1965) regarded partial bleaching as a necessary part of the characterization of any visual pigment. Identification of visual pigments is facilitated by Dartnall's observation (1953) that their absorbance spectra have the same shape when plotted on a scale of frequency rather than wavelength. Any visual pigment may be described therefore by specifying the prosthetic group (retinene, or retinene,) and the wavelength of maximal absorbance (h,,,,,). The absorbance spectrum of any retinene, pigment can be constructed from the nomogram devised by Dartnall (1953). Retinene, pigments have a somewhat broader absorbance spectrum (Bridges, 1967) and require a different nomogram (Munz and Schwanzara, 1967). In favorable cases, appropriate nomogram curves can be fitted to both components in a mixture of visual pigments (Fig. 8). If individuals of a particular species possess mixtures of two known visual pigments (such as a rhodopsin-porphyropsin pair, based on the same opsin) , the proportions of the two can be estimated (Dartnall et al., 1961; Munz and Beatty, 1965). 2. ANALYSISOF RETINENES

Two colorimetric methods are used to analyze retinene. In the CarrPrice reaction, a characteristic blue color appears after an antimony tri-

1. VISUAL

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19

chloride reagent is added to a chloroform extract containing retinene. The blue color quickly fades, necessitating a highly standardized procedure for reproducible results. Water and some other substances can cause turbidity, upsetting the analyses; sometimes acetic anhydride is added to reduce this turbidity. Finally, the spectra of the absorbance bands seem to be variable, A,, reported for the retinene, band varying from 661 to 664 nm, and for retinene, from 705 to 741 nm (Grangaud et al., 1962; Naito and Wilt, 1962; Plack, 1961; Wald, 1939a; Wilt, 1959). The second method uses the results of partial bleaching experiments. The difference spectrum of the product of bleaching (Figs. 6 and 7 ) can be used to characterize retinene, and retinenez (Crescitelli, 1958). The A,, values are less variable than in the antimony trichloride reaction; and the presence of stable, light-absorbing impurities is unimportant. Both methods appear to be reliable in the hands of experienced workers. 3. VISUAL PIGMENTS IN PHOTORECEPTORS

The light absorption of visual pigments is the same, whether they are solubilized in retinal extracts or are in situ in suspensions of the visualcell outer segments (Dartnall, 1961, 1962). When the outer segments are oriented, however, as in the retina, light absorption is quantitatively greater than in randomly oriented suspensions or in extracts (Denton and Wyllie, 1955). A method devised by Denton (Denton and Warren, 1957; Denton and Walker, 1958; Denton, 1959; Denton and Nicol, 1964) uses the intact retina. The light absorption is measured at several wavelengths before and after bleaching the visual pigment. Results are similar to the more precise measurements obtained from retinal extracts but generally lack the essential qualification that homogeneity must be tested by partial bleaching. Nevertheless, they have the advantage of giving information on how much of the light incident on the retina is absorbed there. To anticipate slightly, deep-sea fish retinas can capture a very large fraction of the visual pigment, this may be more of the incident light; at A,, than 90% (Denton and Warren, 1957; Denton, 1959). The intact retina is also used in the method of microspectrophotometry (Section 11, E ) .

C. A Choice of Retinenes: Rhodopsin and Porphyropsin Wald (1936, 1939b) showed that certain freshwater fishes have a different visual pigment from marine fishes. Finding mixtures of both visual systems in some euryhaline species ( Wald, 1941), he proposed an elegant generalization (Wald, 1947, 1958, 1959, 1960) that is widely accepted. According to this view, marine fishes have rhodopsin, the visual pigment based on retinene,. Freshwater fishes have a different prosthetic

20

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group, retinenez; he called their visual pigment “porphyropsin.” Fishes that migrate between the sea and freshwater have mixtures of the two visual pigments, with the pigment which corresponds to the salinity of the spawning habitat predominating in the mixture. Freshwater fish families are not a homogeneous group but have been placed by zoogeographers in three divisions, according to their salinity tolerance and presumed evolutionary history ( Darlington, 1957) . Families in a primary division are restricted to freshwater and “have probably been confined to fresh water so long that their present distributions are the result of dispersal through fresh water, even though their remote ancestors may have lived in the sea” (Darlington, 1957, p. 46). Fishes in a secondary division have greater salinity tolerance and may have dispersed through the ocean. Peripheral freshwater fishes, “although found in fresh water, are somehow closely connected with the sea or have been so recently derived from it that their present distributions may be and often are largely the result of dispersal through the sea” (Darlington, 1957, p. 47). The physiological salt tolerance of a given species, of course, may not fit the probable evolutionary history of the family. The primary division constitutes what should be called freshwater fishes in a strict sense. Two families (Characidae and Cyprinidae) make up a very large proportion of this group. “Euryhaline” is a relative term, which is roughly equivalent to the secondary and peripheral divisions. Many peripheral freshwater fishes migrate between the sea and freshwater, but most secondary species do not. These divisions by zoogeographers seem to provide the most rational background against which to examine the distribution of visual pigments. Wald’s simple pattern does not adequately describe the experimental results obtained by several investigators over the last 20 years; for a competent review, see Schwanzara (1967). The most striking discrepancy is the common occurrence in primary freshwater fishes of rhodopsin, either alone or mixed with porphyropsin (Table I ) . This is evident among the approximately 10 characid and 20 cyprinid species that have been tested. Freshwater catfishes and centrarchids are the most prominent groups having porphyropsin alone. Some species ( mostly cyprinids ) even have rhodopsin alone. Secondary and peripheral freshwater fishes are considered together for their visual pigments have similar distributions. Many species have mixtures and many (notably poeciliids) have rhodopsin alone. It is true that more of the strictly freshwater species have porphyropsin alone and more of the euryhaline species have rhodopsin alone. Perhaps more significant is the unexpected fact that about half of the sample, whether euryhaline or not, has mixtures of the two visual pigments. Marine fishes, at least, seem to be as conservative as predicted

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PIGMENTS

Table I Distribution of Retinenel and Retinenel Pigments in Fishes Number of species” Division

Families

Retinenel

Mixtures

Retinenea

Total

Primary freshwater Secondary and peripheral freshwater Marine

18 20

8 28

28 31

25 3

61 62

27

58

3

2

63

+

(L 184 species 2 subspecies. Included are the published results in which visual pigments have been subjected to a homogeneity test by partial bleaching. Sources: Bridges (1966), Dartnall and Lythgoe (1965), Munz (1964, 1965), and Schwanzara (1967).

(but see Section 11, D ) . The sample of species examined is relatively large and diverse; more than 60 families are represented. A major exception is that very few elasmobranchs have been examined adequately (e.g., Denton and Shaw, 1963; but see Beatty, 1969a, and Crescitelli, 1969). Presumably, most of this largely marine group have retinenel pigments; but a few species are euryhaline or even confined to freshwater ( Potamotrygonidae) . The biological significance of the difference between rhodopsin and porphyropsin remains in doubt. Willmer (1956) suggested that it might be secondary to some role of vitamin A in salt or water balance. Wald (1957, 1958) also doubted that the distribution of rhodopsin and porphyropsin could be attributed to any special visual significance in freshwater and marine environments and proposed some sort of evolutionary recapitulation. Simpson ( 1964), on the other hand, thought that an adaptive significance is extremely probable. The question is not yet settled. So far, this treatment misses a crucial point: in species with mixtures of rhodopsin and porphyropsin, the individuals may actually have a “choice” of retinenes. A migratory lamprey, Petromyxon, undergoes a succession of rhodopsin and porphyropsin that is somehow related to its life cycle ( Wald, 1957); but another, Entosphenus, may have rhodopsin throughout life (Crescitelli, 1956). Dartnall et al. (1961) found that a cyprinid, Scardinius, has more retinene2 pigment in winter than summer. Porphyropsin increased in fish kept in darkness, while daylight caused the rhodopsin to increase. The changes in visual pigment were not influenced by diet and were completely unrelated to salinity, for the fish were maintained in freshwater. These results opened a new approach to the rhodopsin-porphyropsin problem. Another cyprinid, Notemigonus, a poeciliid, Belonesox, and a freshwater gadid, Lota, show the same type

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of seasonal succession of retinenel and retinene, pigments ( Bridges, 1965b; Beatty, 1 9 6 9 ~ ) .Pacific salmon, Oncorhynclzus, undergo somewhat similar changes in their visual pigments during the life cycle (Beatty, 1966). The retinene balance of juvenile salmon is partly controlled by light but is not altered as easily as in the cyprinids. Salinity has no effect on the visual pigments at this stage. Young salmon, even in winter, always have a considerable proportion of rhodopsin. Salmon caught on the high seas (in winter) had rhodopsin alone. Associated with the spawning migration is a nearly complete conversion to the retinene, system. This can start in the ocean, but may be hastened by entry into freshwater. Beatty suggested that sexual maturation may accelerate this process. The sockeye salmon, Oncorhynchus nerka, is best adapted to freshwater for it can complete its life cycle without entering the sea ( landlocked form called “kokanee”). Contrary to expectations, Beatty found that this species, and especially the landlocked form, never has much porphyropsin. The predicted effects of salinity have not actually been demonstrated on the visual pigments of any fishes but probably could be if the right species were used. Further experiments are needed to unravel the nature of environmental, dietary, and hormonal factors that control the proportions of retinene, and retinene, pigments. A clue in this direction was provided by the demonstration that a centrarchid, Lepomis, converts vitamin A, to retinene, in the eye and that thyroxine inhibits the conversion (Naito and Wilt, 1962). Thyroxine has the opposite effect of increasing the proportion of retinene, pigment in the eyes of salmonids; its mechanism of action is not known (Munz and Swanson, 1965; Beatty, 1969b). In a frog, Rana, the immediate precursor of retinene? appears to be retinene,, rather than vitamin A, (Ohtsu et al., 1964). Whether vitamin A, and A, can ever be interconverted directly is not known, but there is sometimes little relation between the forms of vitamin A in the liver and retinene in the eye (e.g., Munz, 1965; Beatty, 1966). The biological significance of retinene, pigments may possibly be related to their frequent occurrence in mixtures with retinene, pigments (Munz, 1965). Proportions of the two pigments can be altered within individual fish ( e.g., Scardinius) in response to environmental light levels. An outstanding feature of freshwater photic environments is their instability, both seasonally and on a geological time scale. An adaptable visual system, therefore, may have selective advantage for some freshwater fishes. Although it is difficult to describe the light in so variable an environment, it is probably richer in long wavelengths (redder) than light in the sea. An increased sensitivity to red light (which would result from a visual system based on retinene,) may be advantageous to many

1. VISUAL

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23

freshwater fishes, but the arguments and uncertainties brought out by Lythgoe ( 1966) could also be applied here (see Section 11, D ) . Schwanzara (1967) found a tendency of surface-feeding freshwater fishes to have retinene, pigments, while bottom dwellers have retinenez pigments. This is consistent with the filtering undergone by sunlight as it penetrates to greater depths. She found that retinene, pigments are more common in tropical fishes than in Temperate Zone species, and the converse; this was also evident within a single primary freshwater family, the Cyprinidae. These trends may be related to some general difference in spectral quality of light in tropical and temperate freshwaters, but comparative data are lacking. At the least, speculation about the possible visual significance of retinenez should provoke experiments that may give insight into this problem in biochemical evolution.

D. A Multiplicity of Opsins The visual pigments of fishes are more conspicuously diverse than those of all other vertebrates combined (Dartnall and Lythgoe, 1965). These differences are referable to a series of species-specific opsins, as well as to the occurrence of retinenee in some fishes. A histogram summarizes the published results (Fig. 9 ) . Only the most abundant visual pigment of each species is presented, except in cases where retinenel-retinene2 pairs have been described. Presumably, these are visual pigments of the rods. The source of less abundant pigments may be either rods or cones but is unknown in most cases. The visual pigments of more than 180 species have been subjected to adequate spectrometric analysis, and the list is growing so rapidly that any table would be obsolete before its publication. The figure should be regarded as a progress report, therefore, and not as the conclusion of a completed survey. Availability affects the choice of species in any survey; as far as possible, however, efforts have been made to sample species from a variety of taxa and of habitats (see Lythgoe, 1966; Schwanzara, 1967). Marine fishes can have any of a series of rhodopsins. The A,, values are not normally distributed about some wavelength such as 500 nm, but there seem to be clusters at several wavelengths as described by Dartnall and Lythgoe (1965). These authors suggested that such a distribution implies a limited number of possible variations in opsin structure. Study of hybrid salmonids, Salvelinus, indicated that a single-factor difference distinguishes the opsins of two species in which the visual pigments have A,,,,, 9 nm apart ( McFarland and Munz, 1965). This first genetic information is at least consistent with the view of Dartnall and Lythgoe. A

24

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470

480

490

500

510

A,,

(nm)

520

530

540

Fig. 9. The distribution of visual pigments in fishes. The histograms give the frequency of occurrence (number of species) vs. pigment x,,. Solid squares represent retinenel pigments; open squares represent retinenez pigments. Half-filled squares are pairs of pigments: retinenel pigments are solid along the bottom; retinene? pigments are open along the bottom. Fishes are grouped by family, according to habitat and phylogeny (see text). Numbers in parentheses indicate the families sampled; the other numbers are the numbers of species. Sources as in Table I. Note: When appropriate, published Amax values of retinenel pigments have been decreased by 1 nm, in accord with a correction of the nomogram (Dartnall, 1967).

similar grouping of both retinene, and retinene, pigments about “preferred positions” was proposed in freshwater fishes by Bridges (1965a, 1966), but the addition of Schwanzara’s data (1967) seems to obscure the relationship that he described. One point made by Bridges (see also Dartnall and Lythgoe, 1965; Munz and Schwanzara, 1967) needs explanation: The ,,A values of paired retinene, and retinene, pigments are correlated. In primary freshwater species there is less diversity of opsins than among marine fishes. The presumed ability of many freshwater fishes to alter the proportions of their retinene, and retinenez pigments (Section 11, C ) may largely eliminate the selective advantage of different opsins (Munz, 1965; Schwanzara, 1967). Thus, the visual system of the individual has a flexibility unavailable to most marine fishes. Attempts have been made to assess the biological significance of the different retinene, pigments of marine fishes. Deep-sea fishes have rhodopsins with A,,,,, values at about 490 nm or less, in evident correlation with the predominant wavelengths in sunlight after it has been filtered

1. VISUAL

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25

by passage through a long column of clear oceanic water (Denton and Warren, 1957; Wald et al., 1957; Munz, 1958a). The generally blue luminescence of deep-sea animals should also favor selection of bluesensitive visual pigments ( Munz, 1958a). There is no obvious correlation between the phylogenetic relationships of fishes and the maxima of their visual pigments. It was tempting, therefore, to generalize from the deepsea fishes and seek ecological correlations instead (Munz, 1958b, 1964, 1965). The predominant colors of sunlight are certainly not the same, as filtered by different seawater types such as oceanic, coastal, and inshore. But the attempt to associate visual pigment maxima with these photic environments has broken down as additional species have been sampled ( Dartnall and Lythgoe, 1965). His own experience as a diver led Lythgoe (1966, 1968) to emphasize that visual contrast, rather than sensitivity, may be the main selective agent. Water acts as a color filter, progressively narrowing the spectrum of transmitted light as well as decreasing its intensity. Light reflected from an object, such as the silvery side of another fish, travels a shorter distance underwater than light scattered back from the water behind it. Background illumination is therefore more monochromatic than light reflected from the object. Its maximum is further from the spectral region dominant in sunlight at the water surface and closer to the wavelength of maximum transmission by the water. This means that the greatest visual contrast between such an object and the background occurs if the visual pigment has its A, at a wavelength removed from the transmission maximum; the object is therefore seen to be brighter than the background. Should the difference be too great, of course, visual sensitivity would be drastically reduced. Lythgoe has shown that the visual pigments of marine fishes from several different photic environments appear to fit the requirements for a balance of visual contrast and relatively high sensitivity. He also pointed out that understanding of these problems is hampered by our lack of definite knowledge of the origin of the various visual pigments in rods or cones and whether the pigments occur in separate receptors or are mixed indiscriminately. In summary, current thinking suggests that the many different visual pigments of fishes probably have been selected for their adaptive advantage in different photic environments.

E. Pigments of Color Vision Of great interest are the mechanisms responsible for color vision, both in ourselves and in other animals, such as teleosts, that can discriminate

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between lights of different wavelengths. How many different visual pigments are involved, one for each primary color? Are they similar to other visual pigments? Does each pigment occur within a different class of receptor cells, or may the visual pigments be mixed together? Direct evidence has recently been obtained by microspectrophotometry of individual retinal cones of fishes; this technically difficult method has also been applied to visual pigments of frogs and primates. Microspectrophotometry was first applied to the cones of carp by Hanaoka and Fujimoto (1957), who reduced a beam of monochromatic light to a diameter of 3 p, small enough to pass through the outer segment. Exposure to bright light changed the absorbance, but the difference spectra obtained in this way were only roughly similar to those of known visual pigments. More sensitive photomultipliers have recently reduced the amount of bleaching caused by absorption of the measuring light (Liebman and Entine, 1964), or the absorbance data have been subjected to computer analysis to compensate for this bleaching (Marks, 1965). These authors have studied the goldfish; they agree that there are three different classes of cones, each possessing a single visual pigment. The data of Liebman and Entine seem more amenable to direct interpretation, but they gave no estimates of A,,,. Marks found that the cone pigments appeared to be generally similar to other known visual pigments, but no good evidence with respect to the product of bleaching has yet been obtained. The approximate A,,,,, values of these pigments are 625 nm ( r e d ) , 530 nm (green), and 455 nm (blue). Each of the pigments occurred in single cones and each in twin cones. In more than 50 single cones, the ratio was approximately 2 red:4 green:l blue. The two members of a twin pair never had the same pigment. Of 30 pairs of twins, 29 were red-green pairs and one was blue-green. No red-blue pairs were found. It is fair to ask, how good is the evidence that goldfish have color vision? Electrophysiological activity in the retinae of goldfish has been found to be consistent with this idea ( MacNichol et al., 1961; Tamura and Niwa, 1967). Potentials have been measured from the inner segments of single cones in the closely related carp by Tomita et al. (1967), who compared their results with those of Marks. Behavioral tests show that goldfish can discriminate between different colors when brightness cues are eliminated (e.g., McCleary and Bernstein, 1959; Muntz and CronlyDillon, 1966) although this capacity has not been investigated systematically (see Yager, 1967). The biochemical and physiological evidence that has been gathered at several different levels should be relevant therefore to the problems of color vision.

1. VISUAL

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PIGMENTS

REFERENCES Abrahamson, E. W., and Ostroy, S. E. (1967). The photochemical and macromolecular aspects of vision. Progr. Bioplays. Mol. B i d . 17, 179-215. Ali, M. A. ( 1959). The ocular structure, retinomotor and photobehavioral responses of juvenile Pacific salmon. Can. J. 2001.37, 965-996. Ali, hl. A. (1962). Influence of light intensity on retinal adaptation in Atlantic salmon (Salmo salar) yearlings. Can. J. Zool. 40, 561-570. Ali, M. A. ( 1964a). Retinomotor responses of the goldfish (Carassius aurutus) to unilateral photic stimulation. Rev. Can. Biol. 23, 45-53. Ali, M. A. (196413). Retina of th6 albino splake (Salvelinus fontinah X S . namaycush). Can. J. Zool. 42, 1158-1160. Baron, J., and Verrier, M.-L. (1951). RCfraction e t cerveau des poissons ?I fovea. Contribution i l’ittude des corrblations organiques. Bull. B i d . France Belg. 85, 105-11 1. Baylor, E. R. (1967a). Vision of Bermuda reef fishes. Nature 214, 306-307. Baylor, E. R. ( 196713). Air and water vision of the Atlantic flying fish, Cypselurus laeterurus. Nature 214, 307-309. Baylor, E. R., and Shaw, E. (1962). Refractive error and vision in fishes. Science 136, 157-158. Beatty, D. D. ( 1966). A study of the succession of visual pigments in Pacific salmon (Oncorhynchus). Can. J. Zool. 44, 429455. Beatty, 1). D. (1969a). Visual pigments of three species of cartilaginous fishes. Nature 222, 285. Beatty, D. D. (1969b). Visual pigment changes in juvenile kokanee salmon in response to thyroid hormones. Vision Res. 9, 855-864. Beatty, D. D. ( 1 9 6 9 ~ ) .Visual pigments of the burbot, Lota lota, and seasonal changes in their relative proportions. Vision Res. 9, 1173-1183. Best, A. C. G., and Nicol, J. A. C. (1967). Reflecting cells of the elasmobranch tapetum lucidum. Contributions Marine Sci., Univ. Texas 12, 172-201. Blaxter, J. H. S., and Jones, M. P. (1967). The development of the retina and retinomotor responses in the herring. J . Marine Biol. Assoc. U . K . 47, 677697. Bogatyrev, P. B. ( 1966). On the visual accommodation of some fish species. (Russian.) Ref. Zh., Biol. No. 21, 156; Biol. Abstr. 48, 9444 ( 1967) (abstr.). Brauer, A. ( 1908). “Wissenschaftliche Ergebnisse der deutschen Tiefsee-Expedition auf dem Dampfer Valdivia 1898-1899,” Vol. 15, Part 2. Fischer, Jena. Brett, J. R. ( 1957). The sense organs: The eye. In “The Physiology of Fishes” ( M. E. Brown, ed.), Vol. 2, pp. 121-154. Academic Press, New York. Brett, J. R., and Ali, M. A. (1958). Some observations on the structure and photomechanical responses of the Pacific salmon retina. J. Fisheries Res. Board Can. 15, 815-829. Bridges, C . D. B. (1965a). The grouping of fish visual pigments about preferred positions in the spectrum. Vision Res. 5, 223-238. Bridges, C. D. B. (1965b). Variability and relationships of fish visual pigments. Vision Res. 5, 239-251. Bridges, C. D. B. ( 1966). Absorption properties, interconversions, and environmental adaptation of pigments from fish photoreceptors. Cold Spring Harbor Symp. Quant. Biol. 30, 317-334.

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Bridges, C. D. B. (1967). Spectroscopic properties of porphyropsins. Vision Res. 7, 349-369. Clarke, W. D. (196‘3). Function of bioluminescence in mesopelagic organisms. Nature 198, 1244-1246. Crescitelli, F. (1956). The nature of the lamprey visual pigment, J. Gen. Physiol. 39, 423435. Crescitelli, F. (1958). The natural history of visual pigments. Ann. N . Y. Acad. Sci. 74, 230-255. Crescitelli, F. (1969). The visual pigment of a chimaeroid fish. Vision Res. 9, 14071414. Darlington, P. J., Jr. ( 1957). “Zoogeography: The Geographical Distribution of Animals.” Wiley, New York. Dartnall, H. J. A. (1953). Interpretation of spectral sensitivity curves. Brit. Med. Bull. 9, 24-30. Dartnall, H. J. A. ( 1957). “The Visual Pigments.” Methuen, London. Dartnall, H. J. A. (1961). Visual pigments before and after extraction from visual cells. Proc. Roy. SOC. B154, 250-266. Dartnall, H. J. A. (1962). The photobiology of visual processes. In “The Eye” ( H . Davson, ed.), Vol. 2, pp. 323-533. Academic Press, New York. Dartnall, H. J. A. (1967). Personal communication. Dartnall, H. J. A., and Lythgoe, J. N. (1965). The spectral clustering of visual pigments. Vision Res. 5, 81-100. Dartnall, H. J. A,, Lander, M. R., and Munz, F. W. (1961). Periodic changes in the visual pigment of a fish. Proc. 3rd Intern. Congr. Photobiol., Copenhagen, 1960 pp. 203-213. Elsevier, Amsterdam. Denton, E. J. (1957). Recherches sur l’absorption de la lumidre par le cristallin des poisons. Bull. Inst. Ocearwg. 1071, 1-10. Denton, E. J. ( 1959). The contributions of the oriented photosensitive and other molecules to the absorption of whole retina. Proc. Roy. SOC. B150, 78-94. Denton, E. J., and Nicol, J. A. C. (1964). The chorioidal tapeta of some cartilaginous fishes (Chondrichthyes). J . Marine Biol. Assoc. U . K . 44,219-258. Denton, E. J., and Nicol, J. A. C. (1965). Direct measurements of the orientation of the reflecting surfaces in the tapetum in Squalus acanthias, and some observations on the tapetum of Acipenser sturio. J. Marine Biol. Assoc. U . K . 45, 739-742. Denton, E. J., and Shaw, T. I. (1963). The visual pigments of some deep-sea elasmobranch. J. Marine Biol. Assoc. U . K . 43, 65-70. Denton, E. J., and Walker, M. A. (1958). The visual pigment of the conger eel. Proc. Roy. SOC. B148, 257-269. Denton, E. J., and Warren, F. J. (1957). The photosensitive pigments in the retinae of deep-sea fish. J. Marine B i d . Assoc. U . K . 36, 651-662. Denton, E. J., and Wyllie, J. H. (1955). Study of the photosensitive pigments in the pink and green rods of the frog. J. Physiol. (London) 127, 81-89. Grangaud, R., Massonet, R., and Moatti, J.-P. (1962). Etude de la vitaniine A et du r&ini.ne des yeux de Gambusia holbrooki Grd. Compt. Rend. S O C . Biol. 155, 2150-2153. Gruber, S. H., Hamasaki, D. H., and Bridges, C. D. B. (1963). Cones in the retina of the lemon shark (Negaprion breuirostris). Vision Res. 3, 397-399. Hamasaki, D. I., and Gruber, S. H. (1965). The photoreceptors of the nurse shark,

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Ginglymostoma cirratum, and the sting-ray, Dasyatis sayi. Bull. Marine Sci. Gulf Caribbean 15, 1051-1059. Hanaoka, T., and Fujimoto, K. (1957). Absorption spectrum of a single cone in carp retina. Japan. J . Physiol. 7, 276-285. Hanyu, I. (1959). On the falciform process, vitreal vessels and other related structures of the teleost eye. I and 11. Bull. Japan. SOC. Sci. Fisheries 25, 595-619. Inouye, K., and Noto, S. (1962). Structure of the retina in Anableps (four-eyed fish). Zool. Mag. ( T o k y o ) 71, 188. John, K. R., Segall, M., and Zawatzky, L. (1967). Retinomotor rhythms in the goldfish, Carassius auratus. Biol. Bull. 132, 200-210. Kahmann, H. ( 1936). Uber das foveale Sehen der Wirbeltieren. Arch. Ophthalmol. 135, 265-276. Kennedy, D., and Milkman, R. D. (1956). Selective light absorption by the lenses of lower vertebrates, and its influence on spectral sensitivity. Biol. Bull. 111, 375-386. Kuchnow, K. P., and Gilbert, P. W. ( 1967). Preliminary in vivo studies on pupillary and tapetal pigment responses in the lemon shark, Negaprion brevirostris. In “Sharks, Skates and Rays” ( P . W. Gilbert, R. F. Mathewson, and D. P. Rall, Johns Hopkins Press, Baltimore, Maryland. eds. ), pp. 46-77. Liebman, P. A., and Entine, G. ( 1964). Sensitive low-light-level microspectrophotometer: Detection of photosensitive pigments of retinal cones. J . Opt. SOC. Am. 54, 1451-1459. Lindeman, V. F. (1943). A comparative study of the oxygen consumption of the vertebrate retina, with especial reference to the nucleo-protoplasmic ratio. Am. J. Physiol. 139, 9-16. Lythgoe, J , N. (1966). Visual pigments and underwater vision. In “Light as an Ecological Factor” (R. Bainbridge, G. C. Evans, and 0. Rackham, eds.), pp. 375-390. Wiley, New York. Lythgoe, J. N. (1968). Visual pigments and visual range underwater. Vision Res. 8, 997-1012. McCleary, R. A., and Bernstein, J. J. (1959). A unique method for control of brightness cues in the study of colour vision in fish. Physiol. Zool. 32, 284-292. McFarland, W. N., and Munz, F. W. (1965). Codominance of visual pigments in hybrid fishes. Science 150, 1055-1057. MacNichol, E. F., Wolbarsht, M. L., and Wagner, H. G. (1961). Electrophysiological evidence for a mechanism of color vision in the goldfish. I n “Light and Life” ( W . D. McElroy and B. Glass, eds.), pp. 795-813. Johns Hopkins Press, Baltimore, Maryland. Marks, W. B. (1965). Visual pigments of single goldfish cones. J . Physiol. (London) 178, 14-32. Marshall, N. B. (1954). “Aspects of Deep Sea Biology.” Hutchinson, London. Marshall, N. B. (1966). Family Scopelosauridae. I n “Fishes of the Western North Atlantic” (G. W. Mead et al., eds.), Sears Found. Marine Res., Mem. No. I, Part 5, pp. 194-204. Yale Univ. Press, New Haven, Connecticut. Morton, R. A., and Pitt, G. A. J. (1969). Aspects of visual pigment research. Adv. Enzymol. 32, 97-171. Motais, R. (1957). Sur I’absorption de la lumibre par le cristallin de quelques poissons de grande profondeur. Bull. Inst. Oceanog. 1074, 1 4 . Munk, 0. (1959). The eyes of Ipnops murrayi Gunther, 1878. Galathea Rept. 3, 79-88.

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Munk, 0. (1963). The eye of Stomias boa ferox Reinhardt. Vidensk. Medd. Dansk Naturh. Foren. 125, 353359. Munk, 0. (1964a). The eyes of three benthic deep-sea fishes caught at great depths. Galathea Rept. 7, 137-149. Munk, 0. (1964b). The eyes of some ceratioid fishes. Appendix: The refraction of light in a spherical lens by K. Steenbury. Dana Rept. 61, 1-15. Munk, 0. (1965a). Ocular degeneration in deep-sea fishes. Galathea Rept. 8, 21-31. Munk, 0. (196513). Omosudis lowei Giinther, 1887, a bathypelagic deep-sea fish with an almost pure-cone retina. Vidensk. Medd. Dansk Naturh. Foren. 128, 341-355. Munk, 0. (1966). Ocular anatomy of some deep-sea teleosts. Dana Rept. 70, 1-62. hluntz, W. R. A., and Cronly-Dillon, J. R. (1966). Colour discrimination in goldfish. Animal Behaviour 14, 351455. Munz, F. W. (1958a). Photosensitive pigments from the retinae of certain deep-sea fishes. J. Physiol. ( L o n d o n ) 140,220-235. Munz, F. W. ( 1958b). The photosensitive retinal pigments of fishes from relatively turbid coastal waters. J. Gen. Physiol. 42, 445-459. Munz, F. W. (1964). The visual pigments of epipelagic and rocky-shore fishes. Vision Res. 4, 441454. 14Un2, F. W. (1965). Adaptation of visual pigments to the photic environment. Ciba Found. Symp., Colour Vision, Physiol. Exptl. Psychol. pp. 2 7 4 5 . Munz, F. W., and Beatty, D. D. (1965). A critical analysis of the visual pigments of salmon and trout. Vision Res. 5, 1-17. Munz, F. W., and Schwanzara, S. A. (1967). A nomogram for retinenez-based visual pigments. ViPion Res. 7, 111-120. Munz, F. W., and Swanson, R. T. (1965). Thyroxine-induced changes in the proportions of visual pigments. Am. Zoologist 5, 683 (abstr.). Naito, K., and Wilt, F. H. (1962). The conversion of vitamin A, to retinener in a fresh-water fish. J. Biol. Chem. 237, 3060-3064. Nicol, J. A . C. (1961a). The tapetum in Scyliorhinw canicula. J. Marine Biol. Assoc. U. K. 41, 271-277. Nicol, J. A . C. (1961b). Photo-mechanical changes in the eyes of fishes. I. Retinomotor changes in Solea solea. J. Marine Biol. Assoc. U . K . 41, 695-698. Nicol, J. A. C. (1963). Some aspects of photoreception and vision in fishes. Aduan. Marine Biol. 1, 171-208. Nicol, J. A. C. (1964). Reflectivity of the chorioidal tapeta of selachians. J . Fisheries Res. Board Can. 21, 1089-1100. Nicol, J. A. C. (1965a). Retinomotor changes in flatfishes. J. Fisheries Res. Board Can. 22, 5 1 3 5 2 0 , Nicol, J. A. C. (1965b). Migration of the chorioidal tapetal pigment in the spur dog Syualus acanthias. J. Marine Biol. Assoc. U . K . 45, 405427. O’Connell, C. P. (1963). The structure of the eye of Sardinops caerulea, Engraulis mordax, and four other pelagic marine teleosts. J. Morphol. 113, 287-329. Ohtsu, K., Naito, K., and Wilt, F. H. (1964). Metabolic basis of visual pigment conversion in metamorphosing Rana catesbeiana. Develop. Biol. 10, 216-232. Pearcy, W. G., Meyer, S. L., and Munk, 0. (1965). A ‘four-eyed’ fish from the deep-sea: Bathylychnops exilis Cohen, 1958. Nature 207, 1260-1262. Plack, P. A. (1961). The colorimetric reaction between vitamin A, aldehyde and antimony trichloride. Biochem. J. 81, 556-561. Prince, J. H. ( 1956). “Comparative Anatomy of the Eye.” Thomas, Springfield, Illinois.

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Pumphrey, R. J. (1961). Concerning vision. In “The Cell and the Organism. Essays Presented to Sir James Gray” ( J . A. Ramsay and V. B. Wigglesworth, eds.), pp. 19S208. Cambridge Univ. Press, London and New York. Rochon-Duvigneaud, A. ( 1943). “Les yeux et la vision des vertibr6s.” Masson, Paris. Rochon-Duvigneaud, A. ( 1958). L’oeil et la vision. In “Trait6 de Zoologie” (P.-P. Grass&,ed. ), Vol. 13, pp. 1099-1142. Masson, Paris. Schwanzara, S. A. (1967). The visual pigments of freshwater fishes. Vision Res. 7, 121-148. Schwassman, H. O., and Kruger, L. (1965). Experimental analysis of the visual system of the four-eyed fish (Anableps microlepis). Vision Res. 5, 269-281. Seliger, H. H. (1962). Direct action of light in naturally pigmented muscle fibers. I. Action spectrum for contraction in eel iris sphincter. J. Gen. Physiol. 46, 333342. Simpson, G. G. ( 1964). Organisms and molecules in evolution. Science 146, 15351538. Tamura, T. (1957). A study of visual perception in fish, especially on resolving power and accommodation. Bull. Japan. SOC. Sci. Fisheries 22, 536-557. Tamura, T., and Niwa, H. (1967). Spectral sensitivity and color vision of fish as indicated by S-potential. Comp. Biochem. Physiol. 22, 745-754. Toniita, T., Kaneko, A., Murakami, M., and Pautler, E. L. (1967). Spectral response curves of single cones in the carp. Vision Res. 7,519-531. Vilter, V. (1951). Recherches sur les structures fovkales dans la retine du Sphenodon punctatus. Compt. Rend. SOC. Biol. 145, 2 6 2 9 . Vilter, V. ( 1954a). DiffCrenciation fov6ale dans l’appareil visuel d u n Poisson abyssal, 1e Bathylugus benedicti. Compt. Rend. SOC. Biol. 148, 59-63. Vilter, V. (1964b). Relations neuronales dans la fovea A bitonnets du Bathylugus benedicti. Compt. Rend. SOC. Biol. 148, 466-469. Wald, G. (1936). Pigments of the retina. 11. Sea robin, sea bass, and scup. J. Gen. Physiol. 20, 45-56. Wald, G. (1939a). On the distribution of vitamins A, and G.J. Gen. Physiol. 22, 391415. Wald, G. (1939b). The porphyropsin visual system. J. Gen. Physiol. 22, 775-794. Wald, G. (1941). The visual systems of euryhaline fishes. J. Gen. Physiol. 25, 235245. Wald, G. (1947). The chemical evolution of vision. Harvey Lectures 41, 117-160. Wald, G. (1957). The metamorphosis of visual systems in the sea lamprey. J. Gen. Physiol. 40, 901-914. Wald, G. (1958). The significance of vertebrate metamorphosis. Science 128, 14811490. Wald, G. (1959). The photoreceptor process in vision, In “Handbook of Wysiology” (Am. Physiol. SOC., J. Field, ed.), Sect. 1, Vol. 1, pp. 671-692. Williams & Wilkins, Baltimore, Maryland. Wald, G. (1960). The distribution and evolution of visual systems. Comp. Biochem. 1, 311-345. Wald, G., Brown, P. K., and Brown, P. S. (1957). Visual pigments and depths of habitat of marine fishes. Nature 180, 969-971. Walls, G. L. (1942). “The Vertebrate Eye and its Adaptive Radiation.” Cranbrook Inst. Sci., Bloomfield Hills, Michigan. Weale, R. A. ( 1955). Binocular vision and deep-sea fish. Nature 175, 996.

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Welsh, J. H., and Osborn, C. M. (1937). Diurnal changes in the retina of the catfish, Ameiurus nebulosus. ]. Comp. Neurol. 66, 349-359. Willmer, E. N. (1956). In “The Comparative Endocrinology of Vertebrates. Part 11. The Hormonal Control of Water and Salt-Electrolyte Metabolism in Vertebrates” (I. C . Jones and P. Eckstein, eds.), p. 101. Cambridge Univ. Press, London and New York. Wilt, F. H. (1959). The differentiation of visual pigments in metamorphosing larvae of Rana catesbeiana. Develop. Biol. 1, 199-233. Wittenberg, J. B., and Wittenberg, B. A. (1962). Active secretion of oxygen into the eye of fish. Nature 194, 106-107. Yager, D. ( 1967). Behavioral measures and theoretical analysis of spectral sensitivity and spectral saturation in the goldfish Carmsius auratus. Vision Res. 7 , 707-727. Young, J, Z. (1931). The pupillary mechanism of the teleostean fish Uranoscopus scaber. PTOC.Roy. SOC.B107, 464485. Young, J. Z. (1933a). comparative studies on the physiology of the iris. I. Selachians. Proc. Roy. SOC. B112, 228-241. Young, J. Z. (193313). Comparative studies on the physiology of the iris. 11. Urarwscopus and Lophius. PTOC.Roy. SOC. B112,242-249.

VISION: ELECTROPHYSIOLOGY OF THE RETINA T . TOMZTA I. Introduction . . . . . . . , 11. Electroretinogram . . . . . A. Electroretinogram as a Mass Response . . B. Component Analysis of Electroretinogram . C. Localization of Electroretinogram Components 111. Response of Single Ganglion Cells . . . . . . . A. Response Types . . . B. Receptive Field . . . . . . . IV. Response of Photoreceptors . . . . . A. Early and Late Receptor Potential . . . B. Response of Single Photoreceptors . . . . . V. Responses in the Inner Nuclear Layer . . . . . . . . A. S Potential . B. Responses in Other Cell Types . . . . VI. Retinal Mechanisms of Color Vision . . . . . . . References .

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33 35 35 37 39 . 4 0 40 41 43 43 4 4 47 47 50 51 53

I. INTRODUCTION

The retina responds to light and signals its presence and pattern to the brain by way of the optic nerve. Information of the surroundings entering the eye mediated by light is first caught by the photopigments in rods and cones and transmitted to the ganglion cells through a complex neural network as shown in Fig. 1. The recent rapid development of electron microscopic techniques has contributed a great deal to the elucidation of ultrafine structures of retinal cells and synaptic contacts between them (Villegas, 1960; Villegas and Villegas, 1963; Sjostrand, 1961; Yamada and Ishikawa, 1965; Stell, 1965; Dowling and Boycott, 1966; Dowling and Werblin, 1969). The morphology shows that the organization of the neural network is much the same in all the verte33

34

T. TOMITA

Fig. 1. Diagrammatic vertical section of ( A ) teleost retina and ( B ) elasmobranch retina. A1-5, amacrine cells; BC1-5, bipolar cells; C, cone; CF, centrifugal nerve fiber; G1-3, ganglion cells; HC1-3 and HS1-2, horizontal cells; R, rod; RS, radiaI supporting cell ( MiiIler cell); SG, steller ganglion cell; and TC, twin cone. The inner nuclear layer consists mainly of the cell bodies of amacrine, bipolar, horizontal, and Miiller cells. From Detweiler (1943) after Franz (1913).

brates. Electrophysiologically also, it is usual that an observation in one animal form applies to the others. For example, the observation of Adrian and Matthews (1927a) that the optic nerve of the eel responds with a burst of impulse discharge at both onset and cessation of light is now known to be common to other vertebrates. As another example, the result

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ELECTROPHYSIOLOGY OF THE RETINA

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of analysis of the electroretinogram (ERG) into three components by Granit (1933) using the cat retina has now been generalized to all the vertebrates. The ERG represents a sum of electrical activities in individual retinal cells, whereas the optic nerve activity represents their integrated result. These two electrical phenomena were therefore utilized for the study of retinal mechanisms and of information sent to the brain along the optic nerve (Adrian and Matthews, 192%; Granit, 1933). A striking advance was made when the microdissection technique was introduced into this research field by Hartline (1935, 1938); th'IS was rapidly followed by the use of the microelectrode technique by Granit and Svaetichin (1939). These two techniques made it possible to record the impulse discharge in single ganglion cells (or in single optic nerve fibers) in response to a variety of photic stimulations, which differ in intensity, wavelength, area, pattern, and so forth. The microelectrode technique has the additional advantage that recordings can be made from within the retinal tissue. The depth recording of the ERG with microelectrodes advanced intraretinally served for more direct localization of the ERG components into retinal layers and for recording potentials at the electrode tip. Since the first use of this method (Tomita, 1950), it has now developed into the technique of intracellular recording from single retinal cells to be described later. It should be emphasized that many of the recent important contributions in the field of electrophysiology of the vertebrate retina have been accomplished by the application of these microtechniques to fish retinas. Readers who are interested in more background information should refer to Granit (1947, 1962) and Brindley (1960).

11. ELECTRORETINOGRAM

A. Electroretinogram as a Mass Response As early as 1865 Holmgren discovered that a pair of electrodes, one on the cornea and the other on the back of the eye, would record a weak electrical change when the retina is illuminated by light. This response, now generally known as the electroretinogram or ERG, can also be obtained from the eye with its anterior half removed, or from the retina isolated completely from the rest part of the eye. Two ERG recordings from the same eye of the carp but at different adaptation states are illustrated in Fig. 2. The upper tracing is from the eye which has been dark-adapted ( scotopic) and the lower after light adaptation ( photopic),

T. TOMITA

36 b

0.2 rnV

Fig. 2. Electroretinograms from the opened eye (in situ) of the carp, immobilized by Flaxedil under artificial respiration. Upper, dark adapted; lower, light adapted. The moments of onset and cessation of light are signaled by up- and downpips in the tracing above the time marking. The c-wave is not discerned because of recording by a capacitance-resistance (C-R) coupled amplifier with a time constant of 0.5 sec. Courtesy of K. Watanabe and Y. Hashimoto, from their unpublished records.

In both the ERG begins with a cornea-negative deflection (a-wave) followed by a cornea-positive one (b-wave), and another deflection (dwave), which is usually cornea-positive, at the cessation of light. Besides the above three fast waves, a very slow comea-positive rise (c-wave) is recorded if the eye is dark-adapted and a dc amplifier is used for the recording. The similarity in configuration of the ERG between fish and other vertebrates is evident from a comparison of Figs. 2 and 3, the latter showing the ERG'S of the frog (solid lines) in the scotopic and photopic state (Granit and Riddell, 1934). In general, in the scotopic state the band c-wave predominate while in the photopic the a- and d-waves are largest. For the obvious reason that the ERG is a mass response of the retinal cells, it has provided since Holmgren the most important means of objective study of the retina function as a whole. Measurement of the ERG threshold, for instance, made it possible to follow the course of dark adaptation (Hamasaki and Bridges, 1965; Witkovsky, 1968) and to plot the spectral sensitivity curve ( Burkhardt, 1966; Witkovsky, 1968)

2.

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ELECTROPHYSIOLOGY OF T HE RETINA

,.--I-

(a 1

rnV +I

0

+o 8

,.-.

*

.

+0.6 +0.4

+0.2 0-0.2 -0.4

Fig. 3. Analysis of the electroretinogram (frog) into three components: PI, PII, and PIII. ( a ) Dark-adapted and ( b ) light-adapted. Duration of illumination shown by a solid line at the base, 2 sec. From Granit and Riddell ( 1934).

of the retina disconnected from the brain. Kobayashi (1962) made an extensive comparative study of fish ERGS in more than 25 species, marine and freshwater, with special reference to ecological aspects.

B. Component Analysis of Electroretinogram Among several attempts to analyze the ERG into components of simpler configurations, the analysis of Granit into three components (PI, PII, and PIII) is most generally accepted. His original analysis was performed using the cat (Granit, 1933) and frog ( Granit and Riddell, 1934), but the result applies to other vertebrates including fish. Minor differences between species are quantitative rather than qualitative. As seen in Fig. 3, the PI11 is a cornea-negative potential change which appears first following the onset of light. Shortly after the start of PIII,

38

T. TOMITA

this is interrupted by the cornea-positive PII. The a-wave of the ERG thus represents the early onset of PIII, while the b-wave the rise of PII. The PI corresponds to the c-wave in the ERG, and, as earlier described, its amplitude depends on the state of adaptation. The d-wave at the cessation of light is, according to this analysis, either a positive or negative deflection, determined by the amplitude and time course of the off-effects of PI1 and PI11 which are opposite in polarity. In most fishes, the PI11 predominates especially in the photopic state, resulting in a large a-wave and a large cornea-positive d-wave. Originally, the component analysis of the ERG was made by isolation of one component from others using various physical and chemical agents. In general, the PI1 is more susceptible than the PI11 to various agents. One exception is alcohol, which results in a marked increase in the bwave and decrease in the a- and d-wave (Bernhard and Skoglund, 1941). Granit (1947) provides evidence that the PI1 is associated with excitatory processes in the retina and the PI11 with inhibitory. This view is consistent with the common observation that a burst of impulse discharge in the optic nerve occurs with the b-wave, which is the rise of the excitatory PII, and another burst with the d-wave, which is the rebound

2W -0

250t

Distance in retina

Q

E

a

-250t \*/ 1 - 500

\

0

\

Fig. 4. Distribution of slow potential response (local ERG) at the receptor surface of the carp’s isolated retina plotted by scanning a small spot of light (60 ,U in diameter) across the recording site at the point 0. Positive responses upward. From Motokawa et al. ( 1959).

2.

ELECTROPHYSIOLOGY OF THE RETINA

39

following release from the inhibitory PIII. The following observations (Motokawa et al., 1959, 1961) also support Granit. The carp retina, detached from the pigment epithelium and mounted receptor side up on the indifferent electrode, gives a characteristic pattern of local ERG (Fig. 4 ) to a micropipette electrode placed on the distal retinal surface when a spot of light is scanned across the site of recording. When the light spot is on the recording site, a positive or PIII-dominant ERG is obtained, but as the spot is moved away from the recording site, the local ERG becomes negative or PII-dominant. In this PII-dominant zone, ganglion cells responding to light with on discharge outnumber those inhibited during light, whereas in the PIII-dominant zone the relation is just the reverse. Meanwhile, the characteristic pattern of local ERG shown in Fig. 4 was recently analyzed by Murakami and Sasaki (1968a,b) in terms of spatial distribution of ERG components, using the carp retina. Concerning the significance of the ERG in the chain of events evoked by light in the retina, however, much remains to be studied. Hamasaki and Bridges (1965) report, for instance, that a second light flash applied within 5 sec after the first to a dark-adapted retina (elasmobranches) fails to elicit an ERG, but the evoked responses at the optic tectum to these two successive light flashes are about the same.

C. Localization of Electroretinogram Components It is generally agreed that the PI1 originates from some cell type in the inner nuclear layer (Granit, 1947). This has been confirmed by the observation that the polarity of the b-wave is reversed after the recording microelectrode has penetrated through this layer ( Tomita, 1950; Brown and Wiesel, 1961). Recent work on the carp retina with intracellular micropipettes (Kaneko and Hashimoto, 1969) shows that some cells in the inner nuclear layer are depolarized during light while some others are hyperpolarized ( cf. Fig. 12). Although their cell types have not yet been identified, it is possible that those depolarized by light are related to the PII. The PI is localized in the outermost retinal structures (Tomita, 1950; Brown and Wiesel, 1961). This localization is also based on the finding from depth recording of the ERG with penetrating microelectrodes. After the retina is detached from the pigment epithelium, the PI is lost both in the retina proper and in the remaining part of the eye covered by the pigment epithelium. Tomita (1950) assumes on this basis that the PI is a phenomenon associated with some metabolic interaction between the receptors and pigment epithelium cells. Noell (1954) and Brown and Wiesel (1961) consider the pigment epithelium cells as the origin of PI.

40

T. TOMITA

The PI11 is of dual nature as earlier suggested by Granit (1947). The separation of PIII into two subcomponents was achieved by a technique of fractional depth recording of the ERG (Murakami and Kaneko, 1966). The one subcomponent is termed the “distal PIII” and localized in the receptors themselves. The other termed the “proximal PIII” originates in some structures in the inner nuclear layer. The latency of the proximal PI11 is several milliseconds longer than that of the distal PIII. The proximal PIII is more susceptible to various agents than the distal PIII. The proximal PI11 may be a transretinal manifestation either of the S potential (Byzov, 1962) or of the activity of the cell types in the inner nuclear layer that respond to light with hyperpolarization (see Section V ) , or probably of both. One aspect of the PI11 that made Granit hesitate to localize it in the receptors was its polarity. The PI11 has a polarity just opposite to what one predicts from other receptors including invertebrate photoreceptors. While it is general that the receptors, when excited, are depolarized to form an electric field making their distal tips negative, the polarity of PI11 makes them positive instead of negative. According to the recent observations on single rods and cones with intracellular micropipettes, however, the unusual polarity of the electric field around the vertebrate photoreceptors is attributed to their being hyperpolarized by light ( see Section IV, B ) .

111. RESPONSE OF SINGLE GANGLION CELLS

A. Response Types

The response types of single ganglion cells in the fish are the same as in other vertebrates such as the frog (Hartline, 1938) and the cat ( KuHer, 1953). They are either on type giving a burst of impulses when the light is turned on, the o# type giving a burst of impulses when the light turned off, or the o*o# type which responds with a burst of impulses at both onset and cessation of light. Intracellular recording reveals that in the on type the cell is depolarized during illumination, while in the off type the cell is hyperpolarized during illumination and depolarized following the cessation of light. In the on-off type a depolarization occurs at both on and off. It is obvious that the ganglion cell functions according to the general plan of neurons which are under excitatory and inhibitory presynaptic controls.

2.

41

ELECTROPHYSIOLOGY OF THE RETINA

The response type of a given unit is not always fixed but can change under certain conditions as described below.

B. Receptive Field The ganglion cell responds only when light falls within a certain circumscribed area of the retina. This area is termed, according to Hartline ( 1938), the “receptive field” of the ganglion cell. Mapping with a small spot of white light, several types of organization of the receptive field are discerned which are characteristic to individual ganglion cells. The two simple types are everywhere on and everywhere off type, but more common are those which are apparently made up of the combination of these two simple types. Figure 5 illustrates an off center-on periphery type obtained in the goldfish (Wagner et al., 1960). This is identical with one of the two typical types first observed in the cat by Kuffler (1953). The reverse type, that is, the on center-off periphery type, is also frequently encountered in the fish. Some ganglion cells in the fish are color coded, responding differently to different wavelengths of light (Wagner et al., 1960; Motokawa et al., 1960). The unit shown in Fig. 6 responds with an on discharge to green but with off discharge to red. Units of the reverse relation (red on-

0 0 00

0 X=OFF

0= O N 0=ON-OFF

-

0

..

0

0

0 I

I MM

I

Fig. 5. Receptive field of a single goldfish ganglion cell. Solid black area indicates region where only off responses were found. Hatched area indicates region where on-off type responses were found. On responses were found only in the periphery of on-off area. Test stimulus, 1 5 3 , ~in diameter; wavelength, 600 mp; and intensity, 18.0 pW/cmz. From Wagner et al. (1960).

T. TOMITA

I

i .--..I

I

4 50

..

! -- . .

!

600

.. 650

,

-

500

5 50

I It

1

1

I

I

0.5 sec

'

(b)

Fig, 6. Variation of response from a single goldfish ganglion cell with change in wavelength of stimulus, Wavelength of stimulus in millimicrons at upper right hand of each record. The duration of stimulus is indicated by the step in the signal trace at the base of each series. From Wagner et aZ. (1960).

W

OFF

l50p

ON

X.500

\

+

+

.++

-_.

I

i

I rnm

I

I

/

*+ i

I rnm

Fig. 7. Receptive field plots of the separate component responses of the same ganglion cell (goldfish), taken with stimuli of ( a ) 500 mp and ( b ) 650 inp. Stimulating spot, 150 p in diameter. From Wolbarsht et al. ( 1961).

2.

ELECTROPHYSIOLOGY OF THE RETINA

43

green off) are also found. The on and off components constituting the receptive field of color-coded ganglion cells can be separately evoked by choosing adequate wavelengths ( Wolbarsht et al., 1961). Figure 7 shows the result of separation by applying green (500 mp) and red (650 mp) light to such a unit. The sensitivity of the red component is generally higher in the center of the receptive field, but it falls off more sharply in the periphery than that of the green component. According to the recent work of Daw (1967), each of the red and green zones is very often surrounded by a zone of the opposite response type, which is detectable only by the use of annular light patch. In units such as illustrated in Fig. 7, for example, the organization of the receptive field as mapped with annular light patch could be on centeroff periphery for green (500 mp) and off center-on periphery for red (650 mp). Such arrangements explain the psychophysically known phenomenon of the simultaneous color contrast. The size of the periphery is very large, being at least 5 mm in diameter for both red and green.

IV. RESPONSE OF PHOTORECEPTORS

A. Early and Late Receptor Potential As already mentioned, the distal PI11 is the earliest potential in the ERG and is considered from its localization to correspond to the receptor potential identified in mammals by Brown et al. (1965). While this response has the latency of milliseconds and is easily evoked by moderate intensity of light, another type of response which has no substantial latency is elicited by very intense light flashes. This response was termed the “early receptor potential” (early RP) to distinguish it from the conventional receptor potential which was accordingly termed the “late receptor potential” (late RP) . The early RP was observed originally in the monkey (Brown and Murakami, 1964a,b), but later this was found to be universal to other animal forms including vertebrates and invertebrates. It is agreed that the early RP is a potential change associated with some steps of bleaching of the photopigment and that this is not generated by changes in membrane permeability but most likely by the net displacement of electric charge resulting from configuration changes in photopigment molecules by light (Pak and Cone, 1964; Pak, 1965; Brindley and Gardner-Medwin, 1966; Cone, 1967). For a net displacement of electric charge, a certain orientation of pigment molecules is necessary. Arden and Ikeda (1966) and Cone (1967) have provided

44

T. TOMITA

evidence that the early RF' is lost by disorientation of the pigment, although the pigment remains. It is probable that the lamellar and rhabdomeric structures in the vertebrate and invertebrate photoreceptors are related to the pigment orientation. Since most works on the early RP have been performed in animals other than the fish, no further description of this potential will be given. The receptor potential to be discussed below is the late RP.

B. Response of Single Photoreceptors Because of the extremely high density in population of photoreceptors in the retina, it is impossible to isolate single receptor responses without using intracellular micropipette electrodes. For intracellular recording, it is necessary to select retinas having large rods and/or cones. The carp has a mixed retina consisting of large cones (10 p across at the cone inner segment) and very slender rods of only a few microns across which are difficult to impale by micropipettes. Penetration into single carp cones has been successful by using pipettes having a tip diameter of less than 0.1 p, and with the aid of a device which jolts the retina at a high acceleration toward a vertically held, slowly advancing micropipette ( Tomita, 1965). Whenever a resting potential is recorded and a response to light is observed, the jolting is stopped to maintain the intracellular position of the pipette for further observations. With an electrode marking method the recording site has been identified as the cone inner segment (Kaneko and Hashimoto, 1967). The intracellularly recorded response to light is sustained and graded, and it is hyperpolarizing irrespective of the wavelengths of stimulating light. When the spectrum is scanned, the response spectra of single cones such as shown in Fig. 8 are obtained. The figure illustrates three response spectra which are maximally sensitive at different wavelengths. Figure 9 is the result of a statistical analysis, showing three average response spectra along with standard deviation curves. Their peaking wavelengths shown by histograms in Fig. 10 are close to those of absorption spectra of single cones measured in the goldfish with a microspectrophotometer ( Marks and MacNichol, 1963; Marks, 1965). No electrophysiological evidence has been provided to implicate lateral interaction between adjacent photoreceptors. The response of single cones is practically the same in size, being independent of the retinal area illuminated, and when tested with a small light spot the response falls off sharply as the spot moves off the recording site. This suggests that the individual receptors are functionally independent, making a

2.

45

ELECTROPHYSIOLOGY OF THE RETINA 1

400

.

1

500

'

1

600

1

4

700

Fig. 8. Sample recordings of response spectra from single carp cones demonstrating three cone types. Scanning of the spectrum was made in steps of 20 mp with monochromatic light adjusted to equal quanta ( 2 X lo5 photons/$ sec) and with a duration of light of 0.3 sec at each wavelength followed by an intermission of 0.6 sec. A downward deflection indicates negatively. Recording was made with a C-R coupled amplifier having a time constant of 0.5 sec. The spectral scale is given in terms of millimicrons at the top of the figure. A dominant peaking occurs at ( a ) blue, ( b ) green, and ( c ) red. From Tomita et al. ( 1967).

100-

-g a,

u 3 + .-

-

Q

-

E

50a m ,

-

0 c

a a m (

L

-

o , , , , , , , , , , , , , , , , , ,

Fig. 9. The averaged response spectra and standard deviation curves of three cone types (carp). From Tomita et al. (1967).

46

T. TOMITA

Mean = 4 6 2 A15 m p

N = 2 3 (16%)

400

500

600

700

’

G 0

Meon = 5 2 9 2 14 m p N=14 (10%)

0

: o

1

n

400

E

500

1

I

600

700

600

700

1

3

40-

3020-

Meon =611? 2 3 m p

N

105 ( 7 4 % )

10-

0-

,

400

I

500

Wavelength ( m p )

Fig. 10. Histograms of the peaking wavelengths of three cone types ( c a r p ) : ( B ) blue type, ( G ) green type, and ( R ) red type. From Tomita et al. (1967).

strong contrast to responses obtained from layers proximal to the receptors. The hyperpolarizing response of vertebrate photoreceptors has been a puzzle, but the latest experiment of Toyoda et al. (1969), although not in the fish, clearly demonstrates that the membrane conductance of single rods (in Gekko gekko) and cones (in Necturus muculosus) is decreased by light according to the degree of hyperpolarization, which is a function of the intensity of light. They also provide evidence that the vertebrate photoreceptors are kept depolarized in darkness and repolarized in light; the amplitude of response to light is increased by extrinsic hyperpolarizing current and decreased or even reversed by depolarizing current. Thus the vertebrate photoreceptor membrane behaves as if it were “excited

2.

ELECTROPHYSIOLOGY O F THE RETINA

47

in darkness and recovering by light toward the “resting state” in a graded manner. On this basis, the unusual polarity of the distal PI11 as the receptor potential can be accounted for in the following way. The electric field around the receptors in darkness is such as to make their distal tips negative owing to a sink existing at or near their distal tips just as in “excited” receptors. With light the sink disappears or becomes weaker, and this brings the distal tips to a relative positivity. The problem remains to be solved of whether or not the electrical response mediates the flow of information from the distal segment, where light is absorbed, to the proximal terminal, where synaptic transmission to secondary neurons takes place. If it does, the amplitude of intracellularly recorded response per photon absorbed should be large enough to meet the extremely high sensitivity of the visual system (some further discussion is given by Tomita, 1968).

V. RESPONSES IN THE INNER NUCLEAR LAYER

A. S Potential

Svaetichin ( 1953) observed in the fish that intraretinal micropipettes record a resting potential of some 40 mV at a certain depth, and, upon illumination with white light, a 20-30 mV hyperpolarization which is sustained and graded. In the belief that the potential was obtained intracellularly from single cones, he termed it the “cone action potential.” However, the response was later relocalized in structures proximal to the receptors (Tomita, 1957; Tomita et al., 1958, 1959; MacNichol and Svaetichin, 1958; Mitarai, 1958; Oikawa et al., 1959). The response had to be retenned accordingly, but different viewpoints regarding the origin and nature of the response brought about different terminologies. For example, the term “glial membrane potential” (GMP) is one of those, reflecting the viewpoint of Svaetichin and his co-workers ( Svaetichin et al., 1961; Laufer et al., 1961; Mitarai et al., 1961; Fatehchand et al., 1966) that the response is a manifestation of interaction between neurons and glia cells. They include the horizontal cells, Muller cells, and amacrine cells as glia cells. Morphological and physiological studies, however, do not always support their view. Structures typical of synapses have been found between the receptors, horizontal cells, and bipolar cells in the outer plexiform layer (Stell, 1965; Dowling and Boycott, 1966), and between the bipolar, amacrine, and ganglion cells in the inner plexiform layer (Dowling and Boycott, 1966). Dowling and Boycott did not

48

T. TOMITA

work on fish but on primate retinas. Under such circumstances, a simple term “S potential,” abbreviated from Svaetichin’s potential, is now most commonly used. The S potentials are classified into two major types from their response patterns to spectral light ( Svaetichin, 1956; MacNichol and Svaetichin, 1958). Those responding only with hyperpolarization to all wavelengths of light are called the “luminosity type” ( L response) and those in which the response polarity is wavelength-dependent are called the “chromaticity type” ( C response). The C response is further subdivided into Y-B (yellow-blue) type and R-G (red-green) type. Examples of these responses are illustrated in Fig. 11. The Lutianidae and other species collected in water 30 to 70 meters deep gave only an L response that had a peak toward the blue end of the spectrum. Of the shallow water fishes, the mullet, Mugil, gave both the R-G and Y-B responses in addition to the L response, but the Serranidae gave L and a

I

.

,

.

,

.

,

.

Fig. 11. Response spectra of S potentials from retinas belonging to different families. ( 1 ) Lutianidae (achromatic vision), which live deeper than 30 meters, giving only L responses with peak at about 490 mp. ( 2 ) Serranidae (dichromatic vision), giving both L and Y-B responses. ( 3 ) Centropomidae ( dichromatic vision ), giving both L and R-G responses. (4) Mugilidae, giving all three types of response. The fishes used to make records 2-4 were all caught in very shallow water. From MacNichol and Svaetichin (1958).

2.

ELECl'ROPHYSIOLOGY OF THE RETINA

49

Y-B responses while the Centropomidae L and R-G responses. It is evident that the types of responses are dependent upon the fish species used. Tamura and Niwa (1967) extended the exploration to some other species and divided the L response into three subtypes (L,-L,) according to the peaking wavelengths, and the C response into four (C1-C4) according to their spectral response patterns. In spite of the difference in classification, the results are similar to those of MacNichol and Svaetichin. From a number of studies (Mitarai, 1960; MacNichol and Svaetichin, 1958; Yamada and Ishikawa, 1965; Byzov and Trifonov, 1968) the site of recording of the S potential was suggested to be the horizontal cell. Recording is easier from retinas having large horizontal cells. The problem is how and where it originates. The localization of S potential in a horizontal cell might not necessarily mean that the membrane of that cell is responsible for the electrogenesis. The recorded potential could be of a passive nature conducted to the recording site from some other structures. In the discussion below concerning the electrogenesis of S potential, let us confine our attention to the L response, just to make the matter simple. After the theory of glia-neuron interaction had been argued for many years without much agreement, Trifonov (1968) presented a new hypothesis that the S cell, which is the horizontal cell in synaptic contact with photoreceptors, is kept depolarized or facilitated in the dark by transmitter substance continuously released from the receptor terminals, and that light acts to suppress the release of transmitter with the result that the S cell is repolarized or disfacilitated. Related to this hypothesis are the observations of Trifonov and Byzov (1965) on the turtle and Byzov and Trifonov ( 1968) on the carp that the S cell which is hyperpolarized or disfacilitated in the presence of adapting light responds to a transretinally applied sclera-positive electric pulse with a depolarization, the amplitude of which is graded according to the intensity of the stimulating pulse, and, if the intensity is h e d , to the degree of hyperpolarization by light. From the polarity of current pulses effective for eliciting the depolarizing response, Trifonov and Byzov consider the receptor endings as the acting site of the pulses which effect a release of transmitter. The new hypothesis of Trifonov is attractive, particularly when the vertebrate photoreceptors are found to function in a similar way: depolarized in darkness and repolarized in light. From this new viewpoint, some of the properties of the S potential, which appeared not to be the kind of electrical activity usually ascribed to neurons, are now better understood. Although not always clearly demonstrated as in the photoreceptors, the amplitude of S potential tends to increase (decrease) by extrinsically applied hyperpolarizing ( depolarizing) current ( Mura-

50

T. TOMITA

kami and Kaneko, cited in Tomita, 1965), and the conductance of the S cell tends to decrease during response to illumination (Toyoda et al., 1969). These are exactly what are observed in the photoreceptors. It should be noted that in spite of the similarities between the S potential and single photoreceptor response there is one distinct difference in that the S potential has a large area effect or a strong dependence of the response amplitude upon the retinal area illuminated, while the single photoreceptor response has substantially no such effect. A convergence of a great many photoreceptors to S cells is suggested. Lateral electric connections between S cells can multiply the effect, and the electron microscope has proved tight junctions between adjacent horizontal cells. The argument might not be complete without reference to the C response. The depolarization in the C cell at certain bands of spectrum appears to be associated with an increase in the conductance of the cell. Conceivably, the C cell is in a half-facilitated state in the dark, and a further facilitation is caused by certain wavelengths of light, while disfacilitation is caused by other wavelengths.

B. Responses in Other Cell Types The intracellular study of single cells in the inner nuclear layer of the fish retina has become possible only recently by the application of a technique developed for single photoreceptors. Figure 12 illustrates three response types recorded in this layer by Kaneko and Hashimoto (1969). They are on, off, and on-off type, being substantially the same as in single ganglion cells. Some of these cells even respond with impulse spikes superimposed on depolarizing phases of slow potentials, confirming the extracellular observation of Brown and Wiesel (1959) in the cat. More common in the carp, however, are those responding to light with slow membrane potential changes superimposed by nonunitary spikelike or oscillatory fluctuations on the depolarizing phases ( Fig. 12). The organization of the receptive field of these cells also resembles that of the ganglion cells, as demonstrated in the cat by Brown and Wiesel (1959). Some have a receptive field of everywhere on, everywhere off, or everywhere on-off, but others have the concentric receptive field such as on center-off periphery or the reverse. The response in the periphery is more easily detectable by an annular light patch. The size of the receptive field is larger than predicted from the dendritic field of bipolar cells and seems to be comparable with that of the S potential. From these observations, Kaneko and Hashimoto suspect that the S cells might intervene between the photoreceptors and these cells to convey information, at least in the periphery of the receptive field.

51

2 . ELECTROPHYSIOLOGY OF THE RETINA

-

-L*bLLLLt 0 I + I S EC -U-iLLU

Fig. 12. Sample records from units in the inner nuclear layer (carp), ( a ) on type, ( b ) off type, and ( c ) on-off type. From Kaneko and Hashimoto ( 1969).

Localization of these cells in the inner nuclear layer has been established by means of the electrode marking, but the identification of cell types is left to future studies [but see the latest work of Werblin and Dowling ( 1969) and Kaneko ( 1970) 1, VI. RETINAL MECHANISMS OF COLOR VISION

The above description of electrical events at each retinal level involved observations related to the color coding. Accordingly, the main task now will be to collect these scattered data along with some others not yet referred to and to arrange them for a brief summary of the present status of understanding of color vision. The behavioral aspects of color vision will be discussed in Chapter 3, by Ingle. Over a century and a half ago, Young (1802) theorized in man that the color discrimination is mediated by three photoreceptor substances, each maximally sensitive to a different region of the spectrum. Two recent studies on single cones of Cyprinidae, one microspectrophotometric ( Marks, 1965) and the other electrophysiological ( Tomita et al., 1 9 6 7 ) , have shown that Young's trichromatic theory also applies to the fish, although it remains to be studied how far the findings in Cyprinidae can

52

T. TOMITA

be extended to other fish species. Results from these entirely different approaches were substantially the same in differentiating three pigments, each contained in different cone groups. Their maximally sensitive wavelengths are compared in the accompanying tabulation.

462 f 15

529 rt 14

611 k 23

455 f 15

530 f 5

625 f 5

Tomita et al. (1967) (carp) Marks (1965) (goldfish)

Coming to the S potential level, the processes of Hering’s opponent color type (1878) predominate. Hering’s theory, which was derived from psychological observations, states that there are four primary colors which are coupled in mutually antagonistic pairs; red-green and yellow-blue. Apparently, the R-G type and Y-B type of C responses in Fig. 11 substantiate these pairs. A transformation from Young’s type to Hering’s type at a certain level of the visual pathway has been suggested by some pioneer workers such as von Kries ( 1905) and Schrodinger ( 1925). It is now obvious that the site of the transformation is the synaptic network in the outer plexiform layer. Concerning the mechanism of the transformation, however, little is known. It might be that the positive component of a C response is related to one cone type and the negative component to another cone type (Orlov and Maksimova, 1965). On this assumption, the absorption maxima of red and green pigments should be determined from analysis of the R-G type of S potential. The result of work along this line by Naka and Rushton ( 1966a,b,c), however, is not consistent with the result of direct spectrophotometric measurements on single cones. Naka and Rushton find that the red-green units peak at 680 and 540 mp, respectively. Marks found a green pigment with maximum near 540 mp a d a red pigment with maximum near 620 mp. In addition, Naka and Rushton find an L-type unit with maximum at 620 mp. Although they have not yet measured a blue component, if present, this means that in Cyprinidae there are four types of cone pigments. [The analysis of C respase by Witkovsky (1967) also suggests a far-red pigment with maximum at 665 mp.] The significance of four cone pigments instead of three, as suggested from the analysis of S potential, remains to be explained. At the ganglion cell level, the rule of Hering’s type is also obvious (Fig. 6), but the component analysis becomes more and more difficult

2.

ELECTROPHYSIOLOGY OF THE RETINA

53

as we go further away from the receptors. Witkovsky ( 1965), working on single carp ganglion cells, concludes that in the photopic state the three pigments of Marks are just suEcient to account for all spectral response types at the ganglion cell level. MacNichol et al. ( 1961), on the other hand, report a red component maximally sensitive at 650 mp in single goldfish ganglion cells. Approaches to the same problem but using the ERG as the index were made recently by Burkhardt (1966, 1968) and Witkovsky (1968). REFERENCES Adrian, E. D., and Matthews, R. (1927a). The action of light on the eye. I. The discharge of impulses in the optic nerve and its relation to the electric change in the retina. J. Physiol. (London) 63, 378414. Adrian, E. D., and Matthews, R. ( 1927b). The action of light on the eye. 11. The processes involved in retinal excitation. J. Physiol. (London) 64, 279-301. Arden, G. B., and Ikeda, H. (1966). Effects of hereditary degeneration of the retina on the early receptor potential and the corneo-fnndal potential of the rat eye. Vision Res. 6, 171-184. Bernhard, C. G., and Skoglund, C. R. ( 1941). Selective suppression with ethylalcohol of inhibition in the optic nerve and of the negative component PI11 of the electroretinogram. Actu Physiol. Scund. 2, 10-21. Brindley, G. S. (1960). “Physiology of the Retina and Visual Pathway.” Arnold, London. Brindley, G. S., and Cardner-Medwin, A. R. ( 1966). The origin of the early receptor potential of the retina. J . Physiol. (London) 182, 185-194. Brown, K. T., and Murakami, M. (1964a). A new receptor potential of the monkey retina with no detectable latency. Nature 201, 626428. Brown, K. T., and Murakami, M. (1964b). Biphasic form of the early receptor potential of the monkey retina. Nature 204, 739-740. Brown, K. T., and Wiesel, T. N. (1959). Intraretinal recording with micropipette electrodes in the intact cat eye. J. Physiol. (London) 149, 537-562. Brown, K. T., and Wiesel, T. N. ( 1961). Localization of origins of electroretinogram components by intraretinal recording in the intact cat eye. J. Physiol. (London) 158, 257-280. Brown, K. T., Watanabe, K., and Murakami, M. (1965). The early and late receptor potentials of monkey cones and rods, Cold Spring Harbor Symp. Quant. Biol. 30, 457482. Burkhardt, D. A. ( 1966). The goldfish electroretinogram: Relation between photopic spectral sensitivity functions and cone absorption spectra. Vision Res. 6, 517-532. Burkhardt, D. A. (1968). Cone action spectra: Evidence from the goldfish electroretinogram. Vision Res. 8, 839-853. Byzov, A. L. (1962). On the origin and some properties of the PIII-component of the frog electroretinogram. Proc. Intern. Union Physiol. Sci., 22nd Intern. Congr., Leiden, 1962 Vol. 1, Part 1, pp. 473-476. Excerpta Med. Found., Amsterdam. Byzov, A. L., and Trifonov, Yu. A. (1968). The response to electric stimulation of horizontal cells in the carp retina. Vision Res. 8, 817-832. Cone, R. A. ( 1967). Early receptor potential: Photoreversible charge displacement in rhodopsin. Science 155, 1128-1131.

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Daw, N. W. (1967). Goldfish retina: Organization for simultaneous color contrast. Science 158, 942-944. Detweiler, S. R. ( 1943). “Vertebrate Photoreceptors.” Macmillan, New York. Dowling, J. E., and Boycott, B. B. (1966). Organization of the primate retina; electron microscopy. Proc. Roy. SOC.B166, 80-111. Dowling, J. E., and Werblin, F. S. (1969). Organization of retina of the mudpuppy, Necturus maculosus. I. Synaptic structure. J. Neurophysiol. 32, 315-338. Fatehchand, R., Svaetichin, G., Negishi, K., and Drujan, B. (1966). Effects of anoxia and metabolic inhibitors on the S-potential of isolated fish retinas. Vision Res. 6, 271-283. Franz, V. ( 1913). Sehorgan. In “Oppels Lehrbuch der Vergleichenden Mikroskopischen Anatomie der Wirbeltiere.” Fischer, Jena. Granit, R. (1933). The components of the retinal action potential and their relation to the discharge in the optic nerve. J. Physiol. (London) 77, 207-240. Granit, R. ( 1947 ). “Sensory Mechanisms of the Retina.” Oxford Univ. Press, London and New York. (hinit, R. (1962). Neurophysiology of the retina. In “The Eye” ( H . Davson, ed.), Vol. 2, pp. 575-692. Academic Press, New York. Granit, R., and Riddell, L. A. (1934). The electrical responses of light- and darkadapted frog’s eyes to rhythmic and continuous stimuli. J. Physiol. (London) 81, 1-28. Granit, R., and Svaetichin, G. ( 1939). Principles and technique of the electrophysiological analysis of colour reception with the aid of micro-electrodes. Upsalu Lakareforenings Forh. 65, 161-177. IIamasaki, D. I., and Bridges, C. D. B. (1965). Properties of the electroretinogram in three elasmobranch species. Vision Res. 5, 483-496. Hartline, H. K. (1935). Impulses in single optic nerve fibres of the vertebrate retina. Am. J. Physiol. 113, 59P. Hartline, 13. K. (1938). The response of single optic nerve fibers of the vertebrate eye to illumination of the retina. Am. J. Physiol. 121, 400415. Hering, E. ( 1878). “Znr Lehre vom Lichtsinne,” pp. 107-141. Carl Gerold’s Sohn, Vienna. Hohngren, F. (1865). Method att objectivera effecten av ljusintryck p i retina. Upsala Lakareforenings Forh. 1, 177-191. Kaneko, A. ( 1970). Physiological and morphological identification of horizontal, bipolar and amacrine cells in goldfish retina. J. Physiol. 207, 623-633. Kaneko, A., and Hashimoto, H. (1967). Recording site of the single cone response determined by an electrode marking technique. Vision Res. 7 , 847-851. Kaneko, A., and Hashimoto, H. ( 1969). Electrophysiological study of single neurons in the inner nuclear layer of the carp retina. Vision Res. 9, 37-55. Kobayashi, H. (1962). A comparative study on electroretinogram in fish, with special reference to ecological aspects. J. Shimonoseki Col2. Fisheries 3, 407-538. Kuffler, S. W. (1953). Discharge patterns and functional organization of mammalian retina. J. Neurophysiol. 16, 37-68. Laufer, M., Svaetichin, G., Mitarai, G., Fatehchand, R., Vallecalle, E., and Villegas, J. ( 1961). The effect of temperature, carbon dioxide and ammonia on the neuronglia unit. In “The Visual System: Neurophysiology and Psychophysics” ( R . Jung and H. Kornhuber, eds. ), pp. 457-463. Springer, Berlin. LlacNichol, E. F., and Svaetichin, G. (1958). Electric responses from isolated retinas of fishes. Am. J. Ophthalmol. [3] 46, Part 2, 2 6 4 0 .

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MacNichol, E. F., Wolbarsht, M. L., and Wagner, H. G. (1961). Electrophysiological evidence for a mechanism of color vision in the goldfish. I n “Light and Life” ( W . D. McElroy and B. Glass, eds.), pp. 795-816. Johns Hopkins Press, Baltimore, Maryland. Marks, W. B. ( 1965). Visual pigments of single goldfish cones. J. Physiol. ( L o n d o n ) 178, 14-32. Marks, W. B., and MacNichol, E. F. (1963). Difference spectra of single goldfish cones. Federation Proc. 22, 2143 (abstr.). Mitarai, G. (1958). The origin of the so-called cone action potential. Proc. Japan Acad. 34, 299-304. Mitarai, G. ( 1960). Determination of ultramicroelectrode tip position in the retina in relation to S potential. J. Gen. Physiol. 43, Part 2, 95-99. Mitarai, G., Svaetichin, G., Vallecalle, E., Fatehchand, R., Villegas, J., and Laufer, M. ( 1961). Glia-neuron interaction and adaptational mechanisms of the retina. In “The Visual System: Neurophysiology and Psychophysics” ( R. Jung and H. Kornhuber, eds. ), pp. 463-481. Springer, Berlin. Motokawa, K., Oikawa, T., Tasaki, K., and Ogawa, T. (1959). The spatial distribution of electric responses to focal illumination of the carp’s retina. Tohoku J. Exptl. Med. 70, 151-164. Motokawa, K., Yamashita, E., and Ogawa, T. (1960). Studies on receptive fields of single units with colored lights. Tohoku J. Exptl. Med. 71, 261-272. Motokawa, K., Yamashita, E., and Ogawa, T. (1961). Slow potentials and spike activity of retina. J. Neurophysiol. 24, 101-110. Murakami, M., and Kaneko, A. (1966). Differentiation of PI11 subcomponents in cold-blooded vertebrate retinas. Vision Res. 6, 627-636. Murakami, M., and Sasaki, Y. (1968a). Analysis of spatial distribution of the ERG components in the carp retina. Japan. J. Physiol. 18, 326-336. Murakami, M., and Sasaki, Y. (198813). Localization of the ERG components in the carp retina. Japan. J. Physiol. 18, 337-349. Naka, K. I., and Rushton, W. A. H. ( 1966a). S-potentials from colour units in the retina of fish (Cyprinidae). J. Physiol. ( L o n d o n ) 185, 5 3 M 5 5 . Naka, K. I., and Rushton, W. A. H. (1966b)).An attempt to analyse colour reception by electrophysiology. J. Physiol. ( L o n d o n ) 185, 556-586. Naka, K. I., and Rushton, W. A. H. ( 1 9 6 6 ~ )S-potentials . from luminosity units in the retina of fish (Cyprinidae). J. Physiol. ( L o n d o n ) 185, 587-599. Noell, W. K. (1954). The origin of the electroretinogram. Am. J. Ophthulmol. 38, 78-90. Oikawa, T., Ogawa, T., and Motokawa, K. (1959). Origin of so-called cone action potential. J . Neurophysiol. 22, 102-11 1. Orlov, 0. Yu., and Maksiniova, E. hl. (1965). S-potential sources as excitation pools. Vision Res. 5, 573-582. Pak, W. L. (1965). Some properties of the early electrical response in the vertebrate retina. Cold Spring Harbor Symp. Quant. Biol. 30, 493-499. Pak, Mi. L., and Cone, R. A. (1964). Isolation and identification of the initial peak of the early receptor potential. Nature 204, 8364338. Schrodinger, E. ( 1925). Uber das Verhaltnis der Vierfarben- zur Dreifarbentheorie. Sitzber. Akad. Wiss. W i e n , Math.-Naturw. Kl. Abt. Ila, 134, 471490. Sjostrand, F. S. (1961). Electron microscopy of the retina. I n “The Structure of the Eye” (G. K. Smelser, ed.), pp. 1-28. Academic Press, New York.

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Stell, W. K. (1965). Correlation of retinal cytoarchitecture and ultrastructure in golgi preparations. Anat. Record 153, 389-398. Svaetichin, G. (1953). The cone action potential. Acta Physiol. Scand. 29, Suppl. 106, 565-600. Svaetichin, G. (1956). Spectral response curves from single cones. Acta Physiol. Scand. 39, Suppl. 134, 1 7 4 6 . Svaetichin, G., Laufer, M., Mitarai, G., Fatehchand, R., Vallecalle, E., and Villegas, J. (1961). Glial control of neuronal networks and receptors. In “The Visual System: Neurophysiology and Psychophysics” (R. Jung and H. Kornhuber, eds. ), pp. 445-456. Springer, Berlin. Tamura, T., and Niwa, H. (1967). Spectral sensitivity and color vision of fish as indicated by S-potential. Comp. Biochem. Physiol. 22, 745-754. Tomita, T. (1950). Studies on the intraretinal action potential. Part I. Relation between the localization of micropipette in the retina and the shape of the intraretinal action potential. Japan. J . Physiol. 1, 11Cb117. Tomita, T. ( 1957). A study on the origin of intraretinal action potential of the cyprinid fish by means of pencil-type microelectrode. Japan. J . Physiol. 7 , 80-85. Tomita, T. ( 1965). Electrophysiological study of the mechanisms subserving color coding in the fish retina. Cold Spring Harbor Symp. Quunt. Biol. 30, 559566. Tomita, T. (1968). Electrical response of single photoreceptors. Proc. IEEE, 56, 1015-1023. Tomita, T., and Kaneko, A. (1965). An intracellular coaxial microelectrode-its construction and application. Med. Electron. Biol. Eng. 3, 367-376. Tomita, T., Tosaka, T., Watanabe, K., and Sato, Y. (1958). The fish EIRG in response to different types of illumination. Japan. J. Physiol. 8, 41-50. Tomita, T., Murakami, M., Sato, Y., and Hashimoto, Y. (1959). Further study on the origin of the so-called cone action potential (S-potential). Its histological determination. Japan. J. Physiol. 9, 6-8. Tomita, T., Kaneko, A., Murakami, M., and Pautler, E. L. (1967). Spectral response curves of single cones in the carp. Vision Res. 7, 519-531. Toyoda, J., Nosaki, H., and Tomita, T. (1969). Light-induced resistance changes in single photoreceptors of Necturus and Gekko. Vision Res. 9, 453463. Trifonov, Yu. A. (1968). Study of synaptic transmission between photoreceptors and horizontal cells by means of electric stimulation of the retina. ( I n Russian.) Biofizika 13, N5. Trifonov, Yu. A., and Byzov, A. L. (1965). The response of the cells generating S-potential on the current passed through the eye cup of the turtle. ( I n Russian. ) Biofzika 10, 673-680. Villegas, G. M. (1960). Electron microscopic study of the vertebrate retina. J. Gen. Physwl. 43, No. 6, Part 2, 1543. Villegas, G. M., and Villegas, R. (1963). Neuron-glia relationship in the bipolar cell layer of the fish retina. J. Ultrastruct. Res. 8, 89-106. von Kries, J. ( 1905). Die Gesicktsempfindungen. In “Handbuch der Physiologie des Menschen” (W. Nagel, ed.), Vol. 3, pp. 109-282. Viewveg, Braunschweig. Wagner, H. G., MacNichol, E. F., and Wolbarsht, M. L. (1960). The response properties of single ganglion cells in the golash retina. I. Gen. Physiol. 43, 45-62. Werblin, F. S., and Dowling, J. E. (1969). Organization of the retina of the mudpuppy, Necturus maculosus. 11. Intracellular recording. ]. Neurophysiol. 32,

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Witkovsky, P. (1965). The spectral sensitivity of retinal ganglion cells in the carp. Vision Res. 5, 603-614. Witkovsky, P. ( 1967). A comparison of ganglion cell and S-potential response properties in carp retina. J. Neurophysiol. 30, 546-561. Witkovsky, P. (1968). The effect of chromatic adaptation on color sensitivity of the carp electroretinogram. Vision Res. 8, 823-837. Wolbarsht, M. L., Wagner, H. G., and MacNichol, E. F. (1961). Receptive fields of retinal ganglion cells: Extent and spectral sensitivity. I n “The Visual System: Neurophysiology and Psychophysics” (R. Jung and H. Kornhuber, eds.), pp. 170-177. Springer, Berlin. Yamada, E., and Ishikawa, T. (1965). Fine structure of the horizontal cells in some vertebrate retinae. Cold Spring Harbor Symp. Quant. Biol. 30, 383-392. Young, T. (1802). On the theory of light and colours. Phil. Trans. Roy. SOC. London 92, 12-48.

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VISION: T H E EXPERIMENTAL ANALYSIS OF VISUAL BEHAVIOR DAVID INGLE I. Introduction . . . . . . . . . 11. Relative Discrimination Weaknesses . . . . 111. Configurational Properties of Shapes . . . . IV. Perceptual Equivalence and Change in Spatial Position V. Selective Attention . . . . . . . . VI. Toward a Unified Outlook on Visual Behavior . . . . . . . . . . . . . References

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I. INTRODUCTION

This chapter reviews some analytical studies of fish visual behavior, laying the chief emphasis upon discrimination abilities demonstrated in the laboratory. Since many fishes depend heavily upon vision to guide their countless daily decisions, a complete review would ramble far and wide without providing a good account of our present knowledge concerning underlying visual mechanisms. Therefore, those studies were selected that seem most useful in analyzing fundamental visual processes -studies that seem most heuristic for a future liason between psychologists and those biologists who probe the visual system with scalpels and electrodes. This restriction in subject matter is not entirely arbitrary, since the best behavioral analyses have often been performed by those workers who have hoped to dissect visual behavior into manageable component processes. Most of the older European studies of fish vision are regretably ignored in this chapter. With a few exceptions, these workers did not use enough subjects or a rigorous enough experimental design to allow firm conclusions. Their aim was apparently to find in the fish kinds of visual behavior that we more readily attribute to higher vertebrates: visual 59

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constancies, susceptibility to illusions, or the ability to view different pairs of stimuli as having the same mutual relationships. Very often these studies stimulate the scientific imagination, only to leave the reader hungry for solid proof of the inherent claims. Nevertheless, the scholarly reader will be interested in the array of observations and experiments reviewed by Herter ( 1953). The first aim of the analytical approach is a physical description of the essential attributes of visual objects which guide their “recognition” or their “discriminability.” Objects can be distinguished along various dimensions-size, brightness, color, distance, orientation, and motionand psychologists have usually assumed that each dimension has a discrete physiological basis. Neurophysiologists have discovered within the visual pathways of various vertebrate species single neurons that are, in fact, tuned in to specific dimensions of the stimulus: brightness, color, orientation, or motion. Some of the data of perception is filtered out within the retina and along the retinofugal pathways, and other data are provided by internal central processes ( or other sensory modalities) whose function can be tenuously and imprecisely inferred. The present studies offer relevent information in approaching the first question of selectivity of the visual system: What features of the visual array do fish particularly notice? One kind of analysis likely to reflect limits of peripheral visual processing is the measurement of minimum separable acuity. Weiler (1966) obtained threshold acuity values of 5.3 min of visual angle, averaging the performance of three “Oscars,” Astronotus ocellatus, required to discriminate finely spaced dot patterns from a solid gray plaque. Taking the calculations of Brunner (1935), this limiting angle approximates the diameter of single retinal cones. Of course acuity of primates and some avian species measures far less than their cone diameters. Perhaps the fish has not evolved a mechanism for extracting this additional information through “temporal integration” of signals from the moving retinal image. A second area of research where retinal physiology ought to prove useful to the psychologist is that of color vision. The analysis of goldfish cones by Marks (1965) reveals three separate retinal photopigments and seems to imply that this species should possess trichromatic color vision. A logical proof that fish had color vision at all awaited the study of McCleary and Bernstein (1959) who showed that generalization between pairs of colored stimuli (red and green) could not be attributed to brightness cues, after first determining their subjects’ judgement as to the relative brightness of the critical stimuli. More recently, Muntz and Cronly-Dillon (1966) obtained evidence that goldfish color vision was very probably trichromatic: their subjects could distinguish reds, greens,

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and blues from one another where reliance on brightness cues was excluded. Furthermore, Yager (1967, 1968) demonstrated the ability of goldfish to detect additions of monochromatic light of any hue to pure white light. The saturation functions so determined in three subjects approximated quantitative predictions that Yager had derived from an "opponent color theory" model, itself based upon studies of human color vision. These lines of research-acuity and color measurements-off er twin rewards: detailed interspecies comparisons of the psychophysical laws of visual function and fruitful correlation with anatomy, biochemistry, and electrophysiology. These studies help to confirm a faith in the existence of discrete units that underly visual behavior. Yet further complexities have simply been avoided thus far by psychologists: measurement of "color constancy" or the interaction of color with form vision. In the following sections we shall review other dimensions of fish vision for which our image of discrete visual analyzers offers a still tenuous and unfulfilled hypothesis.

11. RELATIVE DISCRIMINATION WEAKNESSES

The search for relative weaknesses in shape recognition that might be attributed to limits of peripheral analysis of the visual image is exemplified by Mackintosh and Sutherland (1963), who found that goldfish took longer to acquire a discrimination between 45" and 135" oblique rectangles than to distinguish equivalent shapes set horizontally and vertically (Fig. 1). Sutherland has argued ( 1968) that the ability to distinguish orientations of contours might require orientation-specific visual units-somewhere within the afferent system-such as the units described by Hubel and Wiesel (1962) in the cat visual cortex. Indeed, Westerman (1965) has reported that elongated receptive fields of units recorded in the goldfish tectum do seem to fall more often along horizontal or vertical axes than along oblique axes. If goldfish have a paucity of units sensitive to oblique contours, they may nevertheless notice the horizontal or vertical extents of oblique edges. In fact, fish trained by Mackintosh and Sutherland to discriminate vertical from horizontal continued during transfer tests to distinguish shapes oriented at 30" from those set at 60" If goldfish pay more attention to horizontal and vertical components, even of such tilted shapes, it would be important to see whether fish initially trained on the 30"/60" problem improve their performance when tested with the 0"/90"pair.

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I '

7 - .......

I 1

Fig. 1. Apparatus used by Mackintosh and Sutherland (1963) to test the discriininability of dark rectangles at various orientations. The fish swims from forward chamber through hole and obtains food pellet from trough a t middle of correct shape. The incorrect shape is baited with a stone.

It must be pointed out that even such clear-cut results as these are subject to alternative interpretations. Although Ingle ( 1971) confirmed the conclusion of Mackintosh and Sutherland that goldfish have special difficulties with oblique lines, the suggestion was made that fish might nevertheless detect oblique lines as well as those at any other orientation. They might not remember them from trial to trial, having no built-in central classification scheme for distinctions among obliques. A critical experiment would measure the fish's ability to detect minimally visible edges (or gratings) set at various orientations. Such studies of acuity, although often necessary to interpret specific discrimination weaknesses with shapes, are conspicuously absent from the history of animal studies. Sutherland's recent report ( 1968) that goldfish tend to notice differences at the tops-rather than the bottoms-of a pair of shapes, raises similar problems of interpretation. A goldfish approaching the middle of a figure (where food is placed) might simply be in a better position to view the top of the shape. It would be important to determine whether goldfish detect isolated spots or colors more readily when placed at the top of a blank disc, baited in the center. At least one study, by Matthews (1964) in the blue acara, suggests that sensitivity to the top of a shape is not always the rule: His fish failed to distinguish a triangle with a

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bowed-out base from a circle but easily discriminated this pie-wedge shape from a triangle. Early workers also noted directional biases in fish vision, but they seldom used enough subjects to allow firm conclusions. Yet, in the light of subsequent knowledge, it is interesting that Hager (1938) found minnows discriminating one vs. two stripes, or three vs. four stripes, more readily when these patterns were oriented vertically. As Trevarthen ( 1968a) has emphasized, such frontally approached stimuli generate expanding retinal images, which could be analyzed by retinal units sensitive to image motion. Two studies (Jacobson and Gaze, 1964; CronlyDillon, 1964) have revealed that goldfish ganglion cells sensitive to direction of motion are most often sensitive to horizontal movement of small objects or edges. This mechanism might apply as well to Meesters' finding (1940) that a single fish trained to distinguish large and small squares transferred best to long vs. short rectangles when these shapes were horizontally oriented. That is to say, Meesters' subject was more sensitive to image-expansion (measuring the distance between the two edges) when the differences lay along the horizontal axis. An observation by Saxena (1966) suggests that the trout may-in distinction-pay more attention to size differences along the vertical axis. Her subjects failed to transfer a size discrimination involving outline squares when either the top or bottom side was removed (as did a subject tested in the same way by Meesters, 1940). Unfortunately, control experiments involving removal of a vertical side were not reported. It might be useful to pin down such possible species differences in shape recognition, since we assume that visual analyzing mechanisms are variously adapted to the ecology or social behavior of the species. The trout, for example, must execute h e distance judgments within the upper sagittal plane preparatory to jumping for an insect and might well profit from a mechanism sensitive to distances along the vertical axis, An electrophysiological study by Jacobson and Gaze (1964) provides further information on directional bias within the goldfish visual system. Retinal units were more often sensitive to nasalward-as opposed to temporalward-movement of spots within the visual fields. This built-in asymmetry might explain the observation by Harden Jones (1963) that several species of fish would follow nasalward but not temporalward rotation of a surrounding striped drum. Furthermore, Ingle (1967) has demonstrated a behavioral correlate of this directional bias using a cardiac-conditioning method (Fig. 2 ) . When a small 2" spot-moving at 12"1sec in the lateral field-served as a conditioned stimulus paired with shock, all subjects responded with a stronger cardiac deceleration during nasalward motion of the stimulus. This stimulus was comparable to that

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I

Nasal bias

I

I

Temporal bias

Fig. 2. Apparatus used by Ingle (1967) for cardiac conditioning of goldfish. Single or multiple dots moving behind window in aquarium serve as conditioned stimuli when paired with shock.

used by Jacobson and Gaze ( 1964). However, when the same spot moved at only 3"/sec ( a velocity not systematically studied by the physiologists), each of the six fish showed significant cardiac slowing only to the temporalward stimulus. This reversal of directional sensitivity obtained by changing velocity implies two separate underlying mechanisms (not necessarily to be identified with two different unit populations). A third group of goldfish showed greater temporalward responses when a multispotted stimulus was used, even where velocity was 12"/sec. Experiments on motion detection may be useful in establishing further behavioralphysiological correlations since parameters can be precisely varied in both kinds of study. Furthermore, a detailed knowledge of motion perception seems necessary for full interpretation of shape recognition sincc all procedures utilize moving shapes or moving retinal images.

111. CONFIGURATIONAL PROPERTIES OF SHAPES

The above experiments on discrimination of orientation or motion of objects belong to a psychology geared to physiological models. Yet, traditional concerns about configurational properties ( largely raised by the

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Gestalt school) still guide behavioral research. Since we ourselves recognize many shapes despite transformations of size, position, rotation, or brightness contrast, we are tempted to ask whether our animal subjects also have ways of categorizing shapes that transcend these transformations. For example, it is commonly assumed that shapes are easily recognized by animals, despite changes in size (Sutherland, 1968). However, the usual method of demonstrating “size invariance” is inadequate since an animal approaching the stimulus (for food reward) experiences considerable variation in size of the associated retinal images of each shape. To overcome this difficulty, Ingle ( 1971) trained goldfish to discriminate 14” wide circles from equal-sized squares that were presented at a fixed distance lateral to one eye during a conditioned avoidance paradigm. When well trained, five fish learned the opposing habit using smaller (7”) shapes. For example, fish avoiding the large circle but not the square now learned to avoid the small square but not the circle. When subjects were retested (without reinforcement) using the original large shapes, they retained the initial habit despite the intervening training. We cannot say that these fish found no resemblance between large and small squares, but rather that they used two different sets of rules in distinguishing the two pairs of shapes. These results do not settle the important problem of size invariance, but they do indicate that size is sometimes an important determinant of the way a fish can classify particular shapes. Studies by Bowman and Sutherland (reported in Sutherland, 1968) indicate that goldfish do not generalize the properties of a square through a 45” rotation to a diamond. Following training on a circle vs. square discrimination, their subjects failed to distinguish a circle from a diamond. However, other discriminations may be transposed through rotations. Both Schulte (1957) and Saxena (1966) found good transposition of a discrimination of stripe number (and width) from vertical to horizontal settings (Fig. 3 ) . This task requires attention to number of line elements rather than to their spatial relationship. Saxena noted that a square vs. X discrimination would generalize to a diamond vs. cross pair. Here the discrimination is not one of orientation but a topological distinction between open shapes vs. intersecting lines. These experiments show that fish can judge two stimuli (e.g., squares and diamonds) either as equivalent or as distinctive, depending upon which basis of comparison our training procedure forces upon them. It is the aim of the generalization test to indicate the kinds of “similarity” that may exist. As Kliiver (1933) has documented in elegant detail, it is difficult to disentangle the actual dimensions of visual analysis from even an assortment of such tests. The assumption that fish possess inherently fewer and more rigid classifications than the higher mammals is plausible but is by no means demonstrated. Ingle ( 1971) has speculated that large-scale “gestalt” features such

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nrm X Fig. 3. Stimuli used by Sutherland (1968), Schulte (1957),and Saxena (1966) for studies of discrimination transfer to patterns rotated by 45’. Generalization between the first two pairs of shapes failed while the other two transposition problems were successful with carp and with trout, respectively.

as “parallelness” of left and right sides can be used to distinguish circles from squares. This argument assumes that fish are less sensitive to parallelness along the oblique axis (as with a diamond) than along horizontal and vertical axes as with a square. Therefore, the diamond, with oppositely bent sides, ought to resemble a circle to some extent. In fact, goldfish trained to avoid 14” circles, discriminated squares more easily than diamonds as “no go” stimuli. It would be useful to know whether diamonds are actually more similar to circles than to squares and whether or not such results would be obtained with smaller stimuli as well. In any case, the notion that fish discriminate parallelness of contours has been demonstrated by a more direct procedure. Fish that learned to avoid a stimulus containing a pair of parallel lines (both vertical or both tilted 30” from vertical) readily learned to discriminate a nonparallel pair (vertical plus 30”) as “no go” but did not learn to withhold response to the second set of parallel lines. These subjects did not rely upon the individual orientation of isolated line segments or else they would more easily discriminate between the two parallel sets of lines, where both parts of one differed from both parts of the other. Rather, the relationship between parts of a figure ( parallelness) outweighed the potentially useful information about individual lines. Such visual abilities reflect high order perceptual processes that seem not to depend upon awareness of those

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peripheral mechanisms required for the final classification (i.e., some measurement of individual line orientation must precede or participate in the determination of parallelness ) . The implications of this argument for a theoretical approach to visual coding is discussed in some detail by Sutherland ( 1968). These discussions of the circle-square discrimination have supposed that curved and straight lines are easily distinguished. Ingle (1971) performed a further test to determine whether the recognition of “curvature” is invariant with changes in orientation (Fig. 4).For example, goldfish trained to avoid a shallow horizontal curve easily learned to withhold response to a horizontal line but were confused by a vertical curved line. Similarly, subjects learned to distinguish vertical curves from vertical straight lines but not from horizontal curves. However, without data on generalization to oblique curves, we cannot yet say that goldfish recognize “curvature” per se. Go

No go

No go

(3 C

@GI

Fig. 4. Stimuli used by Ingle (1969a) in studies of relative discrimination difficulty. In each of four problems (a-d) the fish avoids the stimulus on the left. Successful “no go” discriminations were obtained with each stimulus in the center but not with stimuli on the right.

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IV. PERCEPTUAL EQUIVALENCE AND CHANGE IN SPATIAL POSITION

For all vertebrates the retinofugal fibers project to midbrain or thalamus in a spatially ordered manner preserving at the tectum or in the striate cortex a mapping of visual space. Many workers have, therefore, been interested in measuring the extent of an animal's ability to judge shapes as equivalent when they fall upon disparate parts of the retina or are viewed by different eyes. Studies of the invariance of shape recognition with changes in position provide a certain way of excluding factors of retinal coding from considerations of mechanisms underlying shape perception. Other possible transformations of a shape-size, orientation, and color-do not necessarily exclude mechanisms of equivalence at the retinal level. Cronly-Dillon et al. ( 1966) have demonstrated "intraretinal transfer" in goldfish by an ingenious method. Since the dorsal and ventral brachia of the goldfish optic tract diverge to innervate the upper and lower tectum, respectively (i.e., mediate shape discrimination in the upper and lower halves of the field), cutting one branch restricts retinal input to half of one tectum. Subjects trained to discriminate vertical from horizontal rectangles after one brachium had been cut could retain this discrimination several weeks later after the severed fibers had regenerated and the second (formerly used) branch was then cut. This clearly proves that shape equivalence of at least a simple order exists when different retinal inputs are employed. However, it is likely that overlap of tectal receiving neurons occurs at the horizontal margin of these two optic inputs since retinal receptive fields may be as large as 30"-40" of visual angle. A study by Ingle ( 1963) indicates that pattern-discrimination transfer may occur when two sets of retinal images are each projected to two disparate tectal regions. Using a cardiac-conditioning method, goldfish showed good transfer of a horizontal vs. vertical or horizontal vs. diagonal stripe discrimination from a temporal training position to a nasal testing position 120" rostrally. If we allow for the occasional eye movements, ranging up to 30", the minimal intraretinal distance over which equivalence was demonstrated is a respectable 90". Whether or not more difficult shape discriminations ( e.g., circle vs. square) would transfer over this distance is a more critical, but unanswered, question. Ingle noted, rather surprisingly, that intraretinal transfer totally failed with the stripe discrimination in 12 subjects trained and tested by a conditioned-avoidance method even though a red-green discrimination did transfer from back to front in the same fish. It is not known whether the success with

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cardiac-conditioning simply reflects the advantages of an easier task for the subject (heart-rate changes are not all-or-none as is the choice demanded by avoidance training) or whether a more interesting physiological explanation is available. Since each eye of a fish sends visual information exclusively to the contralateral tectum, we might expect incomplete behavioral equivalence between opposite eyes. Indeed, the first relevant study by Sperry and Clark (1949) showed that interocular transfer of a simple up vs. down problem was poor or absent in the majority of subjects. However, Schulte (1957) obtained high levels of pattern transfer with carp following extensive training sessions. Schulte found clear transfer failure only when his fish were confronted via the untrained eye with distorted or rotated versions of the training stimuli. These subjects could transpose their training experience while using the training eye, however. This delicate ability to judge similarity between different sets of patterns might have been disrupted by changing eye covers prior to transfer tests which, at first, made some fish “neurotic.” This interpretation is made plausible by the demonstration of McCleary (1960) that goldfish with eye covers may fail interocular transfer of a simple discrimination that occurs readily when blinders are not used. Other studies, however, have demonstrated limits of interocular transfer that cannot be attributed either to emotional disruption or to the use of a suboptimal response criterion. For example, Ingle (1965) trained goldfish to discriminate a striped from a random pattern, where the stimuli also differed in color (red or green). When these fish were tested with the same patterns, each appearing in opposite colors, they could resolve the conflict by responding to either color or pattern differences. Subjects that were tested first via the trained eye responded on the basis of the (more discriminable) pattern dderences, while those fish initially tested via the second eye behaved in accord with the color differences. Therefore, one concludes that pattern information transfers less well than color information in the goldfish. This method of comparing the “transferability” of various visual discriminations could provide guidelines for eventual recording from units in the various interhemispheric commissures of the fish brain. Although the aforementioned study clearly proved the relative failure of pattern transfer, other studies (Ingle, 1968a) argue that a total failure can sometimes be obtained. This result might be more encouraging for the physiologist who prefers all-or-none results. Goldfish trained to discriminate vertical stripes from those rotated by 23” failed to show any evidence of transfer, unlike successful controls trained with vertical vs. 52” rotation, although both groups were tested with the same stimuli differing by 38”. Even transfer of a horizontal-vertical stripe discrimi-

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nation could be prevented if the second brain half were “occluded by the response to an irrelevant spotted stimulus presented to the second eye on each trial. Thus, we conclude that transfer may fail when ( a ) the commissural system cannot resolve fine stimulus differences, or ( b ) the second brain half is jammed by induced noise and cannot record input from the commissures. The fact that interocular integration is sometimes incomplete suggests that independent visual learning might proceed simultaneously within opposite halves of the brain. Ingle (1968a) has demonstrated that goldfish can, indeed, acquire opposing discriminations of pattern or of color via opposite eyes; for example, avoiding red not green via the right eye and avoiding green not red via the left. Double learning is easily achieved when stimuli are presented either ( a ) to both eyes on each trial, or ( b ) to one eye at a time, alternating eyes on successive trials, but is more difficult to attain when ( c ) long sequences of monocular trials are confined to one eye at a time. Perhaps the difficulties inherent in the “alternating sessions” method account for the inability of Schulte (1957) or Shapiro ( 1965) to demonstrate interocular double learning with carp or goldfish. However, Schulte and Shapiro both used frontally approached (expanding) stimuli, while Ingle used size-restricted stimuli confined to the lateral field. Perhaps interocular integration is favored in the ( overlapping) binocular field, while dissociation is more easily obtained using lateral stimuli. Finally, we consider an interocular transfer problem that is rather peculiar in its very formulation: How can stimuli appearing via opposite eyes be judged equivalent when the discrimination is based upon differences in the directional orientation of the shapes? Since the fish‘s eyes are set upon opposite sides of the head, there is considerable ambiguity in predicting how one hemisphere communicates distinctions of leftright or back-front to the other. As Fig. 5 illustrates, a leftward (nasalward) arrowhead viewed by the right eye casts an image projected to the tectum in a rostral-pointing direction just as a frontward pointing arrowhead seen in the right lateral field. If the arrowhead in the frontal plane-seen by both eyes at once-is to produce two images that map onto one another via the commissural system, one must conclude that a nasalward direction for one eye is “equivalent” to a temporalward direction as seen via the second eye. But if one coding process is applicable to all parts of the two retinas, this logic forces the absurd prediction that an object seen in front of the fish via one eye is more similar to a trailing than to a leading stimulus on the other side. Ingle (1967, 1968b) has shown that both mechanisms coexist and are called forth by different kinds of experimental stimuli. Fish trained on

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Fig. 5. Illustration of left-right mode of interocular transfer obtained with mirror image arrowhead discrimination. Fish trained to avoid forward-pointing stimulus on right (but not backward arrowhead) will avoid backward stimulus but not forward version, when tested via left eye. If these equivalent stimuli were moved into the frontal binocular field, they would come into register.

mirror image discriminations of objects seen in Fig. 6 can show two separate modes of interocular transfer. Subjects discriminating either ( a ) the 135" vs. 45" oblique lines or ( b ) the mirror image arrowheads showed a left-right mode of directional equivalence in that stimuli projecting in the same direction on the frontal plane are taken as equivalent during interocular transfer tests. For discriminations ( c ) and ( d ) , a front-back equivalence is obtained: Similar stimuli project in the same direction on the lateral plane that parallels the sagittal plane of the body. The two pairs of stimuli classified by the left-right mode subtended 8"-1O0 of visual angle, while pairs ( c ) and ( d ) were 15" and 22" in size. Possibly the stimuli of larger size are more easily broken down into two partsthe front and back units-so that the brain can describe the stimulus in

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Group I

Group 2

Group 3

Group 4

Fig. 6. Stimuli used for mirror discriminations by Ingle ( 1967). Fish in Groups 1 and 2 showed left-right interocular equivalence (as illustrated in Fig. 5 ) , while just the opposite relationships were obtained during transfer tests in Groups 3 and 4.

terms of spatial position relative to the body (red in front plus green in back). This is undoubtedly true for the red-plus-green squares since fish trained to discriminate mirror image pairs continue to discriminate well when the back squares are removed from each stimulus. Where local sign seems critical for coding the larger pair of shapes, the smaller stimuli would seem to be taken in as units with a visual direction somehow assigned. As argued elsewhere ( Ingle, 1967, 1968b) , this second kind of transfer cannot be based upon any point-to-point mapping between the two optic tecta, but it must involve a shape recognition process that is based upon a nonspatial code at this level of the brain. This distinction between mechanisms of ( a ) positional labeling and ( b ) shape recognition has been considered as analogous with “orienting vs. identifying” modes of vision, which have different neural substrates in mammals ( Schneider, 1967). Furthermore, both Trevarthen (196813) and Held (1968) make similar distinctions between dissociable visual processes described in cat, monkey, and man himself. Although mechanisms of orienting toward and identifying objects are doubtless more complex among mammals than among fishes, it is important to recognize a fundamental dualism within vision that may have appeared with the first vertebrate. V. SELECTIVE ATTENTION

The human observer takes for granted the ability to glance quickly over a complex visual scene, ready to take in those details that he is

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prepared to see. Observations of fish locomotion and associated eye movement also suggest selective visual operations during behavior. It is even possible that “internalized” selection occurs when the fish rests in place, so that only selected features of the visual array (or selected portions of the visual array) have full access to central processes. If this were so, it would impose still another barrier between the psychologist and the neurophysiologist who is usually restricted to an anesthetized or paralyzed preparation. While there is little conclusive evidence at the behavioral side for selective attention, the issue is so important that a review of the few relevant facts may be of heuristic value. Some fish move one eye at a time in response to a moving object, while the other eye stares indifferently into space. One suspects, without direct evidence, that the redirected eye must be the more sensitive at that moment, in connection with the motor dominance of one hemisphere. Visual behavior toward objects appearing within the binocular field of a moving fish presents the same issue. Harris (1965) has shown for the dogfish that eye movements are asymmetrical during the sideward waggling of the head: The eye toward which the head swings is momentarily stabilized by compensatory reflexes in reference to a locus of points 3 feet to the side of the fish. Trevarthen (1968a) showed that compensatory eye movements in a spinalized goldfish are likewise asymmetrical. It seems that a locomoting fish must either accept double vision within the binocular field or attend selectively to information arriving the stabilized eye. We do know that fish can use both eyes simultaneously during a discrimination task when stimuli appear within the lateral fields. Ingle ( 1968a) trained goldfish to compare stimuli seen via opposite eyes (i.e., to avoid an unlike pair such as horizontal plus vertical stripes) while treating a horizontal or a vertical pair as “no go” stimuli. Another experiment in this series showed that goldfish do not ignore monocular information even where it is irrelevant and where it would be to their advantage. Subjects learning a vertical-horizontal stripe discrimination via one eye also viewed the horizontal stimulus via the opposite eye on each trial. On critical “attention” trials, the “no go” horizontal pair was changed by inserting a novel red-on-white horizontal striped stimulus on one side or the other. Subjects were disinhibited by the red color more often via the irrelevant eye which contradicts the hypothesis that they would suppress visual processes on the nondiscriminating side. Attention might be shifted to particular dimensions of analysis (size, orientation, brightness, etc.) as well as to selected regions of the visual field, as Mackintosh (1965a) has suggested on the basis of experiments with rats. Hemmings (1966) has tested one prediction of this model, using tropical fish: that a difficult discrimination will be more efficiently

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learned by pretraining subjects on an easier version of the problem in order to reward them for “switching in the appropriate analyzer.” Indeed, fish pretrained on a circle-triangle discrimination were able to solve an otherwise impossible circle-square discrimination. A second pretraining series on circle vs. larger square, emphasizing the irrelevant dimension of size, did not facilitate learning of the difficult circle-square problem. However, a study by Sutherlands group (Mackintosh et al., 1966) failed to show evidence of selective attention: Overtrained goldfish reversed a shape discrimination more slowly than controls, unlike rats who reverse more quickly under similar conditions ( Mackintosh, 1965b). Other tests will be required to decide whether fish have evolved this interesting facet of behavior. A final answer will be useful to the physiologist who wonders how radically the visual coding processes of retina and tectum may be altered by centrifugal influences.

VI. TOWARD A UNIFIED OUTLOOK ON VISUAL BEHAVIOR

Aspects of fish behavior which have not yet been analyzed in a sufficiently rigorous manner have been deliberately excluded, although it is clear that vision has been designed for ecological and social functions and not for playing games with psychologists. Analysis of visual mechanisms might be pursued by experiments that describe optimal stimuli for guiding natural movements or eliciting consummatory responses. Briefly summarized below are some hypotheses designed to link observed behavior toward artificial stimuli with the uses of vision in the real world. A more complete discussion of this material has been presented elsewhere ( Ingle, 196813, 1971). The study of motion detection by goldfish (Ingle, 1967, 1968b) reveals two or three independent processes that have been hypothetically identified with aspects of natural visual behavior. The fast-moving single spot is taken as a prototype of predator motion or the motion of a rival’s markings used to direct aggressive attack (an eye or a body spot). The higher probability of catching forward rather than backward moving prey might explain the specialization for nasalward directions with a fast-moving spot. Furthermore, it has been noticed that aggressive mouthbreeders, Tilupiu, seldom, if ever, attack a smaller fish moving in the opposite direction, but they readily chase a rival who moves past in a head-to-head orientation. The slow-moving spot, seen best while moving backward, might correspond to a prey object being pursued; the ternporalward velocity would inform the pursuer how rapidly the gap was being closed and help set

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the timing of the snap, Mouthbreeders do require continuous motion feedback while pursuing food since, under a stroboscopic light at 3 Hz, they badly misjudge the rate of fall and snap too high. The multispotted stimulus might also be an analog of temporally moving features within the visual array through which the fish swims. Here, as well, visual feedback informs the fish of his own progress (in a moving stream, the visual cue to swimming rate would be the most reliable one). Rate of temporalward motion also provides information on distances of objects or surfaces ahead or to the side. In this context, the dominance of horizontal or vertical axes, revealed by shape recognition experiments, makes good sense: These particular axes of image translation are produced by fish that themselves locomote within horizontal or sagittal planes. The interesting ability of goldfish to notice “parallelness” might also serve to discriminate the tilt of surfaces (such as inclining rocks). If a retinal image displacement resulting from body movement maintained the orientation of a contour in the retinal image (i.e., displaced, but still parallel), the fish might infer that the contour is confined to a plane perpendicular to the horizontal plane through which it swims. Nonparallel displacements, on the contrary, represent contours or surfaces that incline toward, or slope away from, the organism. The broad category “curvature” can be seen in this light: A curving retinal displacement must correspond to an object moving independently of the fish. Perhaps as various shapes may have physionomic connotations to man (Werner, 1940), curved lines suggest something “animate” to the fish. These teleological speculations cannot pass for explanations of visual behavior, but they can direct the psychologist toward new methods of measuring specific visual abilities. As yet, we know almost nothing about the sensitivity of fish to the various transformations of the optic array through which they swim. The method of “false feedback used to analyze human sensorimotor abilities could be used with many fish (as Sperry, 1950, achieved by eye rotation). Already, some physiological evidence indicates that concern for differential patterns of motion seen by fish is likely to be fruitful in research. Jacobson (1968) has described directionally sensitive ganglion cells in the goldfish retina that can be suppressed by moving a second object outside of the receptive field in a direction opposite to that of the stimulating object. Discrimination between objects that move together and those that converge may utilize these peripheral intraretinal inhibitions to code differential movement. It is worth adding that the frog-who cannot obtain such parallax information-does not show the kind of direction-specific cross inhibition that Jacobson has revealed in the fish. Perhaps in gaining physiological hints as to the mechanism of spatial vision in higher mammals, the active fish will prove a better model than statuesque amphibians and reptiles,

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REFERENCES Brunner, G. (1935). Uber die Sehscharfe der Elritze (Phoxinus laevis) bei verscheidenen Helligeiten. 2. Vergleich. Physiol. 21, 296-316. Cronly-Dillon, J. R. (1964). Units sensitive to direction of motion in goldfish optic tectum. Nature 203, 214-215. Cronly-Dillon, J. R. Sutherland, N. S., and Wolfe, J. (1966). Intraretinal transfer of a learned visual shape discrimination in goldfish after section and regeneration of the optic nerve branchia. Exptl. Neurol. 15, 455-462. Hager, H. J. ( 1938). Untersuchungen uber das optische Differenzierungsvemogen der Fische. 2. Vergleich. Physiol. 26, 282-302. Harden Jones, R. R. (1963). The reaction of fish to moving backgrounds. J. Exptl. Biol. 40, 437-446. Harris, A. J. (1965). Eye movements of the dogfish, Squalus acanthi- L. J. Exptl. Biol. 43, 107-130. Held, R. ( 1968). Dissociation of visual functions by deprivation and rearrangement. Psychol. Forsch. 31, 338-348. Hemmings, G. (1966). The effect of pretraining in the circle/square discrimination situation. Animal Behaviour 14, 212-216. Herter, K. ( 1953) . “Die Fischdressuren und ihre sinnesphysiologischen Grundlagen.” Akademie Verlag, Berlin. Hubel, D. H., and Wiesel, T. N. ( 1962). Receptive fields, binocular interaction and functional architecture in the cat’s visual system. J. Physiol. (London) 160, 106-154. Ingle, D. (1963). Limits of visual transfer in goldfish. Ph.D. Thesis, University of. Chicago. Ingle, D. (1965). Interocular transfer in goldfish: Color easier than pattern. Science 149, 1000-1002. Ingle, D. (1967). Two visual mechanisms underlying the behavior of fish. Psychol. Forsch. 31, 44-51. Ingle, D. ( 1968a). Interocular integration of visual learning by goldfish. Brain, Behavior Evolution 1, 58-85. Ingle, D. (196813). Spatial dimensions of vision in fish. In “The Central Nervous System and Fish Behavior” (D. Ingle, ed.), pp. 51-60. Univ. of Chicago Press, Chicago, Illinois. Ingle, D. ( 1971). Analyses of shape discrimination abilities in goldfish. Brain, Behavior Evolution ( in press ) . Jacobson, M. (1968). Physiology of fish vision. In “The Central Nervous System and Fish Behavior” ( D . Ingle, ed.), pp. 17-24. Univ. of Chicago Press, Chicago, Illinois. Jacobson, M., and Gaze, R. M. (1964). Types of visual response from single units in the optic tectum and optic nerve of the goldfish. Quart. J. Exptl. Physiol. 49, 199-209. Kliiver, H. ( 1933). “Behavioral Mechanisms in Monkeys.” Univ. of Chicago Press, Chicago, Illinois (reprinted in Phoenix Science Series, Univ. of Chicago Press, Chicago, Illinois, 1961). McCleary, R. A. (1960). Type of response as a factor in interocular transfer in the fish. J . Comp. Physiol. Psychol. 53, 311-321. McCleary, R. A., and Bernstein, J. J. (1959). A unique method for control of brightness cues in the study of color vision in fish. Physwl. Zool. 32, 284-292.

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Mackintosh, N. J. ( 1965a). Selective attention in animal discrimination learning. Psychol. Bull. 64, 124-150. Mackintosh, N. J. ( 196513). Overtraining, extinction and reversal in rats and chicks. J. Comp. Physiol. Psychol. 59, 31-36. Mackintosh, N. J., and Sutherland, N. S. (1963). Visual discrimination by goldfish: The orientation of rectangles. Animal Behauior 11, 135-141. Mackintosh, N. J., Mackintosh, J., Safriel-Jorne, O., and Sutherland, N. S. (1966). Overtraining, reversal and extinction in the goldfish. Animal Behauior 14, 314318. Marks, W. B. (1965). Visual pigments of single goldfish cones. J. PhysioZ. (London ) 178, 14-32. Matthews, W. A. (1964). Shape discrimination in tropical fish. Animal Behauiour 12, 111-115. Meesters, A. ( 1940). Uber die Organization des Gesichtsfeldes der Fische. Zeitschr. Tierpsychol. 4, 84-149. Muntz, W. R. A., and Cronly-Dillon, J. R. (1966). Color discrimination in goldfish. Animal Behavior 14, 351-355. Saxena, A. ( 1966 ) . Lernkapazitat, Gedachtnis und Transpositionsvermogen bei Forellen. Zool. Jahrb., Abt. Allgem. Zool. Physiol. Tiere 69, 63-94. Schneider, J. E. (1967). Contrasting visuomotor functions of tectum and cortex in the Golden Hamster. Psychol. Forsch. 31, 52-62. Schulte, A. ( 1957). Transfer- und Transpositionversuche mit monokulardressierten Fischen. Z. Vergleich. Physiol. 38, 432-476. Shapiro, S. M. (1965). Interocular transfer of pattern discrimination in the goldfish. Am. J. Psychol. 78, 21-38. Sperry, R. W. (1950). Neural basis of the spontaneous optokinetic response produced by visual inversion. J. C o m p . Physiol. Psychol. 43, 4 8 2 4 8 9 . Sperry, R. W., and Clark, E. (1949). Interocular transfer of visual discrimination habits in a teleost fish, Physiol. 2001.22, 372-378. Sutherland, N. S. (1968). Shape discrimination in the goldfish. I n “The Central Nervous System and Fish Behavior” ( D . Ingle, ed.), pp. 35-50. Univ. of Chicago Press, Chicago, Illinois. Trevarthen, C. B. (1968a). Vision in fish: The origins of the visual frame for action in vertebrates. I n “The Central Nervous System and Fish Behavior” ( D . Ingle, ed.), pp. 61-96. Univ. of Chicago Press, Chicago, Illinois. Trevarthen, C. B. (1968b). Two mechanisms of vision in primates. Psychol. Forsch. 31, 299-337. Weiler, I. J. (1966). Restoration of visual acuity after optic nerve regeneration in Astronotus ocellatus. Exptl. Neurol. 15, 377-386. Werner, H. ( 1940). “Comparative Psychology of Mental Development.” Harper, New York. Westerman, R. A. (1965). Specificity in regeneration of optic and olfactory pathways in teleost fish. I n “Studies in Physiology” (D. R. Curtis and A. K. McIntyre, eds.). Springer, New York. Yager, D. ( 1967). Behavioral measures and theoretical analysis of spectral saturation in the goldfish, Carassius auratus. Vision Res. 7, 707-727. Yager, D. (1968). Behavioral analysis of color sensitivities in goldfish. I n “The Central Nervous System and Fish Behavior” ( D . Ingle, ed.), pp. 25-34. Univ. of Chicago Press, Chicago, Illinois.

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CHEMORECEPTION TOSHIAKl 1. H A M I . Introduction . . . . . . . . . . . . . I1. Anatomy of Chemical Sense Organs A Olfactory Organ . . . . . . . . B . Gustatory Organ . . . . . . . . . . . . . . C Free Nerve Endings . . D Morphology of Brain and Feeding Habits . I11. Behavioral Studies of Chemoreceptive Functions . A . Olfactory Sense . . . . . . . . B Gustatory Sense . . . . . . . . IV . Electrophysiological Studies of Chemoreceptor Responses A Olfactory System . . . . . . . . B. Gustatory Receptors . . . . . . . V. Biological Aspects of Chemoreception . . . . A. Chemical Perception of Foods . . . . . B. Reproductive Behavior and Chemical Senses . . C . Discrimination of Body Odors and Schooling . . D Alarm Substances . . . . . . . E . Repellents . . . . . . . . . F. Orientation by Chemical Senses . . . . References . . . . . . . . . . .

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I INTRODUCTION

Chemoreception plays an important and indispensable role in the behavior of fishes. It is involved in the procurement of food. recognition of sex. discrimination between individuals of the same or different species. in defense against predators. in parental behavior. in orientation. and in many other ways. Primarily through conditioning techniques. studies of 79

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chemoreception in fishes have followed two principal directions: (1) the investigation of ecuity and range of the chemical sense organs, and ( 2 ) the investigation of the biological signifkance of chemoreception in the behavior and life cycles of fishes. Chemoreceptive functions in fishes have been well reviewed by Hasler (1957), Teichmann (1962b), and by Kleerekoper (1969). During the last decade there has been a marked increase in the number of studies dealing with the chemical sense in fishes. Electron microscopy has been applied to the fine structure of the chemoreceptor organs; recent electrophysiological investigations of the chemosensory system have added greatly to the understanding of the mechanism underlying chemoreception in fishes. Chemoreception by animals shows a wide range of sensitivities. On the basis of location and structure as well as the central innervation, chemical reception has been divided into three categories: olfaction or smell, gustation or taste, and common or general chemical sense. These sensory modalities overlap somewhat: Some substances elicit responses from both types of receptors. Practically, in terrestrial animals, those receptors which have high sensitivity and specificity, and which are “distance chemical receptors” are distinguished as olfactory, those receptors of moderate sensitivity and stimulated by dilute solutions are gustatory or “contact chemical receptors,” and those receptors which are relatively insensitive and nondiscriminating are considered common chemical sense. In fishes, smell and taste are both mediated by dilute aqueous solutions so that the distinction is made anatomically and physiologically. Olfactory organs are innervated by the &st cranial nerve which contains the axonal extensions of the primary receptor cells to the olfactory bulb. Through the olfactory tracts bulbar activity is related to the rest of the nervous system. Those fishes with well-developed olfactory capacities are called “macrosmatic”; those in which the capacities are less acute are called “microsmatic.” Taste is mediated through the taste buds with the secondary sensory cells. Taste buds lie not only in the mouth and pharynx but also in the gill cavity, on the gill arches, on appendages such as barbels and fins, and also, in some fishes, on all external surfaces of the body. The taste buds are innervated by the VIIth, IXth, and Xth cranial nerves which terminate in much enlarged vagal lobes. Common chemical sense, which was originally named by Parker (1912), is also located on exposed body surfaces of fishes. These are free nerve endings supplied by the spinal nerves. In contrast to the two senses mentioned above, the general chemical sense is relatively low in sensitivity.

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11. ANATOMY OF CHEMICAL SENSE ORGANS

A. Olfactory Organ Extreme variations are found in the morphology of the peripheral olfactory organs depending largely on the degree of development of the olfactory system. In the sharks and rays where the chemical senses are highly important ecologically, the paired olfactory pits or sacs are usually situated at varying distances from the oral opening on the ventral side of the snout. The opening of each pit is divided into two parts by a fold of skin: anterior inlet and posterior outlet; in some instances the latter leads to the mouth. As the fish swims through the water and as it takes water into the mouth to breathe, a current passes through the olfactory sacs. Thus, in the sharks and rays, the olfactory organs are influenced directly by the respiratory current. In the teleost fishes the paired olfactory pits are usually on the dorsal side of the head, somewhat removed from the mouth (Fig. 1).The eels

f

or

on

Fig. 1. Position and internal structure of the nose in the minnow, P h o x i n ~ s phoxinzis ( A ) and eel, AnguiZZu anguillu ( B ) . an, Anterior naris; pn, posterior naris; f, skin flap; and or, olfactory rosette. From H. Teichmann, Umschau Wiss. Tech. 62, 588-591, Frankfurt, Germany, 1962.

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and morays (Anguilliformes), with the most acute demonstrated sense of smell, have large and elongate olfactory pits, extending from the tip of the snout to the orbit of the eye. In contrast, certain puffers (Tetraodontidae ) which are highly visually oriented reef fishes, have completely lost the nasal sacs. Various patterns of intermediate anatomical development can also be seen reflecting the relative role of olfaction in different fishes. Each nasal pit generally has two openings which are separated by an area of skin (Fig. 1).Olfactory currents of water enter the anterior and leave through posterior openings-either passively through the locomotion of the fish in the water, or actively by ciliary action within the pits or by the action of muscles associated with the jaws or gills or by some combinations of these methods (Burne, 1909; Pipping, 1926; Teichmann, 1954). In the lungfish the external nostrils are true anterior nares, whereas the internal nares open into the mouth in a manner corresponding to the choanae of higher vertebrates. Lining the nasal sacs is the olfactory epithelium, which is generally raised from the floor of the organ into a complicated series of folds to make rosettelike arrangements ( Fig. 1).The olfactory folds vary greatly in direction and number. Through these folds the total area of the sensory epithelium is greatly increased. Burne ( 1909) distinguished oval (in most fishes), round (in Esox) and elongate ( Anguillu) olfactory rosettes. Species with elongate rosettes have the most numerous lamellae, which are set at right angles to the longitudinal axis of the nasal sacs; such rosettes can be generally correlated with an acute sense of smell (macrosmatic). Species with round rosettes, on the other hand, normally have only a few lamellar folds and usually show no or minimal behavioral responses to olfactory stimulation (microsmatic). Species with oval rosettes are most common and intermediate between the other two. There have been several attempts to relate the total area of the olfactory epithelia in different species to their particular olfactory sensitivities. Measurements of the surface area of the olfactory epithelia of eleven species of freshwater teleosts (Teichmann, 1954) made it clear that species with round rosettes had the smallest area of olfactory epithelium (Esox, about 0.2%of the whole body surface; Gasterosteus, 0.4%)) and that the broadest olfactory epithelium was not found in fishes with elongate rosettes but in species with oval rosettes (Gobio, 3.6%;Phoxinus, 1.9%).In species with the elongate rosettes, the olfactory epithelium was found to be 1.4%of the whole body surface in Anguillu and 1.3%in Lota hta. However, there is no simple relation between the area of the olfactory epithelium and the number of receptors it contains. It is therefore doubtful whether any simple relation exists between the area of the olfactory epithelium and sensitivity to odors.

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The anatomy of the nose has also been related to the ecological habits of the fishes (Pfeifler, 1963b, 1964, 1965; Teichmann, 1954). On the basis of development of the nose and eye, Teichmann (1954) classified the fishes mentioned above into three groups: ( 1 ) species in which the eye and nose are well developed (Plzoxinus and Gobio ); ( 2 ) species in which the eye is better developed than the nose (Esox and Gusterosteus); and ( 3 ) species in which the nose is exceptionally well developed compared with the eye ( Anguilla and Lotu). The definitions of these groups correspond well with the morphological classification made by Bume ( 1909) : group 1 includes the fish with oval olfactory rosettes; group 2 includes most of the round rosetted species; and group 3 the elongate rosetted species. Olfactory epithelia in fishes generally occurs in isolated sensory areas separated by columnar ciliated cell areas (indifferent epithelium). Three types of arrangements of the sensory cells in the olfactory epithelium have been observed (Holl, 1965): ( 1 ) continuous except for the dorsal parts of the olfactory folds (Anguilla and ZctuZuurus); ( 2 ) separated in large areas between the folds ( E s o x ) ;and ( 3 ) dispersed in small islands, in which the sense cells are arranged somewhat like taste buds (Phoxinus and C yprinus) .

Fig. 2. Diagram of fine structure of the olfactory epithelium of the eel, Anguillu anguille. 1, Receptor cells; 2, supporting cells; 3, ciliated cells; 4, basal cells; 5, goblet cells; 6, club-shaped secretory cells; and 7, olfactory knob with sensory hairs. From Holl ( 1965).

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The olfactory epithelial system consists of receptor cells, supporting or sustentacular cells, and basal cells ( Fig. 2). In some species ( AnguiZZu, MyxocephaZus, etc. ) , large flasklike cells have also been observed; these are probably mucous gland cells (Holl, 1965). In burbot (Lota Zota), furthermore, glial cells have been observed to penetrate into the epithelium from the connective tissue surrounding small bundles of receptor cell axons (Gemne and Doving, 1969). The individual olfactory receptor cells of fishes are similar to those of other vertebrates in general appearance although there is a great variation in details even within a particular olfactory organ. The receptor cell, which is a bipolar primary neuron, sends a slender cylindrical process or dendrite toward the surface of the epithelium. The process terminates in a minute swelling (olfactory knob) which bears a variable number of cilia. Although the receptor cells are not distributed uniformly in all olfactory folds, average numbers of 4 8 x 104/mm2are estimated for the receptor cells in several species (Holl, 196.5). The fine axons arise from the basal pole of the receptor cells. They pass through the basement membrane, become grouped in the submucosa, and form the olfactory nerve fasciculi, which run posteriorly to end in the olfactory bulb. Recent electron microscopic observations have characterized the fine structural organization of the olfactory receptor cells in teleost fishes. Trujillo-Cen6z ( 1961) first studied the ultrastructure of olfactory neurons in fully developed embryos of two cyprinodont species Cnesterodon and Fitzroyia. He found that the olfactory sensory hairs consist of an undetermined number of long cilia which project into the lumen of the olfactory pit and that the dendrite of the olfactory neuron contains profiles of small tubules, aligned parallel to its length. Near the basement membrane of the epithelium, groups of axons, which, as mentioned, arise from the pole of the receptor cells opposite to the distal process, are encased in the surface of the sustentacular cells. Although Trujillo-Cen6z failed to demonstrate in the sensory hairs of receptor cells any common structural pattern which could be related to the chemoreceptor mechanism, he considered these structures as devices serving to enlarge the “active surface” of the cell increasing in this way the effectiveness of the whole receptive system. In earlier reports, Jagodowski ( 1901) working with pike, Esox, and Hopkins (1926) studying Stenesthes claimed that there is a single long apical process on each olfactory receptor instead of the usual group of cilia. To resolve this question, Bannister (1965) compared olfactory receptor cells in two teleosts, a minnow, Phoxinus, and the three-spined stickleback, Gasterosteus. Phoxinus, like all the Cyprinidae, has a welldeveloped olfactory organ. Gasterosteus by contrast is a microsmatic

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ciliated

epithelial cell

receptor ending

-

Receptor with microvilli

Fig. 3. Diagrammatic reconstruction of the olfactory surface of the minnow, Phoxinus. From Bannister ( 1965).

species. Bannister found at least two distinct receptor cells, distinguished by the forms of their distal tips (Fig. 3 ) . The first category consists of the ciliated olfactory receptors, each of which has a ring of 4-6 cilia upon its convex distal end. The ultrastructure of the cilia is generally identical with that of all kinocilia in other organisms; the usual “nine-plus-two’’ pattern of fibrils is present (Bannister, 1965). The second type of receptor endings has been found only in Phoxinus and not previously described in any vertebrate; it bears neither cilia nor microvilli but extends simply as a naked rod from the epithelial surface. Internally, this latter type of receptor resembles the ciliated one in many respects; however, it contains three or more longitudinally oriented bundles of fibers. It may correspond to the single olfactory process of Jagodowski ( 1901) and Hopkins ( 1926). The significance of the presence of more than one type of receptor ending in one species of fish is not clear but suggests a basis for peripheral olfactory discrimination. Confirming the findings of previous investigators regarding the general arrangement of the receptor cells, Wilson and Westerman (1967) have shown certain distinct differences in the receptor cells of the European carp, Carassius carassius. In addition to typical cilia, they noted exceptional ciliary formations on the receptor cells. In these ciliary aggregation,

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the fibrils are grouped together in clusters instead of forming individual cilia and are enveloped by a single limiting membrane. Further, Wilson and Westerman ( 1967) discovered a conspicuous well-differentiated new cell type, the foliaceous cell, which has not been previously described in olfactory mucosa. These foliaceous cells, enclosing prominent leaflike organelles, communicate with the external environment through a small stoma. Their complicated morphology together with their close association with the multifilamentous cilia and the myelinated nerve fibers observed in the stroma suggest that they may be receptor cells; however, further work will be required to provide additional information on their role and neural connection. It is not clear which part of the receptor cell is involved in the initial events of olfactory stimulation. However, it is reasonable to assume that the receptor site lies on the membranes of the olfactory cilia since these are the first points of contact with odorous molecules; moreover, the olfactory knob does not protrude beyond the limiting surface of the mucosa as in other vertebrates. If this is the point of excitation, number, length, and motility of the cilia would be signscant in enlarging the active receptor area and increasing the chance of contact between cilia and molecules. The theory that the olfactory cilia are the locus where electrical excitation in the olfactory organ is initiated by contact with odorous substances has been suggested by Reese (1965), who investigated the fine specialization of olfactory epithelium of the frog. Motile olfactory cilia were first described in Stenesthes by Hopkins (1926). However, the motility of the olfactory cilia in Cyprinus, Esox, and Lampetru has recently been observed ( Vinnikov, 1965; Kleerekoper, 1969). Jagodowski (1901) found only one extremely long, thick cilium on each of the olfactory receptor cells of pike; and Bannister (1965) described a receptor cell with only one rod-shaped process in the minnow. Further works will be needed to determine whether any simple relation exists between the characteristics of the cilia and sensitivity to odors. The supporting cells are polygonal columnar epithelial cells which lie between the receptor cells. They bear a small number of irregular microvilli and have prominent oval nuclei situated basally. The cytoplasm immediately beneath the limiting surface is relatively scant with numerous electron-dense vesicles. In the deeper parts of the cells an endoplasmic reticulum and mitochondria are present as much as the receptor cells. Although the significance of the supporting cells in olfactory perception is not understood, it is likely that they have a significance beyond mere mechanical support. In addition to the olfactory cells mentioned above, free nerve endings of the trigeminal nerve occur in the epithelium (Jagodowski, 1901). Al-

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though the functional significance of the trigeminal nerve is not understood, the concept of a central regulatory control over olfactory afferent inputs has been suggested in rabbits ( Stone et ul., 1968). The olfactory mucosa appears yellow to dark brown or black. Some authors assume that the pigment responsible for this color has a function in olfaction (cf. Moulton and Beidler, 1967; Moncrieff, 1967). However, there is no report on the occurrence of pigments in the olfactory epithelium of fishes. It is, therefore, difficult to assess the role of the olfactory pigment in the function of the olfactory membrane until we know more about pigment location, distribution, and biochemical properties. B. Gustatory Organ

Taste buds of elasmobranchs are restricted to the mouth and pharynx. In teleosts they are also located on the gill rakers and gill arches, on appendages such as barbels and/or fins, and also, in some fishes, on the entire surface of the body (Herrick, 1904). In the roof of the mouth, taste buds are densely packed to form the palatal organ, which is innervated by the palatine nerve in the cyprinids. Based on the histology of the taste buds on the gill rakers and gill arches of 24 species of teleosts, Iwai (1964) concluded that the taste buds are more conspicuous in branchial regions of freshwater fishes than in marine fishes. In some species of hake, Urophycis, and sea robin, Prionotus, taste buds are on the specialized pectoral fin rays, which are often modified into feelers (Scharrer et al., 1947; Bardach and Case, 1965; Bardach, 1967). Taste buds are normally innervated by branches of the n. facialis (VII), n. glossophuryngeus (IX), and n. vugus (X), which terminate in enlarged vagal lobes. Taste buds on the modified fin rays of the hakes are also innervated by spinal nerves. The vertebrate taste bud is typically composed of elongated sensory cells arranged like segments of an orange ( Fig. 4).Recent electron microscopic examination of fish taste buds has revealed the existence of three different cell types: receptor cells, supporting cells, and basal cells ( Trujillo-Cen6z7 1961; Cordier, 1964; Desgranges, 1965; Hirata, 1966). There are also transitional or intermediate forms of cells which may become either sensory or supporting cells. The receptor cell of Corydorar is pear-shaped and occasionally bears few thin and short microvilli ( Trujillo-Cen6z, 1961). Desgranges ( 1965) described various types of microvilli at the apexes of taste receptor cells in the barbels of Ameium; these suggest different functional stages. Hirata (1966) also observed the receptor cells with single or two apical processes of different appearance

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Fig. 4. Cross section through taste bud from the barbel of the sturgeon, Acipeiiser fulvescens. From Bardach (1967).

(spindle, elongated rods, and stalks) in the terminal taste buds on the barbels of freshwater fishes ( Cyprinus, Purusilurus, and Cobitis). The apical region of the receptor cells contain numerous electron-dense tubules sometimes arranged along the cell membrane; there are also abundant vesicles. Some of these vesicles are concentrated at the particular site of cell contact with nerve elements resembling a synaptic contact in the central nervous system ( Hirata, 1966). The supporting cells are provided with a few regular microvilli at their free surfaces. Light and electron microscopy have demonstrated a large number of nerve fibers concentrated beneath the basal pore of the taste buds. These fibers, after entering the bud and forming an intragemmal plexus, terminate on the surface of the receptor cells by mean of a clublike swelling containing mitochondria and a few vesicles. No such specialized contact between neural elements and the receptor cells has been shown in mammalian taste buds. Aside from the taste buds, specialized epidermal “spindle” cells were found on the head and body of minnows and various teleost fishes; in some cases (e.g., sea robins, Triglidue) these occur on the tentacular fin rays (Whitear, 1965; Bardach, 1967). The hypothesis that these cells are

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chemosensory is based on their structural resemblance to the sensory cells of the taste buds.

C. Free Nerve Endings There are numerous free nerve endings in the skin of fishes; these endings are presumed to serve the so-called common chemical sense. The free fin rays of the gourami, Trichoguster, and hake, Urophycis, possess taste buds which are innervated by cranial and spinal nerves, whereas those of sea robins, Prionotus, lack taste buds and the epithelium is richly innervated only by spinal nerves (Scharrer et al., 1947; Bardach and Case, 1965). In behavioral experiments, Scharrer et al. (1947) could not find a clear differentiation between the two; both showed similar positive reactions in response to chemical stimulation. Both the external taste buds and the free nerve endings are sometimes regarded as taste receptors-referred to as taste bud type and free nerve ending type, respectively ( see Bardach, 1967). Electrophysiological evidence indicates that there are two types of nerve discharges (fast- and slow-adapting) following chemical stimulation of hake fin rays; only the fast-adapting type is seen in sea robin fin rays. These findings suggest that the slow-adapting discharges are characteristic of taste buds while fast-adapting discharges may originate from the free nerve endings around taste buds (Bardach and Case, 1965). Reactions following stimulation of the common chemical sense are usually negative or defense reaction. However, there are exceptions and the biological significance of the common chemical sense remains unexplained; some even deny its existence (von Buddenbrock, 1952).

D. Morphology of Brain and Feeding Habits Developmentally, the teleost forebrain is basically different from that of elasmobranchs and the higher vertebrates ( Aronson, 1963). Nevertheless, the morphology of the olfactory bulb and centers are essentially similar throughout the vertebrates although there are minor variations, especially in relative size and position of the different areas. Olfactory nerve fibers (filu olfuctoriu) arise in the nasal mucosa terminate in the olfactory bulb, where they make a special synaptic contact with the bulbar neurons in the glomerulus. The fibers themselves do not branch until they terminate. The length of these olfactory nerves varies greatly in different fishes. They may have either short olfactory

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nerves and a long olfactory tract, pedunculated ( Carassius and lctalurus), or long olfactory nerves and a short olfactory tract, sessile (Anguilla and Esox). In the young goldfish the olfactory bulb is closely located to the olfactory lobe while an increase in the length of the olfactory tract may occur during the growth of the fish (Uchihashi, 1953; Schnitzlein, 1964). Olfactory nerve fibers of the pike ranged from 0.1 to 0.4 p in diameter and showed no obvious size difference from other vertebrates (Gasser, 1956). The average number of axons in the central portion of the olfactory nerve was estimated to be 29/pz in burbots (Gemne and DBving, 1969). The microscopic structure of the olfactory bulb is also essentially similar in all vertebrates. In fishes the olfactory bulb is poorly differentiated and the lamination is not so distinct as in higher vertebrates. The dominant feature of the bulb is the synaptic contact between the olfactory nerve fibers and dendrites of bulbar secondary neurons, mitral and tufted cells. Single mitral cells of fishes often have several dendrites ending in different glomeruli (Allison, 1953). It is significant that the axon of a receptor cell does not terminate in more than one glomerulus, and that each glomerulus receives impulses only from a limited group of several olfactory receptor cells; this is remarkably different from the mammalian mitral cells in which only a single main dendrite ends in each glomerulus. Furthermore, coincident with the lower degree of segregation of glomerular transmission, intrabulbar associational systems such as recurrent collateral also seem to be less elaborately developed in fishes (Allison, 1953; Fig. 5). Information from the olfactory bulbs is conveyed through two main fiber pathways, the lateral and medial olfactory tracts to the basal telencephalic areas. The medial bundle is thicker than the lateral one; both are subdivided into two small bundles. The two main fiber bundles contain myelinated nerve fibers with diameters less than 6 . 5 but ~ ~ in the lateral portion of the medial olfactory tract of Lota Zota the majority of the fibers are smaller than 0.5 p ( DBving and Gemne, 1965). The number of these fibers larger than 0.5 p is estimated to be about lo4. The ratio between receptor cells (primary neurons ) and the myelinated fibers in the tract (secondary neurons) is therefore 1OOO: 1. This is the same order of the convergence ratio between receptor cells and mitral cells in the rabbit (Allison and Warwick, 1949). Some fibers in the medial olfactory tract run directly to the hypothalamus while some cross in the anterior commissure. Ascending or centrifugal nerve fibers running to the olfactory bulbs have also been described (Sheldon, 1912). Recently, Westerman and Wilson (1968) have reported the presence of numerous synapses of two types, axo-axonal and axo-glial, within the medial olfactory tract of the carp, Carassius carassius.

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Fig. 5. Diagrammatic illustration of the essential difference in glomerular transmission between fishes ( a ) and mammals ( b ) : Com. ant., anterior commissure; and Tr. olf., olfactory tract. From Allison (1953).

The morphology of the brain has been related to the ecological habits of fishes ( Uchihashi, 1953; Schnitzlein, 1964). According to the morphological development of the brain, fishes may be classified as forebrain-, mesencephalon-, or medulla oblongata-developed groups. Those species in which the forebrain is well developed (Anguilla) have also a large medulla oblongata and are usually nocturnal; this indicates a dominance of olfactory and gustatory functions. Species in which the facial and vagal lobes are conspicuously developed (catfish and carp) show gustatory feeding behavior.

111. BEHAVIORAL STUDIES OF CHEMORECEPTIVE FUNCTIONS

A. Olfactory Sense

Accurate learning experiments in fish were first made by Strieck ( 1924). Minnows, Phoxinus phorinus, were trained to discriminate' pure odorous (coumarin, skatol, and muscone) and gustatory (glucose, acetic acid, and quinine) substances. These substances were readily detected by minnows. However, trained fishes were unable to discriminate odorous substances after the forebrain was removed although they could still

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perceive taste substances. This clearly demonstrates distinct olfaction and taste receptor functions in fish. Neurath (1949), using essentially similar training technique, determined the threshold values for P-phenylethyl in the minnow Phorinus. and eugenol (6.0 x alcohol (4.3x Similar threshold values for P-phenylethyl alcohol were also determined for the minnow by Teichmann (1959). Teichmann also found that the rainbow trout Salmo irideus detected P-phenylethyl alcohol at dilutions of 1 x this is nearly the same order of sensitivity as in humans. Juvenile sockeye salmon ( Oncorhynchus nerka) detected eugenol at concentrations higher than 1.8 x lo-? (Tarrant, 1966). Hasler and Wisby (1950) used a conditioning technique for the biological assay of pollutants. Upon completion of the training period, blinded bluntnose minnows, Hyborhynchus notutus, discriminated between phenol and p-chlorophenol at dilutions of less than 5 x le4. Similarly, coho salmon fry, Oncorhynchus kisutch, could easily detect (Hasler, 1957). morpholine in concentrations as low as 1 X Using a specially designed conditioning technique, Teichmann ( 1959) trained young European eels, Anguilla anguillu, to detect p-phenylethyl alcohol, Z-menthol, citral, eugenol, and ionon; he established the thresholds for each. Calculations indicate that the limits for detecting a chem( j?-phenylethyl alcohol) to 2 x ical range from dilutions of 3.5 x (ionon). The lowest threshold obtained in the eel is some 100 to 80 times lower than those of the minnow and rainbow trout. This can be compared favorably with the olfactory sensitivity of a terrestrial macrosmatic animal such as the dog. Further, the threshold of the eel for Pphenylethyl alcohol showed seasonal fluctuations; it was lowest in late winter and in midsummer, while about 60 times higher in late fall and in early winter. Such a decrease in the olfactory sensitivity might be explained by central and hormonal regulation ( Teichmann, 1959). Roaches, Leuciscus rutilus, were trained to induce a fright reaction in response to benzol derivatives (Marcstrom, 1959). Thus fishes responded to benzol at dilutions of 2.0 x l W M and to phenol at 9.5 X M . That these responses were olfactory was shown by the failure of responses when the noses were destroyed. The thresholds of the fright reaction to mononitrobenzol and lesolsine were five times higher than those of intact fishes. Recently, Miesner and von Baumgarten (1966) conducted an interesting study of olfactory perception and memory in the goldfish Carassius auratus. Fishes were trained to recognize coumarin, and their swimming movements were registered by means of specially designed technique. Coumarin could be detected at dilutions of less than 1 X Besides the normal positive reaction to the stimulus, the fish exhibited several charac-

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teristic behaviors such as circular and zigzag movements during which the direction of the odor gradient might be searched. Changes of water turbulence at the olfactory mucosa could help to overcome the adaptation of the olfactory receptor cells. When amylacetate was applied instead of coumarin, the fishes generally behaved in a positive way. Furthermore, when coumarin and amylacetate were given simultaneously at the opposite sides of the fish tank, the fish preferred coumarin, to which they were originally trained (primary differentiation). B. Gustatory Sense

As mentioned above, the argument that taste is a different sensory function from olfaction is based on the training of blinded minnows to discriminate certain taste substances even after extirpation of the olfactory lobes, while conditioning for odorous substances was only possible in intact fishes ( Strieck, 1924). Trudel (1929) compared the sensitivity of the minnows for various taste substances-especially saccharides and synthesized sweet substances. Fructose, glucose, galactose, mannose, mannite, arabinose, maltose, lactose, melezitose, raffinose, and two sweet substances, saccharin and dulcin, were all perceived by the minnows as essentially the same quality as sucrose. Trudel also reported that fructose was detected as the sweetest of all and that the relative threshold of the minnows for quinine (as low as 0.0025%)was higher than that of humans ( 0.003%). Krinner (1935) was the first to provide accurate thresholds for sucrose M ) in minnows using his careful trainM ) and salt ( 4 x (2x ing techniques. These thresholds were 512 and 184 times lower, respectively, than those of humans for sucrose and salt. Removal of olfactory lobes caused no change in these thresholds, thus verifying that a true gustatory sense was involved. Recently, by developing Krinner's training technique, Glaser ( 1966) has carefully compared taste sensitivity of the minnow Phoxinus plzoxinus, stickleback Gasterosteus aculeatus, South American salmon Hemigrammus caudovittatus, and Mexican blind cave fish Anoptichthys jordani. The time necessary to learn their task for a given taste substance (e.g., sucrose) differed greatly in various species; in Anoptichthys training was unsuccessful. Differences were also found within a species for various taste substances; for instance, minnows could be most easily trained for sucrose; this was followed by acetic acid, sodium chloride, and quinine. Of four basic taste substances, the reaction time for NaCl was longest in the minnow and that for sucrose in Gasterosteus, Phoxinus, and Hemigram-

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53.0 sec, respectively. No direct correlation between chemical structure and sensitivity was ascertained. According to Humbach (1960), taste sensitivity of Anoptichthys for four basic taste qualities was several thousands times better than that of Phorinus.

mus was 17.8, 19.1, and

IV. ELECTROPHYSIOLOGICAL STUDIES OF CHEMORECEPTOR RESPONSES

A. Olfactory System Electrophysiological studies of the chemoreceptive functions of fishes have not been extensive-partly because of small size of the structure and partly because fish live in water. Adrian and Ludwig (1938) who initiated analysis of the olfactory system in fishes with electrophysiological techniques recorded continuous impulse discharges in the olfactory tract of the catfish, carp, and tench. Such a resting discharge with low frequency and amplitude shifted to a maximum after a latency of 0.5-5 sec in response to mechanical as well as to chemical stimulation of the olfactory sac. The response gradually decreased during stimulation ( adaptation) and was then followed by a refractory period lasting 5-20 sec during which the organ was insensitive to a second stimulus. There has been a marked increase in the number of electrophysiological studies of fish olfactory system in the last few years. 1. MUCOSALPOTENTIALS Resting potentials of the olfactory epithelium were recorded in some teleost fishes with glass microelectrodes (Shibuya, 1960). They were 12.4 mV in Anguilla japonica, 8.7 mV in Misgurnus, 8.6 mV in Parasilurus, and 7.6 mV in Channa, Cyprinus, and Entosphenus. Slow negative potentials were induced in the mucosa during stimulation with odorous fluids such as butyric acid or extract of silkworm pupae (Fig. 6). The shape of the potentials were different in different species of fishes. Generally, however, the potentials showed a fast-rising phase with a slower exponential fall. The potential increased in time with increasing stimulus durations. In Parasilums and Anguilla, it had a short duration (0.4-0.7 sec) with a rapid decline. In the eel it always had a duration of about 0.4 sec regardless of stimulus duration ( Fig. 6e). Corresponding potentials were simultaneously recorded in the olfactory nerves. In addition to the “on-response” (appearing at the onset of stimulation), a distinct shift in potential appeared when stimulus ceased (“off-

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1

0

200pv

I

I

b

200pv

I

I

d

e

I

, -

200pv

200pv

I_

0.5 sec

Fig. 6. Slow potentials induced in the olfactory mucosa when stimulated with extract of silkworm pupae. a, Channa argus; b, carp; c, lamprey; d, catfish; and e, eel. From Shibuya (1960).

response”). The on and off responses occurred singly or successively ( on-off response). Simultaneous recordings with closely positioned microand macroelectrodes showed different types of response. Three different types of response have also been related to the existence of three functionally distinct types of receptors (Shibuya, 1960). Since there has been much argument concerning whether the slow mucosal response is a true generator potential (see Ottoson, 1963; Shibuya, 1964; Ottoson and Shepherd, 1967; Moulton and Tucker, 1964; Takagi, 1967), more precise analyses of the receptor response by recording activity in single receptor cells are required. In this connection, it is noteworthy that there are at least the five chemoreceptors responding to certain kinds of sugars and amino acids ( Adler, 1969), since the olfactory responses to these chemicals can be elicited in receptors and bulbs of some salmonid fish. It has also been suggested that animal receptors have evolved from single-celled flagellated organisms ( Vinnikov, 1965). 2. OLFACTORY NERVEACTIVITY

Information initiated in olfactory receptor sites is relayed through the olfactory nerve to the higher centers in the brain. However, since all

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sensory messages reach the neural centers in the form of impulses, recording activity in the primary olfactory neurons has some advantages from the technical point of view. Recording of the electrical activity of single olfactory nerve fibers may be difficult because of their extremely fine dimensions. Gasser (1956) investigated the properties of the isolated olfactory nerve of the pike Esox estor in a combined electrophysiological and electron microscopic study, He showed that the action potential evoked by electrical stimulation of the olfactory nerve consists of only one single wave of 30 msec duration with a conduction velocity of 0.2 meterslsec. Such a slow conduction velocity of the olfactory nerve has been reported in other animal species ( Ottoson, 1963).

3. ELECTRICAL ACTIVITYOF

THE

OLFACTORY BULB

Spontaneous electrical activities (EEG) have been recorded with bipolar electrodes from the surface of the olfactory bulb of the goldfish Carassius uurutus (Oshima and Gorbman, 1966a, 1968; Hara, 196713; Hara and Gorbman, 1967); EEG's were consistent and characteristic (frequency, 14-16 Hz; amplitude, 70-100 pV) . Infusion of chemical solutions into the nasal cavity induced specific synchronous wave patterns of high amplitude (150-200 pV) in the EEG. Maximal duration of the induced response was %6 sec, despite the continued presence of the chemical stimulant in the olfactory sac (Hara and Gorbman, 1967). The response to NaCl increased in magnitude linearly with increasing concentration within the range tested (1-5 x 1e2 M ) . Qualitatively similar patterns of electrical response were obtained with KCl, LiCl, CaCl,, NaH,PO,, Na,HPO,, Naz-oxalate, and Nag-citrate. No response was induced by choline-chloride at equivalent molar concentrations. Although the nature of the induced response following chemical stimulation of the olfactory sac is still unknown, it seems possible that the responses result from a relatively nonspecific excitation of the olfactory receptor cells. The highly synchronous activity induced in the mammalian and amphibian olfactory bulb by odorous stimulation seems to be analogous. A characteristic potential was also evoked in the olfactory bulb when single electrical stimuli were given to the olfactory mucosa instead of chemical stimuli. The potential at the surface of the bulb recorded through bipolar electrodes, was a biphasic wave with a delay of about 20 msec, a maximum amplitude of about 1.2 mV, and a duration of about 50 msec. The potential consisted of three components with distinctly different properties. The first component lacked a true refractory period and summated to a sustained potential with repeated stimulation. The

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second component had a comparatively long (about 30 msec) refractory period and would not follow repeated stimulation at a frequency higher than about l/sec without undergoing a reduction in amplitude. The third component ( positive-directed afterpotentials ) disappeared after section of the olfactory tract. It has therefore been concluded that the first component is of synaptic origin, the second component represents the activity in the second-order neurons, and that the third component is centrifugal origin. When microelectrodes were inserted into the olfactory bulb, spontaneous unitary activities were recorded in different layers of the olfactory bulb of the goldfish. Oshima and Gorbman ( 1966b) observed three different types of discharge patterns of single cells in the glomerular and subglomerular layers of the olfactory bulb. These spontaneous and evoked unitary activities in response to chemical stimulation were easily influenced by treating with thyroxine and steroid hormones. In the layers about 300-400 p under the surface, large biphasic action potentials were frequently detected ( Hara, 1967a). The discharge frequently varied from cell to cell, but most cells of this type had an average discharge frequency of about 2-6 impulses/sec. Probably much of the activity in this layer is derived from the mitral cells. Different individual neurons responded in different ways to chemical stimulation of the olfactory cavity. The variety of observed responses to 5 x M NaCl from different individual cells included the following: (1)inhibition (decrease in the firing rate) during or after the period of stimulation, ( 2 ) excitation (increase in firing rate) which could outlast for a few seconds the duration of the stimuli, ( 3 ) excitation during stimulus followed by a short inhibition when the stimulus ceased, ( 4 ) excitation at the beginning of the stimulus and inhibition afterward, ( 5 ) a short inhibition at the very beginning of the stimulation followed by excitation, and ( 6 ) no response. More than 60%of the neurons tested were of types (1) and ( 3 ) ,which represent opposite patterns of response. Similar spontaneous activity from single units in the olfactory bulb was recorded with microelectrodes in the burbot Lota lota (Dkiving, 1966a,b). 4. ELECTRICAL ACTIVITY OF THE OLFACTORY TRACTAND CENTRAL REGULATORY SYSTEM Olfactory information filtered in the first relay station, the olfactory bulb, is transferred through the olfactory tract to higher nervous centers. In some teleosts the olfactory tract runs as a long nerve bundle (see Section 11,D ) between the bulb and telencephalon. Such a unique anatomical feature of the olfactory system provides a convenient preparation for elec-

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trophysiological analysis of the messages conveyed in the olfactory tract. As already stated above, the first electrophysiological investigation of this kind was that by Adrian and Ludwig (1938). Similar findings were reported for catfish by Boudreau (1962) who recorded summated activity of the olfactory tract and showed that the increases in tract activity were produced by various chemicals in extremely dilute concentrations. Acetic acid and butyric alcohol remained effective at concentrations of lo-’’ M and M , respectively; at higher concentrations they depressed activity. Electrical properties of the olfactory tract were studied by analyzing compound action potential induced by electrical stimulation in the burbot Lota lota and in some species of the orders Anacanthini and Ostariophysi (Doving and Gemne, 1965; Doving, 1967). The compound action potential of the olfactory tract had three components with different conduction velocities ranging from 0.25 to 5.5 meters/sec at 10°C. Generally, the third component which showed the lowest conduction velocity was present in the medial bundle of the tract. This component, probably nonmyelinated, might be responsible for connecting the olfactory system with the hypophysis ( Kandel, 1964; Jasinski et al., 1966, 1967). Furthermore, the activity of single fibers in the olfactory tract was influenced by stimulating the olfactory epithelium with various chemical solutions in the burbot when efferent inflow was eliminated by sectioning the olfactory tract. Most of the chemicals evoked both excitatory and inhibitory types of response in different individual units. Slow rate of adaptation of the activity of the secondary neurons to continuous stimulation was observed; this confirmed earlier findings in catfish by Adrian and Ludwig (1938). Similar effects on the spontaneous firing of single fibers in the olfactory tract to afferent olfactory stimulation were obtained by Nanba et al. (1966) in Abramis and Carmsius. Recently, Westerman and Wilson (1968) reported the conduction velocity averaging 0.6 meters/sec in the lateral olfactory tract of the carp. The demonstration that afferent nerve impulses in the olfactory system can be directly controlled by influences originating in the central nervous system has been one of the most interesting developments in physiology of the olfactory system. The action is mediated by way of the “centrifugal” fiber system, part of which has long been known anatomically ( Sheldon, 1912; Allison, 1953; Kappers et al., 1960; Aronson, 1963). Section of the ipsilateral olfactory tract caused a marked augmentation of the induced response of the olfactory bulb to NaCl infusion (Hara and Gorbman, 1967; Fig. 7). In the preparation sectioned at the midbrain-hindbrain level ( ceroeau isole‘), cutting the olfactory tract eliminated the similar NaC1-induced bulbar response ( Oshima and Gorbman, 1966a). Bulbar potential waves evoked by electrical stimulation of the

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Fig. 7. Modification of the bulbar response induced by infusion of NaCl solution into the nasal cavity after sectioning the olfactory tract: ( a ) Before tract section and ( b ) after tract section. Calibration, 50 $V and 1 sec. From Hara and Gorbman (1967).

olfactory epithelium were also affected by olfactory tract section; the positive afterpotential almost disappeared and consequently the potential wave became monophasic (Hara and Gorbman, 1967; Fig. 8 ) . These results also verify that the rapid adaptation seen in the olfactory bulb of the intact animal is probably of central origin. Furthermore, electrical stimuli applied to the opposite olfactory bulb or to the anterior commissure, or strong chemical stimuli given to the opposite nostril depressed both intrinsic and afferent-induced electrical activity of the bulb. These findings indicate that olfactory bulbar responses to afferent stimuli can be modulated by influences from the other bulb and from more posterior parts of the brain (Hara and Gorbman, 1967). Spontaneously firing secondary neurons recorded in the olfactory bulb and tract were shown to be similarly regulated by electrical stimulation of the ipsilateral and contralateral olfactory tract ( Doving, 196613; Diiving and Gemne, 1966; Doving and Hyvarinen, 1969; Hara, 1967a).

Fig. 8. Electrically evoked bulbar potential before ( b ) and after ( a ) tract section. Calibration, 0.5 mV and 20 msec. From Hara and Gorbman (1967).

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Central regulation of afferent transmission within the nervous system may be a common process in neural integration. Particularly in fishes, such a mechanism of central regulation, including interbulbar connections, may play an important role in the orientation toward or away from a stimulus source by simultaneous bilateral equating of stimulation.

B. Gustatory Receptors 1. PALATAL ORGANOF CARP

Unlike the olfactory system, taste receptors of fishes are scattered widely over the body surface. For this reason electrophysiological studies on taste function in fishes have mainly involved the recording of potential discharges from the nerve fibers innervating taste buds. Hoagland (1933) first recorded electrical responses from the facial nerve innervating the taste buds on the barbels of the catfish exposed to various taste solutions. Subsequently a more thorough investigation of the electrical responses of the taste receptors was undertaken by Konishi and Zotterman (1961a,b) in carp. Integrated electrical responses to various taste substances were recorded from the glossopharyngeal, facial, and branchial nerves innervating the palatal organ, barbels, and gill rakers. The findings suggest that the palatal organ plays the principal role in gustation in the carp although certain differences exist between Swedish and Japanese carps (Konishi and Zotterman, 1961a,b, 1963). Swedish carp showed a large gustatory response to sucrose (0.5 M ) and acetic acid (0.005M , pH 3.8) and a weak response to quinine (0.01 M ) , while Japanese carp show low sensitivity to sucrose and high sensitivity to quinine. Responses to human saliva were much larger than those to NaCl (0.5 M ) . Single taste fibers from the glossopharyngeal nerve could be qualitatively classified into seven groups according to their response patterns to four basic taste substances and saliva. Acetic acid (0.005M ) stimulated all taste fibers, except for salt fibers which were specifically responsive only to NaCI. The fibers responsive to human saliva were also stimulated by sucrose. Extract of silkworm pupa also induced a marked gustatory response. The final gustatory active compound could not be identified either in saliva or in extract of silkworm pupae. Lytic agents produced an irreversible depression of taste responses. Thus, treatment with 0.3%sodium cholate depressed the response to sucrose while treatment with 0.005%digitonin immediately reduced the responsiveness of the taste receptors. Furthermore, the taste responses of the carp to sucrose, dextrose, levulose, and glycine were analyzed (Hidaka and Yokota, 1967). Re-

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sponses to sucrose, levulose, and glycine increased asymptotically with increasing concentrations, while that for dextrose was sigmoid. Inhibitory interactions may occur when two chemicals are presented to the taste receptors simultaneously or successively. Competitive actions were observed among dextrose, sucrose, and levulose. Mercuric chloride ( 1c4 M) reversibly blocked the taste responses to dextrose, sucrose, and levulose, but not to glycine and NaCl. These results suggest the existence of at least two different kinds of receptors in the palatal organ of carp; one is commonly responsive to all four substances and the other only to glycine. The existence of palatal chemoreceptors responding specifically to dilute solutions (O.OOS0.0005 M for NaC1) of salts with monovalent cations (Konishi and Niwa, 1964; Konishi, 1967) and of various organic compounds (Konishi and Hidaka, 1969) has been demonstrated in carp. Strong responses were produced by various chemicals with polyvalent anions such as Na-citrate, Na2HP0,, Na,Fe( CH) 6 , tetramethylammonium-C1, choline-C1, Na-glutamate, glucose, and glycine. The response decreased with increasing concentration, then increased again at much higher concentrations. In general, the higher the valency of the anion of the compounds, the larger the responses induced. Applications of distilled water, immediately after stimulation with a salt solution at concentrations (O.OOl-O.05A4 for NaCl) where responses were depressed, elicited a marked integrated response (distilled water effect). The effect has been ascribed to the activity of the same receptor as in the response to dilute salt solutions. By analyzing the effects of acid, alkali, and dye salts, a hypothesis which explains underlying mechanism in terms of an interfacial electrokinetic process has been presented ( Konishi, 1967). Such responses to dilute solutions are not restricted to freshwater fishes. Similar responses were also observed in the facial nerves innervating the upper lip of sea catfish Plotosus anguillaris (Konishi and Hidaka, 1967). The biological significance of the response to dilute solution and distilled water is unknown. Similar effects of highly diluted solutions and distilled water on the olfactory bulbar responses have often been observed (Hara, unpublished data). The palatal chemoreceptors of the carp were found to be highly sensitive to carbon dioxide. No detectable responses to oxygen, nitrogen or air were obtained ( Konishi et al., 1969). The responses were confirmed to be independent of pH of the solutions applied. Avoidance behaviors to CO, and/or pH were studied in Atlantic salmon parr, minnow, and roach, in connection with water pollution (Hoglund, 1961; Hoglund and Hardig, 1969). The removal of olfactory tissues and the sectioning of the nerves innervating the lateral line organs did not essentially change the reactions of the fish.

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2. BARBELSOF CATFISH Barbels play an important role in food taking in catfish. Application of taste substances induced a train of spikes in the facial nerves innervating the nasal barbels of the catfish, Ameiurus melas (Tateda, 1961). By stimulating with equivalent molar concentration of various chlorides, the order of magnitude of responses was KC1 > NH,C1 > NaCl > Lick thresholds for these salts were about 0.1 M . Solutions of NaC1, NaN03, NaBr, and Na,-citrate in equivalent normal concentration elicited responses of similar amplitude. Hydrochloric acid induced a large initial burst of impulses, which declined rapidly in 10-20 sec, at concentrations of 0.0005-0.001 hl. Taste responses of isolated barbels of the catfish, Parasilurus asotus, were also analyzed by recording potential discharges from single nerve fibers (Tateda, 1964). The majority of single fibers responded well to hydrochloric acid and salt but not to sucrose and quinine. No simple classification of the taste fibers could be obtained. In this connection, it must be pointed out that one single taste fiber may innervate several taste receptor cells of different natures. Treatment with KC1 and CaC12, although in themselves stimulating, caused a depressive effect on the receptors and resulted in irreversible changes in the taste responses at higher concentrations of 0.25-1.0 M . By short application of urethane ( 13%for 20 sec) and cocaine (0.25%for 30 sec), taste responses of the barbels to HC1 and NH,Cl were reversibly abolished. Furthermore, from the analysis of stimulating effects of hydrochloric acid and several organic acids (formic, acetic, propionic, and butyric) on the catfish barbels, it was shown that the responses to organic acids were greater than those to hydrochloric acid at an equal hydrogen ion concentration and that the responses increased with increase in molecular size of acids tested (Tateda, 1966). In sea catfish Plotosus anguillaris single taste fibers, responsive to both NaCl and quinine, were most commonly found (Konishi et al., 1966). Sucrose did not produce any appreciable response. It is surprising that distilled water gave no positive response in most fibers despite the fact that the receptors were rinsed with seawater between applications of test solutions. Many fibers specifically responded to natural gustatory substances such as extract of a marine worm and blood sera of other animals. The active components of these stimulants are not yet known. Electrical response to cysteine of the taste fibers of the barbels of yellow bullhead ( Ictalurus natalis) was demonstrated to be impaired after exposure to low concentrations (0.5 ppm) of detergents. Histological examination revealed erosion of the taste buds. Affected fish did not fully recover after 6 weeks in detergent-free water (Bardach et al., 196.5). Recently, Mann (1969) reported that the deposition of phenolic sub-

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stances and oils, released from aqueous flora, and their impairing effect on taste and smell in carp was intensified by the presence of detergents in water. 3. MODIFIEDFINSAND OTHERFORMS OF MARINE FISHES

It is important to ascertain whether or not there are differences between external and oral chemoreceptors and to compare the taste sense of marine and freshwater fishes. A series of observations have been carried out with appendages of several species: (1) those whose barbels have cranial innervation only (bullheads, lctalurus) , ( 2 ) those in which fins have both cranial and spinal innervation ( tomcods, Microgadus tomcod), and ( 3 ) those with modified fins innervated only by spinal nerves (sea robins, Prionotus carolinus) (Bardach and Case, 1965; Fujiya and Bardach, 1966; Bardach et al., 196713). Acetic acid produced responses in most nerve fibers of the barbel of the bullhead at a concentration of 0.0008 M , while in tomcod and sea robin about ten times the concentration was needed to evoke a response. This, however, does not imply that marine fishes are less sensitive to acids; on the contrary, it means that marine fishes are less able than freshwater fishes to perceive acids at the same concentrations. Responses to acids depend on the presence of free hydrogen ions in the solutions, and the above two acid solutions produce about the same amount of hydrogen ions. Upon application of NaCI, nerve discharges from both marine and freshwater fishes increased only at concentrations higher than those of their natural ecvironments. Reduction of nerve discharges in tomcods and sea robins was observed at concentrations between 0.4 and 0.3 M NaCl. Complete inhibition occurred when freshwater was applied; this was followed by a transient augmentation of nerve discharges. Responses to choline-chloride were similar to those to NaCl in three species. Varying numbers of fibers responded to quinine hydrochloride. Only a few fibers (less than 10%)responded to sucrose, and no response was observed in sea robins where the fins have not taste buds but only spinal nerve endings. All three preparations responded well to flesh extracts. An attempt has been made to extract the active component. Of various amino acids which were identified in the extracts, only cysteine elicited strong responses; some of the fibers responded selectively only to cysteine. More precise investigation will be required to resolve a question as to whether or not there are taste qualities other than the four basic ones in fishes. Katsuki et al. ( 1969) studied electrophysiological response to chemical stimulation of pit organs of the nurse shark, Ginglymostoma cirratum.

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They responded readily to NaCl and KCI solutions (1M ) . Divalent cations such as Ca and Mg were inhibitory. Responses to acid, sugar, and quinine were either slight or inhibitory.

V. BIOLOGICAL ASPECTS OF CHEMORECEPTION

A. Chemical Perception of Foods There are numerous observations of the searching behavior for foods, and it has been recognized that all senses (e.g., optic, amustic, and chemical) are involved. Clues which initiate searching behavior depend on the species, history, and schooling tendencies as well as environmental conditions; some fishes rely more on chemical senses and others more on optic or acoustic senses. The importance of chemical senses, especially sense of smell, has been demonstrated in the following observations. Parker (1910) placed five normal catfish, Ameiurtu, in an aquarium in which were hung two wads of cheesecloth, one of which contained minced earthworm. The wad containing the worms was seized and tugged eleven times by the fishes, while the wad without worms failed to excite any noticeable reaction in the course of an hour. Fishes whose barbels were removed but had normal olfactory organs reacted in the same manner as intact fish; fishes with cut olfactory tracts never seized the wad containing worms. These findings indicate that the olfactory apparatus of the catfish is useful in sensing food at a distance; catfish truly scent their foods. Barbels are also very valuable to Ameiurus in procuring foods only by coming into direct contact with it (Olmsted, 1918). The eyes of the killifish, FunduZus heteroclitus, in strong contrast with the catfish, play an important role in the initial stages of procuring food but actual swallowing of the food depends on other sense, probably olfactory. Fishes whose olfactory tracts were cut or whose anterior olfactory apertures were stitched never discriminated between two packets of clothone with dogfish meat hidden in it and the other without the food (Parker, 1911). The importance of sense of smell in finding foods has long been demonstrated in Selachians. Dogfish, Mustelus canis, failed to recognize and determine the location of food substances such as crab meat when the olfactory capsules were occluded with cotton. Food recognition was regained when the cottom was removed; plugging of one nostril did not seriously affect that ability ( Sheldon, 1911). A similar directive influence of the sense of smell in the dogfish was observed by Parker ( 1914). Normal dogfish found the foods by making combined right- and left-handed

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movements; this resulted in continuous and characteristic courses in the form of figure eight. Dogfish with one nostril occluded found the food by predominant movements toward the side of the open nostril. Their movements were essentially circular in such a way that the open nostril is toward the center of the circle. Parker explained these movements on the basis of the osmotropotaxis as in the circus movements of invertebrates. Gilbert et al. (1964) reported that electrical potential recorded from the forebrain reflected olfactory processes in three species of sharks ( N e gaprion, Sphyrw, and Ginglymostom) . Both the amplitude and frequency of the potential clearly increased during chemical stimulation of the olfactory sac with extracts of crabs and tuna. Blinded bluntnose minnows, Hyborhynchus notatus, were able to discriminate odors of aquatic plants at extreme dilutions. They also could discriminate between odors of certain aquatic invertebrates ( Hasler, 1957). These findings support the view that aquatic plants may play an important role in the life of fish. Juvenile sockeye salmon, Oncorhynchus nerka, responded by evoking exploratory and feeding behavior to aqueous extracts of foods to which they had been previously conditioned but failed to respond to similar foods which had not previously been in their diet ( McBride e t al., 1962). Responses were characterized by breaking up of the school, increased swimming speeds, and swimming into lighted areas. Fish probably became conditioned to some component( s ) of their foods, but they might be attracted by extracts of foods that they had not previously ingested. As already mentioned, such a complicated feeding behavior is accomplished by successively separated individual behaviors. For instance, once a fish has found food by visual, mechanical, or chemical clues, it may still have to test before eating it. Various environmental stimuli are integrated in the central nervous system, probably hypothalamus, and subsequently organized into a unified expression of the behavior. Electrical stimulation of olfactory areas in the central nervous system of goldfish elicited stereotyped feeding activity indistinguishable from normally induced behavior ( Grimm, 1960). Stimulation of vagal lobes produced no feeding arousal. It is suggested that the olfactory rather than the peripheral gustatory system plays the predominant role in the arousal of feeding activity. Further studies of the central integration of feeding behavior are to be encouraged. B. Reproductive Behavior and Chemical Senses In contrast to work with visual cues, the role of chemical senses in reproductive behavior has been poorly investigated. The role of chemical

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factors was described in the courtship behavior of the catfish Ameiurm by Breder ( 1935), and of gobiid fish Bathygobius by Tavolga (1956). Bathygobius males showed courtship behavior in response to the introduction of a small amount of the water in which a gravid female had been placed for a few minutes. Of various internal body fluids tested only the ovarian fluid elicited a courtship response at extreme dilutions. Whether the stimulating ovarian substance is secreted by the eggs or the ovary has not been determined. The male detected the rapidly diffusing substance by the olfactory sense, since anosmic males whose nostrils were plugged failed to respond to any amount of the courtship-stimulating substance ( Tavolga, 1956). Furthermore, it was demonstrated that anosmic males tended to show fighting behavior and that castrated males never exhibited such behavior (Tavolga, 1956). These results may indicate that the olfactory stimulus by the ovarian substance not only elicits courtship but inhibits fighting. It is also possible that the male sex hormone affects the sensitivity of the olfactory organs in some ways. In this connection, it has been shown that afferent-evoked bulbar electrical responses produced by NaCl infusion into the olfactory sac and by electrical stimulation of the olfactory epithelium in goldfish were markedly influenced by administrations of sex hormones (Hara, 196713; Oshima and Gorbman, 1968, 1969). C. Discrimination of Body Odors and Schooling

Wrede (1932) apparently was the first to demonstrate that a minnow has an individual body odor recognizable by other members of the same species. GO, (1941) later trained the blinded minnow to discriminate the body odor of the catfish Ameiurus and proved that minnows could no longer discriminate the odor after the olfactory lobes were removed. The minnows could be trained to discriminate the body odors of 15 different species of fish from eight families; they could also recognize odors of different individuals of their own species. Furthermore, they could specifically discriminate olfactorily between two different species of frogs ( R a m esculenta and R. temporaria) and between two species of salamanders ( Triturus and Salamandra) , but they could not discriminate between two individuals within the same amphibian species. Yellow bullheads, Ictalurus notatus, also recognized individuals of their own species by means of intraspecific chemical stimuli (Todd et al., 1967). Through conditioning, blinded bullheads were able to discriminate between the odors of two donor fish, but they lost this ability when deprived of their sense of smell. The main source of the intraspecific chemical stimuli in-

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volved in recognition was the integumentary mucus. A change in status after fighting was chemically communicated to other bullheads. Oshima et al. (1969b) reported that chinook salmon (Oncorhynchus tshawytschu) showed olfactory bulbar responses to the water, which had previously evoked only a slight response, by placing other individuals of the same species in it. Keeping coho salmon under the same conditions did not make the water as stimulatory. It has been suggested that olfactory communication plays some part in maintaining the coherence of schools. Keenleyside (1955) found that blinded rudd, Scardinius erythrophthalmus, could detect and preferred water that had contained other rudd but showed no such preference after their olfactory epithelium was destroyed. Stevens (1959) observed in two pelagic schooling fishes, Hepsitia stipes and Bathystoma rimator, that during the day schools were maintained visually; the fish were more active and did not swim in schools at night; and their behavior was clearly directed toward the investigation and sampling of potential food. It seems reasonable to assume, therefore, that schools can be guided to their foods from long distances by olfactory perception. Finally, it may be expected that fishes swim up concentration gradients of substances excreted by their food organisms and that schools of the same species may find each other by this means (Stevens, 1959). Recently, Hemmings (1966a) studied the schooling behavior of the roach, Rutilus rutilus, in relation to the role of olfactory and visual senses; he suggested that school structure is maintained by balanced attractive and repulsive forces, the attraction modalities involved are vision by day and olfaction by night, and the repulsion modality is the lateral line sense. D. Alarm Substances

Von Frisch (1938) discovered that a school of minnows, Phoxinus laevis, showed a strong fright reaction when an injured minnow was introduced into the school. Since then alarm substances, which communicate the presence of danger and which are produced by members of the same species, have been extensively studied by von Frisch and his students. In a series of observations it was shown that the alarm substance is released only from injured skin. Dead minnows with undamaged skin were ineffective. Pieces of their stomach, gut, liver, spleen, and muscle were equally ineffective. Although absolute values of the potency of the extract could not be obtained, a solution of 0.1 g of fresh skin in 100 ml of water was sufficient to induce fright reaction in aquaria of 25-150 liters

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capacity, Little is known concerning the chemistry of the alarm substance. It was suggested that the alarm substance was a pterine and close to isoxanthopterine ( Huttel, 1941; Huttel and Sprengling, 1943). These authors called it ichthiopterine. There is evidence, however, that ichthiopterine is not identical with alarm substance (Schutz, 1956; Pfeiffer, 1967). It was also pointed out that alarm substance was not volatile although very soluble in water. Several amino acids and amines perceived by parasitic Petromyzon marinus were chemically separated from the body odor of brook and brown trout (Kleerekoper and Mogensen, 1959, 1963). From histological observation, on the other hand, Pfeiffer (1960) showed that the alarm substance is produced in specialized epidermal cells (alarm substance cells), which do not open onto the surface but only release their contents when the skin is injured. These cells are found in all species which show a fright reaction. The fright reaction appears at a certain stage of the growth of fishes regardless of their prior experience and does not develop until some time after the alarm substance is formed in the skin (Schutz, 1956; Pfeiffer, 1983a). A predator odor capable of eliciting a fright reaction in local prey species was found in three North American predatory fishes (Lepomis macrochirus, Mkropterus punctulutus, and Esox niger) and in two South American fishes ( Astronotus ocellatus and Cichhoma sezlerum) . No damage to predator skin was necessary to release the substance which caused the alarm response (Reed, 1!369). Only the olfactory sense is involved in the detection of the alarm substance. Minnows never responded to the alarm substance after the olfactory nerve and olfactory bulb were removed (von Frisch, 1941). Schutz (1956) observed that if the excited movements of fish showing the fright reaction could be seen by other minnows, even though separated by glass walls, a typical reaction could be visually transferred without the presence of the alarm substance. Thus, under certain conditions, the movements associated with the fright reaction could initiate a reaction in fish which had not been exposed to the alarm substance. From observations with 60 species of Ostariophysi and 91 species (44 families) of non-Ostariophysi ( von Frisch, 1941; Schutz, 1956; Pfeiffer, 1962, 1963a, 1967)) it may be concluded that the fright reaction exists only in the Ostariophysi and is associated with this group of fish and not with any particular habitat or type of social behavior. The reaction is certainly absent in some species in Serrasalminae and Mylinae. The Mexican blind cave fish, Anoptichythys jordani, does not react to its own skin extract, although the fish does have the alarm substance. Furthermore, although the fright reaction is species specific, a marked interspecific reaction has been observed (Pfeiffer, 1963a). However, the

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strongest response was always obtained with the alarm substance from the same species. Interspecific reactions bear a definite relation to the taxonomic position of the species ( Pfeiffer, 1963a). Skinner et al. (1962) isolated the alarm substance of the top smelt Atherinops afinis by extraction with methanol or ether from suffocated top smelt. These extracts, when introduced into an aquarium containing top smelt, induced a strong alarm reaction in the fish. Reaction was species-specific. Later, however, this was denied by Rosenblatt and Losey ( 1967). Two functions, that is, warning against predators and prevention of intraspecific predation, have been postulated to attribute to the alarm substance, though these have been questioned recently ( Verheijen, 1962).

E. Repellents Brett and MacKinnon (1952) found that striking reduction in the rate of upstream migration of adult sockeye Oncorhynchus nerka, coho 0. kisutch, and spring 0. tschawytscha salmon occurred when human hands were rinsed in the salmon ladder. Later this phenomenon was demonstrated in all five species of migrating Pacific salmon. Of various substances tested only dilute water rinses of mammalian skins had distinct repellent action (Brett and MacKinnon, 1954; Alderdice et al., 1954). Upon detecting the repellent odors, salmon swam excitedly moving in a circle in the enclosed area, exhibiting an alarm reaction. Chemical analysis of the properties of a repellent from human skin indicated that only L-serine elicited a strong repellent action at extremely high dilution (8 x lO-'O); however, the effects were neither so dramatic nor for so long a duration as the response obtained by a hand rise (Idler et al., 1956, 1961). Recently it has been observed that rainbow trout respond to handdipped water and to L-serine at concentrations as low as M by recording electroencephalographic responses from the olfactory bulb (T. J. Hara, unpublished data).

F. Orientation by Chemical Senses Numerous investigations have attested the high acuity of the chemosensory organs in most fishes. Olfactory cues are important in the orientation of migrating fish (eels, Anguilla anguilla, and salmon, Oncorhynchus sp.) and in the localization of spawning grounds; gustatory cues are important in the location of distant chemical stimuli (bullheads,

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Ictalurus) . Many theories have been proposed to explain the mechanisms of migration of anadromous salmon to their spawning grounds (Jones, 1968). It has been suggested that the orientation is mediated through olfaction, and reference has been made to a home-stream substance; some investigators have opposed these views (Ramsay and Hasler, 1961). Since the problems pertaining to the orientation of migrating salmon are dealt with in Volume VI (also see Hara, 1970), only current topics will be mentioned here. Spontaneous electrical potentials ( EEG ) were recorded from several different parts of the brains of adult spawning salmon (Oncorhynchus tschawytscha and 0. kisutch) when they arrived at their home pond (Hara et al., 1965). Activities in the olfactory bulb and in the posterior cerebellum consistently had a much higher amplitude than those of other parts; amplitudes of potentials in the optic lobes were especially low; in some fish the optic lobes were electrically “silent.” In contrast to adult salmon, young salmon exhibited high activities in the olfactory bulb and in the optic lobe. Cerebellar electrical activity in these younger fish was not yet developed. Spontaneous electrical potential records were also made from brains of adult, nonmigratory rainbow trout, Salmo gairdnerri, and of goldfish, Carassius auratus. In both of these nonmigratory species, EEG activity in the olfactory bulbs was relatively low while that of the optic lobes was much higher than in adult spawning salmon. Infusion of “home water” into the nasal cavity of migrating adult salmon produced a clear stimulation in EEG patterns recorded from the olfactory bulb; various natural waters from other nearby sources produced virtually no response ( Fig. 9 ) . These findings suggest that olfaction is an important factor in guidance during the final phases of homeward migration of salmon and that such olfactory discrimination occurs at the level of either the olfactory bulbs or the olfactory epithelium. However, there remains the possibility that certain nonspecific odorous substances, such as foods, were merely in higher concentration in the home water than in any other water used. Apparent specificity of the response to home water was confirmed by treating salmon from three different termini of migration with a series of natural waters (Ueda et al., 1967; Oshima et al., 1 9 6 9 ~ )The . high amplitude EEG response of characteristic pattern recorded from the olfactory bulb by infusion with home water is specific; little or no response can be evoked by water from spawning sites of other groups of breeding salmon (Table I). This implies that each spawning area has its own specific stimulant or specific combination of stimulants recognized and responded to by the anadromous salmon. Furthermore, weaker but definite responses could be evoked by waters (1)traversed by the salmon migrating toward the spawning site, ( 2 ) from the bypassed branch of a

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B

E

' I

J

Fig. 9. Effects of infusion of different waters into the nasal cavity upon EEG activity in the olfactory bulb of an adult spawning salmon: A, Distilled water; B, tap water; C, home water; D-F, waters from three different lakes. Lines below each of the records indicate duration of stimuli. Calibration, 50 p V and 1 sec. From Hara et al. (1965). Copyright 1965 by the American Association for the Advancement of Science.

stream near the spawning site, and ( 3 ) from a point upstream of the spawning site. It is suggested that on returning from the sea, the adult salmon retrace a trail of stimulatory factors, presumably the ones to which they were imprinted as young fish on their seaward migration (see Hara, Table I Specificity of Response to Home Water in Fishes from Three Spawning Systems" Water from

Fish from Fisheries pond (Chinook) Issaquah Creek (silver) Soos Creek (silver)

Fisheries pondb

100 (4) 39.1 f 1 4 . 5 (3) 0 (4)

Issaquah Creekb

0 (4) 100 (3) 0 (4)

soos Creekb 2 . 3 f 3 . 1 (4) 6 . 4 f 3 . 5 (3) 100 (4)

From Ueda et al. (1967). The magnitude of response is represented as a percentage of that to home water [mean k S.E. (number of animals)]. a

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1970). However, this question requires further study before a definitive statement can be made. Intracranial injection of antimetabolites, puromycin, actinomycin D, or cycloheximide in the homing chinook salmon markedly inhibited 01factory bulbar discrimination between home water and other natural waters ( Oshima et al., 1969a). Earlier, Rappoport and Daginawala ( 1968) showed that olfactory stimulation with morpholine induced an increase in brain nuclear RNA and a change in base ratios in both intact and split-brain preparations in isolated heads of marine catfish, Gabichthys felis. Thus, there seems to be agreement that RNA synthesis is part of memory-establishing mechanism. By monitoring and three-dimensional photographic techniques, Kleerekoper ( 1967a,b) analyzed certain aspects of orientation through olfaction in some marine ( Scyliorhinus stellaris, Mustelus mustelus, and Diplodus sargus) and freshwater ( Zctalurms nebulosus and Lepomis gibbosus) fishes. Single specimens were placed in an experimental tank, which was divided into 16 partial compartments by radially placed walls. Direction of the fishes’ movement, left- and right-hand turns, speed of locomotion, the frequency of entries into any one compartment, the frequency of pathways, and the diurnal distribution of total activity were continuously and automatically recorded with or without odorous stimulation. None of the fishes studied moved randomly under experimental conditions devoid of directional cues. The radius of the curve in changing direction was fixed within relatively narrow limits and seemed to be species-specific; left- and right-hand turns were not evenly distributed. Such a locomotor behavior resulted in a nonrandom pattern of relatively rigid parameters. Introduction of an odor without directional cues caused a drastic change in these parameters; the radius of the curve in changing direction decreased and the ratio of left-handed to right-handed turns was greatly changed. When an attractant odor was introduced unidirectionally, none of the species studied could locate the source, unless the odor was associated with a differential in the rate of water flow. It is, therefore, suggested that an attractant odor releases rheotactic response so that the localization of the source of the odor takes place through rheotaxis rather than through osmotropotaxis. Similarly, Hemmings (196613) made an analysis of the mechanism of orientation of roach Rutilus rutilus in an odor gradient. The swimming speed of the fish was not directly related to odor concentration but was low when fish swam into decreasing concentration and much higher in the opposite direction; turning was more frequent at the high concentration end of the gradient. These findings cannot be explained simply in terms of ortho- or klinokinesis. Hemmings suggested that orientation of

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this fish is by spatial and temporal comparison of intensities, that speed is related to the rate of change in stimulation, and that variation in turning serves to maintain fish at high odor concentrations. As is evident from Fig. 7, however, there occurred an immediate adaptation in the olfactory bulbar response in salmon by infusing the home water. It is impossible to assume that there exists such large odor gradients as to overcome the adaptation. Hence, it is probable that the orientation of these fish is not by osmotropotaxis but by “trial and error.” Creutzberg (1958, 1959) reported that the elvers of eels, Anguilla vulgaris, most probably used the tidal streams for their migration; they are transported in the direction of the inland waters by the flood tide and go to the bottom during the ebb in order not to be carried back to sea. From the evidence obtained by preference experiments for the waters, it is further suggested that elvers are guided from the open sea to the inland water by olfactory cues and like adult salmon are thus guided to their parent stream. Eels showed a clear preference for natural freshwater originating from inland sources. The water lost its effectiveness when passed through a charcoal filter. Recently, Miles (1968) reported that elvers of the American eel Anguilla rostrata show a stronger positive rheotaxis to freshwater than to saltwater. By the various treatment of the water he also demonstrated that the attractive components dissolved are biodegradable, heat stable, and nonvolatile; this seems to differ from the substance that attracts salmon to their home stream (Fagerlund et al., 1963; also see Volume VI, and Hara, 1970). Furthermore, the presence of adult eels in the water made it more attractive, while the presence of elvers made it less attractive. However, differences exist between the salmon and the eel; knowledge of the cues seems to be acquired by imprinting in the salmon, whereas it is most likely to be inborn in the elvers. Recent studies of homing by tagging showed no clear evidence that European eels (Anguilla anguilla) utilize olfactory cues to locate their home area after transplantation over long distances up to a few hundred kilometers off the coast (Tesch, 1967, 1970; Deelder and Tesch, 1970). Bullheads, Zctalurus nebulosus and 1. natalis, have an extremely welldeveloped taste sense. Bardach et al. (1967a) have established that taste alone can guide these fish to chemical clues. Bullheads seem to exhibit true gradient searching in the absence of a current b y spatial and temporal comparison of concentrations. Deprivation of the sense of smell did not impair their searching ability, but unilateral deprivation of taste receptors caused pronounced circling toward the intact side. Detailed electrophysiological analysis of neural activity of the barbels in response to chemical stimuli will greatly help to understand the mechanism under-

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5 TEMPERATURE RECEPTORS R. W. MURRAY I. Introduction . . . . . . . 11. Thermal Sensitivity of Fishes . . . 111. The Sense Organs Involved . . . IV. Electrophysiology . . . . . . A. Teleosts . . . . . . . B. Elasmobranchs . . . . . . V. Thermal Responses of Other Sense Organs References . . . . . . . .

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I. INTRODUCTION

Because of the all-pervading nature of heat and its influence on chemical reactions and physical processes there are many kinds of effect of “temperature” on animals, including variations in metabolic processes, temperature optima, acclimation processes, and so on, which do not come within the scope of this chapter. Even among sense organs there are a number whose responses may be different at different temperatures, or which may respond to changes of temperature, but which are not thermoreceptors in the strict sense of of receptors responding to thermal stimuli and initiating or controlling behavior relevant to the thermal properties of the environment. Clearly behavioral rather than electrophysiological experiments are needed to identify thermoreceptors thus defined, but the latter techniques provide the different evidence required for the understanding of the receptor mechanisms.

11. THERMAL SENSITIVITY OF FISHES

Fishes, or at least bony fishes, often behave as if they were aware of environmental temperatures, and this sensitivity has been well established 121

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R. W. MURRAY

experimentally (Table I). Thresholds have been found as low as 0.03"C (Bull, 1936) in experiments in which the fish were trained to swim up a long sloping trough against a water stream when the temperature of that water was suddenly increased. An increase in temperature can be distinguished from a decrease (Dijkgraaf, 1940), and although it is normally changes of temperature rather than absolute levels that are important, the rate of change found in other experiments (Bardach and Bjorklund, 1957) was so slow that, to the sensory physiologist, tonic rather than phasic receptor mechanisms would seem to be involved. In these experiments the conditioned stimulus was the slow warming of a well-stirred tank, and food was added when the temperature had risen 2°C. When fully trained, the majority of fishes showed characteristic and identifiable food-seeking behavior within %l min of the onset of the Table I Sensitivity of Fishes to Temperatiire Change in Conditioning Experiments Threshold Species

("C)

Reference

Ulennius pholis, Centronotus gunnellus, Cottus bubalis, Gadus mei,langus, Liparis montagui, Onos muslela, Zoarces viviparus

+0.03

Bull (1936)

Colt us scorpius, Crenilabws melops, Cyclopterus lumpus, Gadus callarias. Gobius jiavescens, Platichthys jlesiis, Pleiironectes platessa, Raniceps ranintis, Spinachia v ulgaris

+0.05

Bull (1936)

Gadus virens .Yer.ophis 1umbricifot mis

+O.OG +0.07

Bull (1936) Bull (1036)

Phozinus laevis, Awiiurus nebulosus

<+I

< -1

Dijkgraaf (1940)

('arassaus auiatus

+0.1

Bardach (1956)

Caiassaus auiatus, Ictaluius natalis, Seiiiotzlus at, oniaculatus

+0.05

Bardach and Bjorklund (1957)

Lepomas gibbosus

+0.l

Ictalurus natalis

+0.1 -0.1 -0.5'

Bardach and Bjorklund (1957) Bardach and Bjorklund (1957) Ba.rdach and Bjorklund (1957)

An escape response, not the conditioned movement.

5.

TEMPERATURE RECEPTORS

123

warming, which corresponds to a change of 0.05"-0.1"C. Although Bardach and Bjorklund did not specifically test for this, it is probable that it is the change away from the previous temperature (albeit a slow one) to which the fish responded, rather than to an absolute temperature level, since responses of trout, Salvelinus, in temperature gradients are known to depend on their previous thermal history (Sullivan, 1954). From Table I it appears that the threshold for marine fish is lower than that of freshwater fish, but the differences are more likely to be caused by the interests, or patience, of the experimenters. For example, Dijkgraaf (1940) did not attempt to find absolute thresholds. The comparable evidence for the sensitivity of elasmobranch fishes is lacking. Amphioxus has an escape response to warming of the skin in the region of the dorsal fin, which can be elicited by a rise of 0.75OC in 3 sec or by 3°C in 20 sec (Lele et al., 1958).

111. THE SENSE ORGANS INVOLVED

Two alternative sensory systems have been proposed as the thermoreceptors responsible for the results described in the previous section; namely, the lateral line organs and the general, cutaneous, segmental innervation. Historically, the lateral line came first, following the pioneering electrophysiological investigations of Hoagland ( summarized in Hoagland, 1935). He had found that the "spontaneous" discharge increased at higher temperatures, and in the absence of cutaneous sensitivity, he attributed thermoreception to the lateral line. This suggestion was followed up by behavioral experiments (Rubin, 1935; see below). Since that time, much of the effort of later workers has been devoted to the demonstration that the lateral line is not involved, but rather the segmental cutaneous system. A thermoreceptor function of the lateral line is unlikely in view of its known mechano- and electroreceptor functions (see chapters by Flock and Bennett, this volume), although arguments based on strict, single-modality functioning of receptors are not so valid today as was once thought. However, there is clear evidence on this point from conditioning experiments (Dijkgraaf, 1940). Minnows, Phoxinus laevis, which had been trained to respond differentially to streams of warm or cold water applied by pipette to the flank, responded as usual if the lateral line nerves had been cut; but they failed to respond if the site of stimulation was

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R. W. MURRAY

posterior to a deletion of the spinal cord over 1-2 segments in the region of the anal fin. Confirmation of the role of the cutaneous nerves has been provided by tests in which fish were trained to swim away from a normal resting position in the center of an experimental tank when touched by a warmed rod and not to move if the rod was at tank temperature (Bardach, 1956). When the rod was 2°C warmer than the tank, consistent responses were obtained from almost the whole of the body surface, including the fins, and they were not restricted to the region close to the lateral line. A similar conclusion follows from Sullivan’s records of the behavior of trout in a temperature gradient (Sullivan, 1954); the rate and extent of turning and the frequency of moving vary with temperature in such a way that the fish remain mostly in a certain “optimal” region. This optimum is maintained even if the lateral line nerves are cut, but it is lost after treatment of the skin with cocaine. If temperature reception in teleosts is normally mediated by the general cutaneous innervation, as indeed it is in most other vertebrates (see, for example, Murray, 1962), then Rubin’s implication of the lateral line has to be explained. He found that the temperature at which a gradual warming of the water elicited an escape response in five different species was raised from about 27°C to over 34°C (in fact to a lethal level) by cutting both the trunk lateral line nerves (Rubin, 1935). The first point which can be made is that the response is clearly a nociceptive one (i.e., “painful”) and does not involve the kind of sensitive thermoreception that is involved in threshold experiments; and it may well be that excessive activity of all lateral line organs, however initiated, is a stimulus eliciting escape and there need be no identification of the stimulus as thermal. Second, the responses were unconditioned, and in such an experiment there is always the possibility that it is the responsiveness of the animal which has altered and not the stimulus. The removal of the tonic effect of the trunk lateral line system might well be expected to depress the responsiveness of the central nervous system (CNS), and there was no control showing that escape responses to other modalities of stimulus were unchanged. There is also a third possibility (Dijkgraaf, 1940), that detection of convection currents by the lateral line could have been involved. A further series of experiments ( Andrews, 1952) which apparently connect the lateral line with thermoreception can be criticized along the same lines. A number of species lose their normal light avoidance behavior when the water gets too hot (the exact level depends on the acclimation temperature), and this critical level was raised by about 4°C when the trunk lateral line nerves were cut.

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TEMPERATURE RECEPTORS

125

IV. ELECTROPHYSIOLOGY

A. Teleosts It is likely that a number of unreported attempts have been made to identify impulse activity from the thermoreceptors in teleost skin, but so far none has proved successful (Hoagland, 1935, reported such a negative result). One possible reason for this is that surviving preparations cannot normally be isolated from the body for electrophysiology in the way that they can be in elasmobranchs or amphibians. In most teleosts the sense organs must be left intact and the fish maintained under artificial respiration during the experiment. But there may also be a much more serious theoretical reason, namely, that there are no specific thermoreceptors at all, i.e., no nerve fibers mediating responses to temperature or temperature change alone, but only mechanoreceptors whose responses to touch are sensitively altered according to skin temperature and its changes (Spath, 1967). In the redeye, Leuciscus rutilus, immobilized with curare and under artificial gill ventilation, Spath found no impulse discharge in response to thermal stimuli alone, in cutaneous nerves, but the phasic impulse responses to standardized single or repeated mechanical stimuli were digerent at different temperatures, and they varied following sudden temperature changes. Figures 1-3 illustrate his results. An example of the response to a standard mechanical stimulus and the way in which it can be modified is shown in Fig. 1. The nature of the temperature effect in this experiment will be described later. The simplest situation in which temperature affects the response to mechanical stimulation is illustrated by Fig. 2, where the standard stimulus is applied at a number of steady temperatures. The curves show that there is an “optimum” ( i.e., the response consists of the greatest number of impulses) at a temperature in the middle of the range covered by the experiment, in this instance between about 20” and 22°C. The optimum temperature depends to some extent on the temperature to which the fish had previously been adapted (15°C for 4 weeks in this example). The optimum for fish adapted to 5°C was at 18”C, but for fish adapted to 25°C it fell above the range tested, i.e., the response increased all the way to 30°C. On changing the temperature suddenly by means of the water flowing over the stimulus point, the responses do not merely alter at once to the level characteristic of the new temperature. There is a specific effect of change. Mechanical stimuli applied within a few seconds of a rise of temperature produce a smaller response than at the previous steady

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R. W. MURRAY

Fig. 1. Responses in a cutaneous nerve of Leuciscus to standard mechanical stimuli (monitored on the upper traces) when the stimulator was warmed above the temperature of the fish and the surrounding water (which was 5OC ). The temperatures given on the right of each trace refer to the stimulator. From Spath (1967).

temperature, and those applied after a drop in temperature are more effective than before (Fig. 3). One of the complexities of this particular experimental method results from the fact that the response to stimuli repeated at l / se c falls off even at steady temperatures, and disappears completely after about 30 sec, probably owing to viscoelastic movement of the skin away from the point of stimulation. This accommodation can be seen by interpolating a curve between curves A and C in Fig. 3, which would represent the effect of stimulation at constant temperature. It means that the effect of change of temperature cannot be followed all the way through until a steady level is reached. It can be seen now that Fig. 1 shows the same effect, for the skin is in this case touched by a warm stimulator rather than by a neutral stimulator (glass) while warm water is flowing over the stimulated point.

5.

127

TEMPERATURE RECEPTORS

& 62

0

I 200

CT

0 u)

5

E

150

.-

50 -

1

I

I

I

I

I

Fig. 2. Differences in the responses of cutaneous receptors of Leuciscus to standard mechanical stimuli, tested at different temperatures. The fishes had previously been adapted to 15OC for 4 weeks. The temperature of the skin was changed by the gentle flow of water of the appropriate temperature, and mechanical stimuli were applied with a small glass probe. All impulse frequencies are expressed as a percentage of the response to the first stimulus at 6OC. The upper curve ( a ) sho\vs the responses to single stimuli (mean values from 10 experiments). The lower curve ( b ) shows the adapted level to which the frequency falls with repeated stimuli at l/sec (mean of 11) . Vertical lines indicate standard errors. From Spath ( 1967 ).

In fact, warm objects result in fewer impulses, and cold objects in more, than do thermally neutral objects. The results are roughly quantitative in that the greater the change of temperature the more rapid the change, and thus the greater the effect on the response to mechanical stimuli. Clearly, the question now arises as to whether such a system can underlie the known temperature responses of fishes. There are two problems, first, whether the CNS can disentangle the two modalities, touch and temperature, and obtain useful information about both, and, second, whether the recorded sensitivity can account for the known thresholds (e.g., Table I ) . A single bimodal, touch-temperature receptor can, in theory, give information about temperature as long as there is independent evidence

128

R. W. MURRAY

Seconds

Fig. 3. The responses in cutaneous nerves of Leuciscus to repeated mechanical stimuli applied while the skin temperature was being changed by water flowing over it. At time = 0 sec the stimulation at l/sec began, and the water temperature changed. Impulse frequencies are expressed as percentages of the first response. A and B show the effects of cooling, A from 10' to 6'C, and B from 30' to 6'C; C and D show the effects of warming, C from 6' to 10°C, and D from 6' to 30'C. ( A and B are mean values from six experiments, C and D from seven.) The fish had been adapted to 15'C. Data from Spath (1967), redrawn.

about the touch to act as a reference, i.e., if the fish knows how strong the mechanical stimulus is and thus how vigorous the impulse discharge ought to be under any particular thermal conditions. If the responses to a repeated stimulus get weaker, it may be that the stimulus is smaller; but if the responses to a stimulus known to be constant become weaker than they ought to be, either that stimulus object is warmer than the environment or the fish has just swum into warmer water. If the responses now adapt to a different level and stay there, then the fish must be in water of a certain, calculable temperature (or at one of two temperatures on either side of the optimum). Ambiguities of this kind can be avoided by making use of a population of receptors having different optima. The strength of the mechanical stimulus can best be derived from the output of temperature-insensitive mechanoreceptors, but in fact this is only the extreme instance of the more general solution using bimodal receptors

5.

TEMPERATURE RECEPTORS

129

having a range of relative touch-temperature sensitivities. An alternative method could be to depend on stimulation arising from the fish‘s own movements, in which case reafference (von Holst and Mittelstaedt, 1950) could form the basis for the comparison. The idea that one modality of stimulation can be detected by an animal, using multimodal receptors only, is relatively recent. For instance, the inadequacies of the old doctrine of specificity of receptors (and especially of anatomical specificity) have been stressed for mammals in various “pattern of stimulation” hypotheses ( e.g., Sinclair, 1955; Lele and Weddell, 1959). However, modern views retain a considerable degree of functional specificity, while admitting that especially within one modality the information from a single receptor may be ambiguous, and positive identification of the stimulus may only be possible by comparison of the responses in a number of channels (e.g., taste: Cohen et al., 1955; and the review of cutaneous sensitivity in mammals by Melzack and Wall, 1962). In some cases, however, simpler explanations for multimodal units may be adequate, either that one stimulus is mistaken for the other (“seeing stars,” or the explanation of Weber’s Deception-cold weights feel heavier-in terms of the stimulation of cold-touch units) or that the two modalities can be separated by reference to coexisting specific units for each of the modalities. But neither of these can apply in teleosts, for temperature is evidently not confounded with touch, and there are as yet no specific thermoreceptors known. Thus the computing problem for the CNS must be complex, and the methods used are completely unknown. One particular difficulty not found in the mammalian touch-temperature units is that the fish mechanoreceptors are rapidly adapting, not tonic. But it is a reasonable hypothesis, all the same, since, as Spath points out, an aquatic animal is warmed or cooled by convection or conduction, never under natural conditions by radiation, and the former two necessarily involve mechanical stimulation. In fact, when Dijkgraaf (1940) tested the effect of radiant heat on the exposed fish, he found that they responded to the warming with a much higher threshold than in his other experiments. The large number of touch-temperature units in mammals supports the idea that the vertebrate CNS can make suitable computations, and recently Bailey (1969) has shown that the most sensitive thermal responses in lizard skin are given by touch-temperature units very similar to those of the teleost. There is one extra source of information for the lizard, namely, that the adaptation time for the phasic response is temperature dependent, as well as the initial impulse frequency. The fact that the teleost touch-temperature units are “cold sensitive,” i.e., have an increased response on cooling and decreased in warming,

130

R. W. MURRAY

does not preclude them from mediating responses to both warming and cooling. The idea of either increase or decrease in a resting frequency forming the basis of sensation has been implicit in sensory physiological theory since Lowenstein and Sand's demonstration of the resting discharge from semicircular canals ( Lowenstein and Sand, 1936), although strict experimental proof is rare ( e.g., Lowenstein, 1937). As for the question of sensitivity, here again Spath's system would change in the impulse response seem to be just adequate, He records a 10% to standard mechanical stimulation on changing the temperature by about 1.5"C. Clearly this 10%is well below threshold for a single receptor, owing to the small number of impulses involved in a single response (see Fig. l ) ,but it works out as a possible, although optimistic, threshold when the results of a number of units are summed, as in his published results. It is known experimentally that spatial summation occurs in fishes (Bardach, 1956), the threshold falling from about 10°C for point stimulation, to 2°C with a stimulus area of 2 mm2, and to less than 0.1"C for the whole fish. Spath's stimulator had an area of about 1 mm2; thus, his results are close to those obtained behaviorally, when an allowance is made for some temporal summation and for the possibility that his recording did not show the activity of all the fibers which were in fact stimulated. The greater sensitivity obtained by summation results from the statistical advantages to be gained from using a larger sample.

B. Elasmobranchs Cutaneous nerves are known with sensitivities to either warming or cooling ( Murray, 1961). However, Murray's experimental method, while demonstrating the existence of bimodal touch-temperature units analogous to those of teleosts, did not allow the possible existence of pure thermoreceptors to be explored fully. The method involved recording single unit activity by means of a wire electrode resting on the surface of the damp skin (in Raja sp.); thus, the touch of the electrode which was, of course, necessary to ensure electrical continuity in the recording circuit, provided a continuous, controlled mechanical stimulus. Details of three individual units were given. In the first, where there was complete adaptation to the mechanical stimulus, rapid radiant warming (at lS°C/sec) elicited impulse activity, with a threshold 2"-3°C above room temperature. This unit could have been either bimodal, i.e., hot-touch, or possibly a specific warm unit, lying within range of the recording electrode. The second was a cold-touch unit, responding to warming with a decrease in the nonadapting response to the presence of the electrode. The third, the most sensitive, was also a cold-touch unit, for the phasic

5.

131

TEMPERATURE RECEPTORS

response to repeated mechanical stimulation was inhibited by warming the skin from 20" to 20.8"C but returned when the temperature stayed steadily at 20.8"C.

V. THERMAL RESPONSES OF OTHER SENSE ORGANS

A number of other sense organs in fishes exhibit either phasic responses to temperature changes or tonic effects of steady temperatures. The chief examples are the lateral line organs in teleosts and elasmobranchs and the ampullae of Lorenzini in the latter. The ampullae, in fact, are so sensitive that Sand (1938) was forced to attribute a thermoreceptive function to them, but there is no evidence that they are so used in the life of the fish, and on anatomical and physiological grounds it now seems most unlikely. For example, it is hard to account for the ramification of the tube system in rays, and the deep location of the ampullae themselves which are the temperature-sensitive regions, if thermoreception was their function. Moreover, there is no evidence of any special thermal conductivity in the jelly (Murray, 1960). It is, of course, possible that the tonic increase in the resting discharge at high temperatures in the ampullae and lateral line organs could have a nonspecific stimulatory effect on the CNS (especially if the latter were still not warmed), but there is no good evidence even for this function. [See chapters by Flock and Bennett, this volume, for a discussion of the function of the ampullae, and also Murray ( 1967).] The basic pattern of the response (Fig. 4 ) is a phasic increase in frequency on cooling or decrease on warming, which adapts to a new tonic level normally higher, the higher the temperature (Sand, 1938). The

i 150 \

-z2

100 16.25OC 15.75OC \

3? - 35O0

g:

6

t-

10

'0

0

3 6 9 12 15 18 21 2 4 2 7

E...'

.o,.-.

2i2 20 30 4 & 10

e G20

-*

I

.-2

0

1

2

Time (min)

Time (min)

(0)

(b)

3

4

Fig. 4. The impulse response from the ampullae of Lorenzini in response to temperature changes: ( a ) A single unit from Scyllium [from Hensel (1955)], and ( b ) a single unit from RU~U [from Murray (1962)l.

132

R. W. MURRAY

phasic response, called "paradoxical" by Sand to emphasize its negative sign, can be extremely sensitive, reaching -90 impulses/sec/ "C for example in the ampullae of Scyllium. (Hensel, 1955); but in the lateral line the sensitivity is much less (-0.3 impulse/sec/"C; Sand, 1938). NO phasic response has been described in teleosts (Hoagland, 1935). The tonic response is usually described as positive only, i.e., faster when hot (e.g., Hoagland, 1935; Sand, 1938) with Qlo values ranging between 1.5 and 3. But in ampullae of dogfish from the warmer waters of the Mediterranean (Hensel, 1955) this positive relationship is seen to be merely the lower part of a peaked curve having its maximum around 20"-25°C and falling back to zero again by 30°C. Thus the responses of the ampullae, both phasic and tonic, are seen to parallel almost exactly those of the cold fibers in mammalian skin. The stretch receptors from the pelvic fin of rays, when put under tension to establish a continued impulse discharge, showed tonic and insensitive phasic responses virtually identical with those of the lateral line organs ( Sand, 1938). If the ampullae are not temperature receptors, then it may be possible to turn the argument the other way around, namely, that the fish are not in fact normally subjected to temperature changes fast enough to elicit significant impulse frequency responses from the ampullae. The normal functioning of the system in response to its proper stimuli (e.g., electroreception) is not therefore distributed. However, the thermal sensitivity is clearly of interest to sensory physiologists for it provides a model of the action of mammalian cold fibers. In this connection, for example, Murray (1966) has stressed the very dissimilar anatomical organization of the many nervous structures which exhibit the same kind of negative phasic response superimposed on a less sensitive positive tonic change. Ampullae of Lorenzini have sensory synapses on receptor cells, mammalian cold fibers have free nerve endings, motor nerves in crustaceans have no natural terminal structures when excised, and so on. Murray argues from this diversity that the thermal responses represent one of the basic properties of nerve membrane, and temperature receptors are those nervous structures in which this property is most highly developed. REFERENCES Andrews, C. W. (1952). Sensitivity of fish to light and the lateral line system. Physiol. ZOO^. 25, 240-243. Bailey, S. E. R. (1969). The responses of sensory receptors in the skin of the green lizard, Lacerta uiTidis, to mechanical and thermal stimulation. Comp. Biochem. PhysioZ. 29, 161-172.

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Bardach, J. E. ( 1956). The sensitivity of the goldfish (Carussius auratus L.) to point heat stimulation. Am. Naturalist 90, 309-317. Bardach, J. E., and Bjorklund, R. G. (1957). Temperature sensitivity of some American fresh water fish. Am. Naturalist 91, 233-252. Bull, H. 0. (1936). Studies on conditioned responses in fishes. VII. Temperature perception in Teleosts. J . Marine Biol. As~oc.U . K . 21, 1-27. Cohen, M. J., Hagiwara, S., and Zotterman, Y. (1955). The response spectrum of taste fibres in the cat: A single fibre analysis. Actu Physiol. Scand. 33, 31633.2. Dijkgraaf, S. (1940). Untersuchungen iiber den Temperatursinn der Fische. Z Vergleich. Physiol. 27, 587-605; but see also corrections to this in Dijkgraaf, S. ( 1943). Berichtigung und Erganzung zu meiner Arbeit “Untersuchungen iiber den Temperatursinn der Fische.” Z. Vergleich. Physiol. 30, 252. Hensel, H. ( 1955). Quantitative Beziehungen zwischen Temperaturreiz und Aktionspotentialen der Lorenzinischen Ampullen. Z. Vergleich. Physiol. 37, 509-526. Hoagland, H. ( 1935). “Pacemakers in Relation to Aspects of Behaviour.” Macmillan, New York. Lele, P. P., and Weddell, G. (1959). Sensory nerves of the cornea and cutaneous sensibility. Exptl. Neurol. 1, 334-359. Lele, P. P., Palmer, E., and Weddell, G. (1958). Observations on the innervation of the integument of amphioxus, Branchiostoina lanceolatuni. Quart. J. Microscop. Sci. 99, 421440. Lowenstein, 0. (1937). The tonic function of the horizontal semicircular canals in fishes. J. Exptl. Biol. 14, 473482. Lowenstein, O., and Sand, A. (1936). The activity of the horizontal semicircular canal of the dogfish, Scyllium cunicula. J. Exptl. Biol. 13, 416-428. Melzack, R., and Wall, P. D. (1962). On the nature of cutaneous sensory mechanisms. Bruin 85, 331-356. Murray, R. W. (1960). The response of the ampullae of Lorenzini of elasmobranchs to mechanical stimulation. J . Exptl. Biol. 37, 417424. Murray, R. W. (1961). The initiation of cutaneous nerve impulses in elasmobranch fishes. J. Physiol. ( L o n d o n ) 159, 546-570. Murray, R. W. (1962). Temperature receptors. Aduan. Cornp. Physiol. Biochem. 1, 115-175. Murray, R. W. ( 1966). Nerve membrane properties and thennal stimuli. I n “Touch. Heat and Pain” (A. V. S. de Reuck and J. Knight, eds.), pp. 164-182. CIBA Found., London. Murray, R. W. (1967). The function of the ampullae of Lorenzini of Elasmobranchs. I n “Lateral Line Detectors” (P. Calm, ed.), pp. 277-293. Indiana Univ. Press, Bloomington, Indiana. Rubin, M. A. (1935). Thermal reception in fishes. J. Gen. Physiol. 18, 643-648. Sand, A. (1938). The function of the ampullae of Lorenzini, with some observations on the effect of temperature on sensory rhythms. Proc. Roy. Soc. B125, 5 2 4 4 5 3 . Sinclair, D. C. (1955). Cutaneous sensation and the doctrine of specific energy. Brain 78, 584-614. Spath, M. (1967). Die Wirkung der Teniperatur auf die Mechanoreceptoren des Knockenfishes Leuckcus rutilus L. Z. Vergleich. Physiol. 56, 431462. Sullivan, C. M. (1954). Temperature reception and responses in fish. J . Fisheries Res. Board Can. 11, 152-170. von Holst, E., and Mittelstaedt, H. ( 1950). Das Reafferenzprinzip. Naturwissenschaften 37, 464-476.

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SOUND PRODUCTION AND DETECTION WILLlAM N . TAVOLGA I. Introduction

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135 136 136 138 142 154 162 162 164 170 182 183 189 192

I. INTRODUCTION

The field of aquatic bioacoustics has grown rapidly in many directions, involving many allied areas of research. Major recent reviews of the subject include those by Moulton (1963), Protasov (1965), and Tavolga ( 1965). Three international symposia on aquatic bioacoustics have been held and their proceedings published (Cahn, 1!367; Tavolga, 1964a, 1967a). Sound is probably the most effective channel for long-range communication under water, and it has become clear over the past 20 years that many fishes utilize this channel. The mechanisms of sound production and the sounds themselves have formed an active area of research, aided by recent technical developments in underwater acoustics. Although sound production may be restricted to some as yet unknown fraction of 135

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all fish species, it is apparent that all fishes must be capable of receiving acoustic stimuli. Sound detection in an aquatic medium presents certain problems not normally encountered by terrestrial organisms, e.g., the separation of pressure from displacement detection seems to be characteristic of aquatic animals. 11. SOUND PRODUCTION

A. Historical Background Prior to the 1940’s published reports appeared sporadically on sounds produced by fishes and other aquatic animals. Aristotle and Pliny (cited by Moulton, 1963) gave several examples, but all such reports up through the nineteenth century were based upon what the unaided human ear could detect. Some primitive techniques used by fishermen possibly since prehistoric times have included pipes, oars, and other objects that would transmit underwater sounds to the ear. Moulton (1963) reviewed several instances of fishermen who rely on detecting the presence of desirable fish by listening for them. An important nineteenth century contribution was the report of Dufoss6 (1874), in which many instances of sound production by fish were described, including several marine species. In addition to such essentially descriptive studies ( Geoffroy St. Hilaire, 1829; Smith, 1927; von Ihering, 1930; Dijkgraaf, 1932; and others cited by Moulton, 1963, and Tavolga, 1965), there was an early interest in the mechanisms by which fish produced sounds. Agassiz (1850) suggested the swim bladder as a sonic organ, and Moreau (1876) made certain deductions as to the sonic function of the swim bladder in the sea robin, Trigla, based upon anatomical studies. An extensive anatomical and experimental investigation of swim bladder sonic mechanisms was reported by Tower ( 1908), in which he was probably the first to suggest that the vibration frequency of the bladder was equivalent to the fundamental frequency of the emitted sound. The production of sound by certain catfishes was reported by SIdrensen (1894), and he provided a highly detailed description of the skeletal and muscular apparatus. Although these structures, such as the “elastic spring” in catfishes, had been described earlier by Muller (1842, 1843), their function in sound production was not known until Sgrensen published his thesis in 1884, and his conclusions were confirmed by Bridge and Haddon (1894). In the following 40 years, accounts were published sporadically on further investigations of sonic mechanisms in fishes, notably those of Smith (1905) on sciaenids, Greene (1924) on

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Porichthys, and Hardenberg ( 1934) on Therapon. In addition, structures such as stridulating teeth, fin spines, and other hard parts were found to be involved in sound production in many species (Sgrensen, 1894; Burkenroad, 1930, 1931 ) . Among small aquarium species, the croaking gourami, Trichopsis vittatus, is probably the best known sound producer ( Stampehl, 1931; Reickel, 1936; Meder, 1953; J. A. Marshall, 1963). The behavioral significance of fish sounds also occupied the interest of many investigators. Dufossk (1874) remarked on the possible communicative functions of these sounds. Most workers reported these sounds to be a sign of alarm or fright (Greene, 1924; Burkenroad, 1930, 1931), although the fact that some fish sounds, notably of sciaenids, are associated with the spawning season or with schooling had apparently been known to fishermen since ancient times. As is often the case, major scientific and technological advances occur as a byproduct of the search for more efficient means of making war. The field of marine bioacoustics serves as a good example. Motivated by a concern for detection of submarines and other means of undersea warfare, new and efficient mechanisms for detection of underwater sound were developed during World War 11. Hydrophones and their associated electronic and recording equipment also proved capable of detecting sounds produced by undersea animals. Shortly after the war, reports were made public that sounds produced by undersea animals often formed an ambient, interfering noise (Loye and Proudfoot, 1946; Knudsen et al., 1948). Some progress was made in identification of sonic species and their seasonal occurrences (Dobrin, 1947, 1948), and in the use of sonics as a tool in the study of marine ecology (Johnson, 1948). An important contribution to this field is represented by the reports of Fish et al. (1952) and Fish (1954). These publications described sounds produced by a wide variety of marine fishes, representing many families, and showed that sound production was much more common than had been previously supposed. Most of the examples given were recorded under artificial, aquarium conditions, and the sounds emitted were often from animals under duress. Such reports demonstrated that many fishes were potentially capable of producing sounds. Spectral analyses of the sounds showed a significant amount of species distinctiveness. In 1953, Kellogg published a bibliography of sounds of marine organisms. Although not complete, this listing included a large majority of references available at that time. Over the past 15 years, the number of relevant articles and books issued totals at least 10 times the 53 listed by Kellogg. In addition, several reviews of the field have been published, notably by Backus (1958), Maliukina and Protasov (1960), N. B.

1x5

W l L L l A M N. IAVULLm

Marshall ( 1962), Moulton ( 1963), Protasov ( 1965), Schevill et al. ( 1962), Schneider ( 1961), and Tavolga ( 1960, 1965, 1967~).Two international symposia on marine bioacoustics covered many aspects of the field, including sound production in fishes, cetaceans, and other marine organisms ( Tavolga, 1964a, 1967a). A serious problem encountered in obtaining recordings of aquatic animals under natural conditions is that the listener is often working blindly and thus the identification of the sound producer often becomes impossible. A major approach to the solution of this problem has been to accompany sonic observations with visual observations by means of underwater television. A unique installation of this sort is now located off Bimini, Bahamas (Steinberg et al., 1962; Steinberg and Koczy, 1964; Kronengold et al., 1964). This system has been undergoing constant improvement and modification and has provided a large body of data on sonic species of fish (Cummings et al., 1964, 1966; Steinberg et al., 1965).

B. Underwater Acoustics Any study of sound production or sound detection in fishes necessitates an understanding of the acoustic properties of water as a medium. Since water is much denser than air, the velocity of sound in water is almost 1500 meters/sec while in air it is about 330 meters/sec. In air, sound velocity is affected slightly by humidity, temperature, and barometric pressure. In water, temperature and pressure are independent variables in shallow areas, while at greater depths pressure affects the temperature. The curves that relate sound velocity to depth, therefore, become quite complex (Albers, 1965; Tschiegg and Hays, 1959; MacKenzie, 1960) (Fig. 1). Salinity increases sound velocity, and in the oceans sound velocity may attain 1540 meters/sec. As a corollary to this almost fivefold difference in sound velocity, c, between air and water, the wavelengths, h, of underwater sounds are almost five times the length of those in air for the same frequency, f, i.e., h = c/f.

Since water is about a thousand times denser than air, more input energy is required to initiate the propagation of sound in water. However, once the sound is propagated, the acoustic energy will be transmitted faster in water. This transmission is further enhanced by the reflection of sound from the water surface (up to 99.9%is reflected back), from the sea bottom, and from interfaces that are formed by layers of water at different temperatures (Vigoreux, 1960; Albers, 1965). If we measure the sound level under ideal conditions, that is, with

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Sound velocity ft/sec

0

50

100

200

Fig. 1. Velocity of sound in the sea. The two series of determinations show that velocity varies from about 5070 ft/sec (1546 meters/sec) at the surface, to about 4800 ft/sec (1464 m e t e d s e c ) at depths below 500 fathoms (915 meters). After Tschiegg and Hays (1959).

the sea at dead calm and with no vessels or sound-producing animals about, there is still an ambient noise whose pressure is about 0.18 to 0.20 dyne/cm2. This is usually expressed in terms of decibels (dB) with respect to a reference level of 1dyne/cm2 [ = 1pb (microbar)], and, in this case, the noise level would be about 15 dB below this reference level. In air, the reference level is usually taken to be 0.0002 pb, since this is the standard threshold of human hearing for a frequency of 1000 cps ( H z ) . This reference level has little objective meaning in underwater acoustics. Thus the 1-pb level is now almost universally used as the reference level in water and all the sound pressure values given in this report will be in decibels re 1 pb (abbreviated to dB pb) (Table I ) .

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Table I Comparative Chart of Approximate Acoustic Pressure Levels of Common Sounds in Air and in Watera Acoustic pressure (dB)

Sounds in air Jet aircraft takeoff (at 75 meters)

60

Threshold for human aural discomfort (discomfort at 1000 Hz) Loud auto horn (at 1 meter)

50

Small propeller aircraft. (at 5 meters) New York subway train (at 10 meters) Noisy business office Home high fidelity set

40

30 20 10 0

Average conversation (at 1 meter) Private business office

- 10 -20

Average residence

-30 -40

Quiet country residence Quiet whisper Human hearing threshold (at 1000 Ha) a The reference point is set a t 0 dB point of 0.0002 pb add 74 dB.

=

Sounds in water Underwater dynamite explosion (at 100 meters) 25 h p outboard motor (at 15 meters) Toadfish boat-whistle sound (at 1 meter) Rough sea (state 6 ) Large chorus of marine catfish Noise of ships in busy harbor Large chorus of snapping shrimp (at 100 meters) Calm sea (state 0) Squirrelfish hearing threshold (at 800 Hz) Threshold of hearing of ostariophysine fishes

-50 -60 -70 -80 1pb ( = 1dyne/cm2).To convert to a reference

Ambient noise in the sea normally includes the sounds produced by wave motion on the surface, friction of moving water currents against the bottom and against each other, the noise of shipping traffic, and, superimposed on all that, the noises of marine animals (Wenz, 1962, 1964) (Fig. 2). The average level of ambient sea noise is about 10 or 15 dB pb. In air, sound is usually defined as a more or less periodic form of compression waves that can be detected by the human ear. The frequency range, again with reference to human hearing, is normally considered to be from 20 to 20,0000 Hz. At 20 Hz, the sound is “felt” rather than heard, and most people cannot detect sounds above about 16,000 Hz ( = 16 kHz). This is a subjective and anthropomorphic definition of sound and does not apply to acoustic energy in water. Since water is highly resistant to compression, the propagation of sound in water usually involves particle displacement as well as compression. This displacement is partic-

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Wand -dependent bubble and spmy m s e Heavy precipitation

+Oceanic

traffic

L"

I

10

10'

lo3

lo4

lo5

Frequency (Hz)

Fig. 2. Typical ambient noise spectra in the sea. Horizontal bars show approximate band of influence of various sources. Sound pressure level on the ordinate is given in decibels with reference to 0.0002 dyne/cmZ; to convert to reference level used in this paper subtract 74 dB from all values. After Wenz (1964),with permission of Pergamon Press.

ularly evident at close range to the sound source, and the phenomenon has been termed the "near-field effect, as opposed to the "far-field" compression waves. The relationship between these two forms of acoustic energy has been discussed extensively by van Bergeijk (1964, 1967a), Harris (1964), and Harris and van Bergeijk (1962). Under most conditions in the field and in aquarium tanks, both near- and far-field energy occur together and are difficult to separate with standard equipment, since a hydrophone is basically a pressure transducer and will respond to compression produced at its surface as a result of particle displacement, i.e., to near-field energy as well as to far-field energy. The energy propagated by the fins of a fish, the flow of water along the body of a

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WILLIAM N. TAVOLGA

moving fish, and even the currents of water flow in rivers and seas are essentially acoustic phenomena, and a line of demarcation between such energy and that of a distinctly audible hoot of a toadfish is difficult to draw. Some investigators have referred to these extremely low frequency or steady state displacements and pressures as “unsound or “pseudosound” ( Parvulescu, 1964, 1967; Ffowcs-Williams, 1967). It is apparent, therefore, that in water the usual definitions of sound are not entirely applicable, and the distinction between rheotaxis and hearing is not clear, As evidenced by the problems and discussions at a recent conference on the lateral line of fishes (Cahn, 1967), the concern over underwater bioacoustics has extended far below what is ordinarily considered the audiofrequency range. C. Sonic Mechanisms in Fishes

There appears to be little relationship between the morphology of sound-producing mechanisms and phylogenetic position in fishes. At the present time, however, only a few hundred out of more than 20,000 species have been clearly identified as sound producers. Many species, such as certain deep-sea forms ( N . B. Marshall, 1967), appear to possess the means for sound production but have not yet been recorded. Three general types of sonic mechanisms are present in fishes: stridulatory, hydrodynamic, and swim bladder. Stridulatory sounds are produced by friction of teeth, fin spines, or bones. Hydrodynamic sounds result from swimming movements, especially during rapid changes of direction or velocity. The swim bladder acts as a sound projector when it is vibrated by contiguous or attached muscles. 1. STRIDULATORY MECHANISMS

A large majority of teleost fishes possess opposing patches of denticles in the pharynx and are at least potentially capable of sound production during feeding. Some species stridulate pharyngeal denticles in connection with other activities such as alarm and territoriality. The best known sound producers of this type are members of the family Pomadasyidaethe grunts (Fig. 3 ) . Burkenroad (1930) described the sounds and the mechanism in some detail for the white grunt, Haemulon plumieri. He also noted that if the swim bladder were deflated, the character of the sound became “dry” and lost its “gruntlike” quality. He concluded that the swim bladder acts as a “resonator.” A similar arrangement has been described for other members of the family, and the margate fish, Haemulon album, has been observed to emit sounds, probably produced

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AND DETECTION

143

Fig. 3. Dissection of the pharyngeal region of a grunt, Haemulon, shows the location of pharyngeal denticles ( a and b ) and the swim bladder ( c ) . After Tavolga (1965), with permission of the U. S. Naval Training Device Center.

by this mechanism, in connection with feeding and schooling behavior (Cummings et al., 1966). The function of the swim bladder as a possible resonator was also demonstrated in triggerfishes (Salmon et al., 1968). The occurrence of pharyngeal tooth stridulation has been reported from a wide variety of species and families of teleostean fishes by Burkenroad (1931), Dobrin (1947), Dorai Raj ( 1960a), Fish ( 1954), Knudsen et al. (1948), Moulton (1958), Tavolga ( 1964b), and Taylor and Mansueti (1960). The courtship and territorial sounds of the croaking gourami, Trichopsis vittatus, are evidently produced by pharyngeal denticles (J. A. Marshall, 1963), and this small species offers many possibilities as an experimental animal in this field. It is also probable that the sounds of priacanthids are produced in this manner (Salmon and Winn, 1966) . Almost any predatory species of fish is likely to produce sounds when feeding, and even some herbivorous forms that browse on sessile plants and animals can emit sounds when crushing rocks and corals. Incisor types of teeth are capable of biting through the exoskeletons of crustacea and thus produce strong metallic sounds (Fig. 4). Given any food of moderate hardness, the action of teeth will produce sounds. Sometimes fish will gnash their teeth without the direct presence of food. Fish (1954) even listed some sharks and rays that produce sounds when feeding, despite the fact that elasmobranchs are not known to be sound producers in any specialized sense ( Backus, 1963).

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WILLIAM N. TAVOLGA

Fig. 4. The sharp incisorlike teeth ( a ) of the triggerfish, Bulistes, can produce sounds during feeding. The erection of the dorsal fin spines ( b ) generates a stridulatory sound. After Tavolga (1965), with permission of the U. S . Naval Training Device Center.

Burkenroad (1931), Fish (1954), and Moulton (1958) listed a number of species that produce sounds by means of modified molariform teeth. Sound production of this type is particularly common among the members of the order Tetraodontiformes (puffers, filefish, etc.) and the family Scaridae (parrot fishes). Molariform teeth are also present in the family Sparidae (porgies), and Fish ( 1954) included the scup, Stenotomus chrysops, as a sound producer. Another common sparid is the pinfish, Lagodon rhomboides, whose sounds have been described by Burkenroad (1931) and Caldwell and Caldwell (1967). Knudsen et al. (1948) listed over a dozen additional species that were found to produce sounds when feeding. Stridulation by the movement of special fin rays and spines has been reported in a few species. The sea caffish, Gabichthys felis, and the gafftopsail catfish, Bagre marinus, produce high-pitched squeaks when the enlarged pectoral fin spines are moved ( Burkenroad, 1931; Tavolga, 1960), and several species of freshwater catfishes produce sounds in a similar fashion ( Pfeiffer and Eisenberg, 1965; SGrensen, 1894; Mahajan, 1963). The erection of the specialized dorsal fin spines in triggerfish produces a stridulation (Schneider, 1961) (Fig. 4), and it is possible that low intensity sounds may be produced by sticklebacks, Gasterosteus acubatus and Apeltes quadracus, when dorsal fin spines are moved (Fish, 1954). Sounds produced by the friction of adjacent bones were reported from the clown fish, Amphiprion, by Schneider (1961, 1964a) and for the sea horse, Hippocampus, by Fish (1953, 1954). In both these cases, the prob-

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able mechanism is the stridulation of the posterior margin of the skull against some vertebral element. A similar mechanism was described for the pipefish, Syngnathus louisiunae, by Burkenroad ( 1931). Klausewitz (1958) described sounds produced by the Indian loach, Both hymenophysu, which he attributed to the stridulation of Weberian ossicles, although this function of the Weberian ossicles should be considered highly doubtful.

2. SWIM BLADDER MECHANISMS-INDIRECT As noted above, Burkenroad (1930) observed that pharyngeal denticle stridulation in the grunts (family Pomadasyidae) was affected by deflation of the swim bladder. In many such cases, the swim bladder may act as a resonator and thus change the quality of the emitted sound. Stridulation of bones of the pectoral girdle has been found in a triggerfish, Balistes, by Schneider (1961). Bone stridulation in a sculpin, Myoxocephalus (Fish, 1954)) generates a sound at a point adjacent to the anterior end of the swim bladder. In a study on seven species of triggerfish, Salmon et al. (1968) found sounds were produced by the rubbing of pectoral fins against the body sides where skin thinly covered lateral evaginations of the swim bladder ( Fig. 5 ) . The swim bladder has also been thought to function literally as a drum. Triggerfish, Balistes, were reported to produce a sound by beating their pectoral fins against the areas of the body wall that cover the swim bladder (Moulton, 1958), and some species of serranids appeared to beat their opercula in sound production ( Fish, 1954; Tavolga, 1960 ) . Schneider

Fig. 5. Sound production in a triggerfish, Rhinecanthus rectagulus, takes place by rubbing the pectoral fins against the swim bladder: ( 1 ) pectoral fin spine, ( 2 ) drumming muscle, ( 3 ) pectoral fin rays, and (4)fleshy muscular lobe of pectoral fin. After Salmon et al. (1968).

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WILLIAM N. TAVOLGA

(1961), however, stated that the drumming in triggerfishes was probably produced only out of water. Hazlett and Winn (1962) described the sonic mechanism of a serranid, Epinephelus striatus, as consisting of a pair of sonic muscles (see below). 3. SWIM BLADDER MECHANISMS-GAS EXPULSION

In some fishes, where there is a pneumatic duct between the swim bladder and esophagus, sounds can be detected when air bubbles are eructed. Fish (1954) and Dufossk (1874) reported this type of sound production in some eels and catfishes. A characid fish, Glandulocauda inequalis, is reported to produce sounds by gulping air at the water surface (K. Nelson, 1965). 4. SWIMBLADDER MECHANISMS-EXTRINSICMUSCLES Among the best known of the sonic fishes are members of the drum and croaker family (Sciaenidae). The sonic mechanism consists of a pair of large muscles (sometimes present only in males) that are derived from the lateral body wall musculature (Fig. 6 ) . These muscles are contiguous to the lateral walls of the swim bladder, and their vibrations occur in short bursts producing a series of drumlike beats or knocking sounds. There are a number of variations in the size and position of the sonic muscles among the species of the family, but the fundamental arrangement is the same in all the sonic forms (Dijkgraaf, 1947a; Fish, 1954; Schneider, 1961, 1967; Schneider and Hasler, 1960; Tower, 1908). A type of sonic mechanism common to a wide variety of teleosts con-

Fig. 6. The swim bladder ( a ) in the drumfish, Aplodinotus, is vibrated by a pair of sonic muscles (b). Redrawn from Schneider and Hasler (1960) after Tavolga (19f%), with permission of the U. S. Naval Training Device Center.

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147

sists of a pair of short muscles that originate on the occipital region of the skull and insert on the ribs and connective tissue that forms the anterior-dorsal surface of the swim bladder (Figs. 7 and 8). This mechanism has been described in some detail for the Nassau grouper, Epinephelus striatus, the squirrelfish, Holocentrus rmfus, the tiger fish, Therapon, and a scorpaenid, Sebasticus, by Hazlett and Winn (1962), Winn and Marshall ( 1963), Schneider ( 196413 ), and Dbtu ( 1951), respectively.

Fig. 7. The sonic muscles ( b ) in the squirrelfish, Holocentrm, extend from the rear of the skull and insert on the first pairs of ribs. The ribs are tightly laced to the swim bladder ( a ) . After Tavolga ( 19sS), with permission of the U. S. Naval Training Device Center.

A modification and specialization of the above mechanism is present in some of the catfishes (Siluroidea). First noted by Muller (1842, 1843), the catfishes commonly possess a fused shelf of bone over the anterior and dorsal surfaces of the swim bladder. The many variations of this arrangement were described by Bridge and Haddon (1889, 1893). In some species, this shelf of bone is extremely thin and springlike and has

0

Fig. 8. The swim bladder ( a ) in the tigerfish, Therapon, is vibrated by a pair of sonic muscles ( b ) . Redrawn from Schneider (196413) after Tavolga ( 1%5), with permission of the U. S. Naval Training Device Center.

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W W M N. TAVOLGA

been called the “Springfederapparat” ( Muller, 1842) and “elastic spring” (Sdrensen, 1894). This structure is derived from the transverse processes of the first few vertebrae and was first reported to function in sound production by Sgrensen ( 1894) and later confirmed by Tavolga (1962). In marine catfishes, Bagre marinus and Galeichthys felis, the sonic mechanism has been described in some detail as consisting of a pair of sonic muscles that originate from the epiotic and neighboring bones of the skull and insert on the upper surfaces of the elastic spring. Vibration of these muscles causes the swim bladder to vibrate, and the elasticity of the bony shelf acts as the antagonist to the contraction of the sonic muscles ( Tavolga, 1962). Many species of fishes have been found to possess a pair of muscles connecting the skull and swim bladder, and, on the basis of morphology,

sb

I

idm

[

sb

Fig. 9. Swim bladder and presumed sonic mechanism of a brotulid, Monornitpus. Left: The complete mechanism showing t w o pairs of muscles originating on the rear of the skull and inserting on the ribs; upper right: insertion points of the sonic muscles on the ends of the ribs; lower right; side view showing attachment of sonic muscles to ribs and swim bladder. Key: idm and odm, inner and outer drumming muscles; I, 11, and 111, vertebrae with corresponding ribs, r l , r2, r3; sb, swim bladder; lsb, ligament; so, saccular otolith. After N. B. Marshall (1967), with permission of Pergamon Press.

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it is probable that these are sonic muscles. Such structures have been found in many brotulids, macrourids, and other deep-sea forms ( Walters, 1960; N. B. Marshall, 1967; Schneider, 1961) (Fig. 9). It is probable that future deep-sea exploration will identify these forms as sound producers. 5. SWIM BLADDERMECHANISMS-INTRINSIC MUSCLES In some families of fishes the sonic muscles are completely attached to the walls of the swim bladder. In such cases, the bladder can be dissected out of the body cavity and can function as a sound-producing mechanism just by stimulation of the nerves leading to the sonic muscles (Fig. 10). The sonic mechanism of the toadfish, Opsanus, is typical and probably the best known. The swim bladder and its role in sound production has been described by Tower (1908), Rauther (1945), Fish (1954), and Tavolga ( 1960, 1964b). The bladder is heart-shaped with its apex pointed

Fig. 10. Sound producing mechanisms in many species use a swim bladder with intrinsic sonic muscles: ( a ) Opsanus (Tower, 1908), ( b ) Porichthys (Greene, 1924), ( c ) Prionotus (Tower, 1908), ( d ) Trigla (Rauther, 1945), ( e ) Sebasticus (DBtu, 1951), ( f ) Zeus ( DufossB, 1874), ( g ) Dactylopterus (Dufosst., 1874). After Schneider ( 1967), with permission of Pergamon Press.

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WILLIAM N. TAVOLGA

posteriorly. Two broad bands of muscle are firmly attached along the lateral surfaces, with the fine, striated fibers running obliquely (Fig. 10a). The interior of the bladder contains an extensive vascular network and a pair of “red glands.” A thin transverse membrane with a small sphincter separates the secretory and absorptive chambers. The remarkable gas exchanging properties of this organ has been extensively investigated by Fange ( 1966) and Fange and Wittenberg ( 1958). The first experimental studies on the sonic function of the swim bladder of toadfish were those of Tower (1908). His observations, confirmed by Tavolga (1964b), showed that the fundamental frequency of the emitted sound is exactly equivalent to the vibration frequency of the sonic muscles. Thus far, although differences in the character of the cell exist among populations and different species of toadfishes, no structural differences in the swim bladder mechanism have been found to account for these variations in pitch, duration, and quality ( Tavolga, 1958c, 196413). In Porichthys, a genus of fish closely related to the toadfishes, the sonic mechanism appears to be virtually identical ( Greene, 1924) (Fig. lob). The sea robins (Triglidae) possess essentially the same mechanism of sound production as the toadfishes except for the shape of the swim bladder. In the sea robins (Trigla in Europe; Prionotus on our Atlantic coast), the swim bladder is composed of two dirigible-shaped chambers side by side, and attached to one another by a narrow passageway (Figs. 1Oc and 10d). The arrangement of the sonic muscles and their function appears to be similar to that of the toadfish (Moulton, 1960a; Moreau, 1876). A family related to the sea robins is the Dactylopteridae-the flying gurnards. Although little is known of their ability for sound production, the mechanism seems to be identical to that of the sea robins (Tower, 1908; Fish, 1954; Tavolga, 19641,) (Fig. log). Simultaneous electromyograms and sound recordings in the midshipman, Porichthys notatus, showed that sound pulses and muscle potentials corresponded in a one-to-one fashion (Cohen and Winn, 1967). 6. MECHANICS OF SWIM BLADDER SOUNDPRODUCTION If we consider the swim bladder and its associated skeletal and muscular systems as an underwater loudspeaker, the energy source for the emitted sound is the contraction of specialized muscles. In no case is there a muscular antagonist to the action of the sonic muscles. In the catfishes, the “elastic spring” maintains tension on the sonic muscles and returns the fibers to their normal condition after each contraction ( Tavolga, 1962). In the squirrelfishes and groupers, the sonic muscles

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insert on the first pairs of ribs. These muscles contract against the tension of hypaxial muscles, vertebral ligaments, and connective tissue lacings to the roof of the swim bladder (Hazlett and Winn, 1962; Winn and Marshall, 1963). In the sea robins and toadfishes, where the sonic muscles are intrinsic to the swim bladder, the elasticity of the bladder walls and the internal pressure of the distended bladder will tend to return the muscle fibers to normal length after each contraction. In the sciaenids, the return of the sonic muscles to normal shape after contraction is a function of the swim bladder tension, in part, and probably a function of the elasticity of the lateral body wall musculature. In catfishes, Tavolga (1962) showed that the fundamental frequency of the sound is a direct translation of the frequency of contraction of the muscle. Packard (1960) recorded sounds and muscle action potentials simultaneously in the pigfish, Congiopodus, from New Zealand, and he found that the contraction frequency coincided with the sound frequency. A similar observation was made by Barber and Mowbray (1956) in the sculpin and Schneider (1964b, 1967) in Therapon. Winn and Marshall (1963) found that each sound from a squirrelfish, Holocentrus rufus, consisted of up to 5 pulses, about 10 msec apart. This corresponds to a fundamental frequency of about 100 Hz. Muscle action potentials coincided with the pulses. Artificial stimulations of nerves leading to the sonic muscles can show that the muscles are capable of unusually rapid contraction and recovery cycles. In the gaff-topsail catfish, Bagre marinus, the sonic muscles took up to 12 sec to tetanize at 150 pulses/sec, and up to 3 sec at 200 pulses/ sec ( Tavolga, 1962). Using the same stimulating equipment as described earlier (Tavolga, 1962), the nerves leading to the sonic muscles were stimulated in the toadfish, Opsanus tau, the slender sea robin, Prwnotus scitulus, the squirrelfish, Holocentrus ascensionis, and the red hind, Epinephelus guttatus. Opsanus and Priorwtus muscles were found to be most resistant to tetanization, but all could be stimulated at pulse frequencies over 100/sec without tetanization ( Tavolga, 1964b). Among vertebrates, the occurrence of such fast-acting muscles is unusual. Extrinsic eyeball muscles are known to reach 350 contractions/ sec, but the majority become tetanized when stimulated at 50 pulseslsec. The sonic muscle of Opsanus has a contraction-relaxation cycle of 10 msec ( Skoglund, 1959), which would theoretically limit its response to about 100 contractions/ sec, but Skoglund's observations were limited to single twitches. Sonic muscles in the squirrelfish show fusion at frequencies above 200/sec, while normal white muscle begins to show summation at 50 and fusion at 100 pulses/sec (Gainer et al., 1965). Electron microscopy showed the presence of an unusually developed sarcoplasmic reticulum which Fawcett and Revel (1961) have related to the muscle's fast-acting

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properties. Swim bladder muscles in nonsonic fish may also possess this reticulum and its associated fast-acting properties ( Kilarski, 1964). In the catfishes, squirrelfishes, groupers, and sciaenids ( drumfish and croakers), the sonic muscles are characteristically red in color as a result of a high degree of vascularization, and this may enhance the resistance to tetanization and fatigue. Histological examination of the sonic muscles of these species showed uniformly thin fibers and numerous polyaxonal innervations ( Gainer and Klancher, 1965). The innervation of the sonic muscles shows features common to many species. In the catfishes, the sonic muscle is supplied by a branch of the occipital nerve (Tavolga, 1962). Except in certain Ostariophysi, there are normally two pairs of occipital nerves in teleosts. Their homology is uncertain, but they are presently thought to be homologous to the hypoglossal ( XIIth) cranial nerve of the tetrapods. Based upon dissections, stimulation experiments, and serial cross sections, the nerve supply to the sonic muscles was traced in the squirrelfish, toadfish, sea robin, and red hind. Both pairs of occipital nerves were found to innervate these muscles in all of the above species (Tavolga, 1964b). Except for the sciaenids, in which sonic muscles are supplied by spinal nerves (Schneider and Hasler, 1960), it appears that the innervation of the sonic muscles in widely divergent teleosts is the same and that the muscles in all these forms must be homologous structures, despite the gross differences in appearance and location (Tavolga, 196413). In the catfishes, if the swim bladder is damaged, deflated, or filled with water, the sound output of the sonic muscle and the elastic spring is greatly lowered, but the sound quality, i.e., its harmonic content, remains unchanged ( Tavolga, 1962). Winn and Marshall ( 1963) found that in the squirrelfish, they could no longer detect the sound if the swim bladder was completely filled with water. Partial deflation of the bladder reduced the sound amplitude but did not affect the fundamental frequency or other properties (also true for other species: Salmon et al., 1968). In toadfishes, if the swim bladder was partially deflated, the spontaneous grunting sounds continued to have the same spectral characteristics but showed a reduced amplitude by as much as 20 dB (Tavolga, 196413). It does not appear likely, therefore, that the resonating role of the swirn bladder is an important one, especially since the excess internal pressure in the swim bladders of most fishes is low (Alexander, 1959a,b). The pulsation of the bladder should be essentially that of a large air bubble and would be affected by the low compressibility of the medium and a variety of other physical factors. The net result is a damping of the resonance and a decrease in the resonant frequency. Based on the formula given by Meyer (1957), the resonant frequency of a bubble of air

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of radius 3 cm at atmospheric pressure would be about 100 Hz. If we treat the swim bladders of fishes as if they were spherical air bubbles, the bladder dimensions in most individuals of squirrelfish, red hind, etc., would give resonant frequencies in the 50-100-H~ range. Even such rough calculations indicate that the fundamental frequencies of most fish sounds are in the same order of magnitude as the resonant frequencies of the swim bladders. The resonance peak of fish swim bladders was found to be flat, with a maximum rise of about 3-4 dB (Tavolga, 1964b). Conceivably, this would enable the fish to gain some efficiency from resonance, but this gain would be quite small. Harris (1964) investigated the properties of mathematical and physical models of vibrating swim bladders. He concluded that the pulsating air bubble, such as a swim bladder, would have the elastic and resonant qualities for a highly efficient underwater sound producer in the low frequency range that fishes usually produce. The toadfish, for example, develops sound pressures of up to 35 dB pb at distances within 5 meters, and many species produce sounds powerful enough to be heard distinctly above the sea surface. An underwater loudspeaker was constructed along the physical principles of a toadfish swim bladder and was found to be quite efficient in the 100-2000-Hz range (Tavolga and Wodinsky, 1963). The mechanics of swim bladder sound production can be summarized as follows: The fundamental frequency of the sound emitted is a direct translation of the contraction frequency of the sonic muscles. The contractions of the sonic muscles produce volume and pressure changes in the swim bladder, and, therefore, the entire surface of the bladder pulsates. The fact that the natural frequency of the swim bladder as an air bubble is in the same order of magnitude as the vibration frequency of the sonic muscles undoubtedly aids the efficiency of the system, and the mechanical energy of the moving muscle is transmitted by way of air pressure changes to the entire bladder surface. These pulsations are transmitted through the tissues of the fish to the outside with little loss, since the fish is virtually transparent to water-borne sound. In acoustic and electronic terms, therefore, the swim bladder is an impedance-matching device analogous to the large surface cone of a high fidelity loudspeaker.

7 . HYDRODYNAMIC AND SWIMMING SOUNDS The movement of any object through the water will create displacement. Such displacements and compression waves may be rhythmic subsonic vibrations when produced by the fins and body of a swimming fish. Such phenomena have been classified as hydrodynamic sounds by Shishkova ( 1958a,b) and Moulton ( 1960b). Moulton also considered

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the possibility .that some of the sounds produced by swimming fishes are of internal origin, i.e., produced by muscular and skeletal movements within the animal. The most intense of these sounds are emitted when the animal turns rapidly or changes its velocity. In his review of fish propulsion, Nursall (1962) pointed out that the main thrust in a swimming fish arises from transverse movements of the contralateral waves. Turning is accomplished by a unilateral wave as the head of the fish turns, and the caudal portion is used as a fulcrum. It is clear that lateral displacement of the medium will arise during straight-line swimming, and these will tend to be rhythmic since the body movements are rhythmic. The turning of the head will produce a strong displacement of the medium. These displacements will, of course, result in compression waves, which can be detected as sound by most hydrophones. Skudrzyk and Haddle (1963) defined flow noise as turbulences and concomitant pressure fluctuations produced by the motion of a body through water. This is primarily a near-field phenomenon and, as such, would not be propagated over large distances. Although the above study was concerned with the noise generated by the motion of rigid objects, such as vessels, it is obvious that surface turbulence and eddies would be produced by swimming fish. The locomotion of fish, therefore, produces pressure and displacement effects in three possible ways: the more or less rhythmic effects of undulatory movement; the turbulence generated by flow noise; and, possibly, internally generated locomotor sounds. D. Characteristics of Fish Sounds

It has long been recognized that a verbal description of a sound is inadequate and often misleading. This problem has been especially troubling to observers attempting to describe sounds of animals such as birds and amphibians. With the advent of high fidelity tape recording equipment, the problem of preserving the data seemed to be solved, although precautions still had to be taken against artifacts resulting from overloaded amplifiers, incorrect equalization, and other electronic difficulties. Photographs of oscilloscope tracings of sounds were frequently used, and these analyses are still especially valuable for short, rapidly pulsed sounds. Fish (1954) presented her data on sounds of fishes in the form of frequency analysis graphs that were made with an octave band filter. An important improvement over the octave band filter was the de-

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velopment of the sound spectrograph. This instrument is now well known in many areas of acoustic research, including virtually all phases of bioacoustics. In brief, the sound spectrograph takes a sample of the sound and produces a graph of frequency against time. Other displays such as frequency against relative intensity are also possible. Many examples of its use in bioacoustics can be found in Lanyon and Tavolga (1960) and Busnel (1963). Spectrographic displays of various fish sounds are shown in practically all current publications in this field. As applied to underwater bioacoustics, the use of the sound spectrograph has been carefully evaluated by Watkins (1967). The capabilities and limitations of this instrument are often not fully understood by biologists. The display of apparent harmonics, for example, can result from repetition of pulses, and other complexities can be introduced if the sound consists of short, repeated bursts of pulses, with each pulse consisting of a brief tone or complex of tones. Often the so-called fundamental frequency is actually a pulse repetition rate. In addition, the verbal descriptions of bioacoustic phenomena have had to be standardized, and attempts at this standardization resulted in glossaries compiled by Broughton (1963) and Bondesen and Davis (1966), although only a minority of the terms they listed apply to the sounds of fishes. A serious source of difficulty in making original recordings of animal sounds is the effect of the reverberations, reflections, absorptions, and other acoustic phenomena in the environment. Field recordings are always plagued by such problems, including the presence of background noise. Such problems are particularly troublesome in underwater recordings. The spectral and other characteristics of sea noise and man-made noise have been summarized by Wenz ( 1964), but the acoustic properties of the ocean bottom, surface, suspended particles, and air bubbles are still under intensive study ( Albers, 1965; Richardson, 1957). The problems of recording sounds of captive fishes are even more complex, and the acoustic field generated in an aquarium tank, for example, virtually defies analysis (Parvulescu, 1964, 1967). Any spectral analysis of an underwater sound, therefore, must be cautiously interpreted, especially if the acoustic conditions are not controlled and not specified (Schneider, 1967; Tavolga, 1965). These difficulties are especially evident in attempts to identify unknown sound sources. Sounds of marine animals are often recorded without any visual confirmation of the source of the sound, and it is tempting to make comparisons between such field recordings and recordings of known species in captivity. This problem was discussed in detail by Tavolga ( 1965). Tables of data on pitch and other characteristics of fish sounds are

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given in papers by Fish et al. (1952) and Fish (1954). The tables are based primarily on recordings made under laboratory conditions, and, in many cases, the stimulus for sound production was some noxious stimulus such as electric shock, These data, therefore, must be treated cautiously and specific conclusions as to sonic characteristics of a given species need to be corroborated with field recordings under natural conditions. Much of the following discussion is based upon reviews by Fish ( 1954, 1964), N. B. Marshall (1962), Moulton (1958, 1963), Schneider ( 1967), Tavolga (1960, 196413, 1965), and Winn (1964).

SOUNDS 1. STRIDULATORY Sounds produced by the grating of pharyngeal denticles, jaw teeth, fin rays, or bones are essentially nonharmonic in structure, i.e., they do not resolve, in a spectrogram, into a series of horizontal parallel bars at harmonic intervals. Such sounds contain many harmonically unrelated frequencies (Fig. 11). Two general categories of stridulatory sounds can be distinguished on the basis of predominant pitch. High frequency stridulations are usually produced by the gnashing of jaw teeth and, sometimes, the patches of pharyngeal denticles during feeding. The component frequencies extend continuously from below 100 to over 8000 Hz. Predominant frequencies are in the 1000-4000-Hz range, and durations are extremely variable.

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Fig. 11. Spectrogram of stridulatory sounds produced by a jack, Carunx. A frequency-intensity section shown on the right was taken at the point indicated by the arrow. This is characteristic of short, broad-band pulsed sounds. After Tavolga (1965),with permission of the U. S. Naval Training Device Center.

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Such sounds have been described verbally as rasps, scratches, clicks, chirps, and scrapes. The second category of stridulatory sounds are those in which the swim bladder plays some part in determining the quality of the sound. Verbally, the sounds have been called grunts, croaks, thumps, knocks, etc. The frequency range is generally from 1000 to 8000 Hz, and the predominant frequencies are below 1000 Hz. Comparison of the analyses made by Fish (1954), Moulton (1958), and other authors shows a considerable divergence of data as to frequency range and predominant pitch. It is probable that the different types of equipment, different methods of eliciting the sonic behavior, and, most important, the different conditions under which recordings were made can account for the discrepancies in the data. 2. SWIM BLADDER SOUNDS Sounds produced by the vibration of muscles around or attached to the swim bladder are usually recognizable by their harmonic structure. If accompanied by stridulation, the harmonics can sometimes be partially masked in a spectrogram, but to the ear these sounds seem vibrant and possess a tonal quality. A number of parameters of such sounds can be defined and measured by the use of spectrographic and oscillographic analysis. The fundamental and predominant frequencies usually can be determined. If the sound is a sustained call, its duration usually varies within narrow limits. Often the sound consists of a number of rapidly repeated pulses, and in such instances it is necessary to know the duration of each pulse, the pulse repetition rate, and the usual number of pulses within the sound complex. The number and relative strengths of harmonics is extremely variable. In studies on the sounds of marine catfish (Tavolga, 1962), it became evident that the harmonic components could be varied by changing the character of the surroundings, the type of hydrophone, the distance from the second source, and the recording and analyzing levels. The hypothesis was advanced that the actual emitted sound was virtually a pure tone. In the process of listening to the boat whistle calls of toadfish, Opsanus tau (Fig. 12), Tavolga ( 1964b) recorded a long series of sounds emitted by single individuals that remained in the same position for many hours. Several environmental variables were found to affect the number and relative intensities of harmonics, e.g., the distance of the hydrophone from the sound source was of prime importance. Schneider (1967) found that the construction material of an aquarium tank affected the quality of the recorded sounds of Therapon. The midshipman, Porichthys notutus, is also a member of the toadfish

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Fig. 12. Spectrogram of a boat-whistle sound of a toadfish, Opsanzls. The fundamental frequency of this harmonic sound is about 250 Hz, and the frequencyintensity section on the right was taken at the point indicated by the arrow. After Tavolga (1965), with permission of the U. S. Naval Training Device Center.

family (Batrachoididae) and is known as a sound producer (Greene, 1924). Sounds recorded both in the field and in the laboratory were either short grunts or long buzzes (Cohen and Winn, 1967). Both types of sounds consist of bursts of sound pulses. To use the terminology recommended by Watkins (1967), the pulse frequencies were 175-200 Hz, the pulse repetition rates were about 50/sec, and the burst durations varied from about 77 msec (grunts) up to 3 sec (buzzes). The short bursts sometimes occurred in trains. Unlike the toadfish, the majority of fish swim bladder sounds are short pulses, with a fundamental frequency of from 75 to 100 Hz. This is true for the members of the Sciaenidae, the largest family of sonic fishes, including croakers and drumfish. Fish (1954) described the sounds of several species, primarily under captive conditions, and Kellogg ( 1955) presented several recorded examples. The sounds of the squirrelfish, Holocentrus ( Winn and Marshall, 1963) (Fig. 13), triggerfishes of several species (Schneider, 1961; Moulton, 1958; Vincent, 1963a), some of the groupers and sea basses (Fish, 1954; Tavolga, 1960; Hazlett and Winn, 1962), the sea catfish (Tavolga, 1962) (Fig. 14), and many other species fall into this acoustic category. Some species produce sounds of lower fundamental frequency, as, for example, the codfish and haddock with a fundamental of 40-50 Hz ( Brawn, 1961; A. D. Hawkins and Chapman, 1966). Others exhibit higher

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Fig. 13. Spectrogram of a burst of eight sound pulses produced by a squirrelfish, Holocentrus. The fundamental frequency within each pulse is about 75-100 Hz. After Tavolga (1965), with permission of the U. S. Naval Training Device Center.

frequencies. The yelps of the gaff-topsail catfish range over 250 Hz (Tavolga, 1962), and the calls of toadfish may reach above 300 Hz ( Tavolga, 1 9 5 8 ~ ) . Some figures on the duration of swim bladder sounds in various species

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Fig. 14. Spectrogram of two gruntlike sounds produced by a marine catfish, Galeichthys. These are short pulses with a fundamental frequency of about 200 Hz. After Tavolga (1965), with permission of the U. S. Naval Training Device Center.

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were given by Moulton ( 1958), Tavolga ( 1960), and Winn ( 1964) in spectrograms. In the majority of species, the sounds consist of single pulses with a duration of 20-100 msec each. Some forms characteristically produce a train of such pulses, and the repetition rates are probably species-specific. Sounds of the black grouper, Mycteroperca bonaci, usually occur in volleys of 4 or 5 pulses each, while other species normally emit one sound pulse at a time (Tavolga, 1960). Squirrelfish, Holocentmcs, produce rattling volleys of up to 20 pulses in quick succession (Winn and Marshall, 1963). Many species of croakers and drumfish also produce such trains of pulses, and Winn (1964) classified such emissions as “iixed-interval signals.” He proposed that this kind of temporal patterning can serve as a primitive means of communication. Long, sustained tones are unusual. The boat-whistle sounds of toadfish vary from 350 to 450 msec in length, and occasional yelps of the gafftopsail catfish may reach 500 msec in duration (Tavolga, 1960). The low-pitched pulses of many species are extremely difficult to distinguish from one another on the basis of acoustical characteristics alone. Harmonic structure is greatly affected by the conditions of recording and the equipment used. There is little known as to the consistency with which certain species produce characteristic pulse trains. The problem of identification of sound sources, therefore, is one that will require considerably more data than are now available.

3. HYDRODYNAMIC AND SWIMMING SOUNDS The character of sounds produced by the motion of fishes through the water has only recently been recognized and described. Thus far, Moulton (1960b) has been the only investigator to report any spectral analyses on such sounds. He has found that the sounds are nonharmonic with the dominant frequencies extending far below 100 Hz. The main sound output from individual or schooling fish occurred when there was a rapid change in direction or speed. The sound resembled a low roar or that of a wooden mallet striking the side of a boat under water. Such sounds could be detected from single predatory fish such as jacks or barracuda (Fig. 15). Sounds of veering schools of sardines, herrings, and anchovies were of lower amplitude and appeared to have more high frequency components (Tavolga, 1964b). The pressure fluctuations produced by flow noise are affected by a wide variety of factol’s such as surface roughness, shape, and velocity. The predominant frequencies, upon spectral analysis, are in the range below 500 Hz, and the frequencies below 100 Hz are least affected by changes in the above variables (Skudrzyk and Haddle, 1963).

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Fig. 15. Spectrogram of a hydrodynamic sound prpduced by a small school of jacks, Caranx. The sound begins almost explosively and contains many harmonically unrelated frequencies, mostly below 200 Hz. After Tavolga (196Fi),with permission of the U. S . Naval Training Device Center.

All of these hydrodynamic phenomena generate primarily near-field displacements and, with the usual pressure-sensitive hydrophones, can be detected only at short range and when emitted at a high intensity. 4. SOUNDSOF UNKNOWNMECHANISMS Tavolga (1956, 1958a,b, 1960) reported sounds produced by several species of small tidal zone fishes-gobies and blennies. These were low frequency thumps with a fundamental well below 100 Hz and with no harmonic distribution of frequencies. Acoustically these sounds resemble hydrodynamic pulses. Kinzer (1961) and Protasov et al. (1965) described similar sounds from European species of gobies. Tavolga (1960) proposed that a possible sonic mechanism here might be an explosive ejection of water through the gill slits. Romanenko and Protasov (1963) found that the beluga sturgeon, Huso huso, produced at least four kinds of sounds: (1) whistles with a dominant frequency of about 3800 Hz, ( 2 ) a broad-band hissing, ( 3 ) sharp pulses with a dominant frequency of 100-125 Hz, and ( 4 ) short clicks with acoustic energy predominant around 4000 Hz and possibly higher. The electric ray, Torpedo marmorata, was reported to produce low frequency grunts (below 100 Hz) in connection with its electrical dis-

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charges (Vincent, 1963b). Despite efforts to electrically isolate the hydrophone, the acoustic nature of these signals is still doubtful. If true, this report could become the only definite record of an elasmobranch producing sounds other than hydrodynamic ones. 111. SOUND DETECTION

A. Historical Background Since fish have been known, from early times, to be capable of sound production, it was logically assumed that they could also hear. The earliest study of significance in this field was the classic report by Weber (1820) in which he not only described the morphology of the fish ear but also postulated the function of the small ossicles that in the Ostariophysi connect the swim bladder with the inner ear. He compared these structures, since known as the Weberian ossicles, to the middle ear bones of mammals and concluded that they functioned in a similar fashion, i.e., to conduct sound from the swim bladder to the fluids of the inner ear. It was not until after the turn of the century that the ability of fish to hear was finally established. Several experimenters, notably Kreidl (1895), had concluded that fish were deaf or, at best, could receive some vibrations through a cutaneous sense. However, the investigations of Bigelow (1904), Parker (1902, 1910a,b, 1918), and Parker and van Heusen (1917) proved conclusively that fish receive sound stimuli both through the inner ear and the lateral line. Numerous reports confirming Parker’s conclusions followed, in which many other species of fish were shown to possess the ability to detect water-borne sound. The significant studies and reviews of the time included those by Bull ( 1928,1929, 1930), Evans ( 1935), von Frisch ( 1923, 1936, 1938a), Froloff (1925), LafiteDupont ( 1907), McDonald ( 1922), Marage ( 1906), Moorhouse ( 1933), and Warner (1932). The first attempts at any quantitative study of fish hearing consisted of determinations of the highest frequency to which fish would respond. In a summary of the results of most of these studies, it is clear that the upper frequency limits of members of the Ostariophysi are significantly higher than representatives of other orders (cf. Tables in Lowenstein, 1957, and Moulton, 1963). In most of the listed reports, little or no data were given on the intensities of the signals used, and, indeed, in many cases the sound stimuli were poorly controlled, e.g., whistles, plucked strings, and other crude sound makers. Stetter (1929) was the first to control stimulus intensity and attempt to measure it.

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The relation of the inner ear to sound reception was demonstrated by means of extirpation techniques. Manning (1924) and von Frisch and Stetter (1932) localized the main sound detection sense in the sacculus and lagena, and von Frisch (1938b) further demonstrated the importance of the Weberian ossicles in transmitting the sound from the swim bladder to the sacculus. The morphology of the inner ear and swim bladder of fishes has been extensively studied, beginning with the report of Weber (1820) and detailed works by Bridge and Haddon (1893, 1889) on the Weberian apparatus of siluroid fishes. The 1930 decade seemed to be one of the most productive periods, with many major anatomical contributions (de Burlet, 1934; Farkas, 1938a,b; Froese, 1938; Tomaschek, 1936, 1937; Wohlfahrt, 1938). The report by Froese (1938) covered 68 species in 42 families and stressed the various types of connections existing between the swim bladder and the inner ear. This connection and its possible function in clupeid fishes was explored by Wohlfahrt (1936, 1938) and in mormyrids by Stipetih (1939). The course of the VIIIth nerve and its connections with the inner ear in fishes was described by Pearson (1936), including data on the connection of Mauthner‘s cells and the swim bladder. By means of electrophysiological techniques, the microphonic response of the fish labyrinth and action potentials in the VIIIth nerve were demonstrated (Adrian, 1938). Controversy as to the function of the lateral line system has existed for a long time. Parker (1902, 1905) theorized that the lateral line system is capable of detecting only shock waves, currents, and possibly low frequency sounds. Whether these periodic and nonperiodic phenomena can be called sound, and their detection hearing, depends upon one’s definitions of sound and hearing. This question is related to the distinction between near-field and far-field acoustics and is taken up elsewhere in this chapter. Several investigators confirmed Parker’s contention that the lateral line is a low frequency sound (sensu latu) detector (Rode, 1929; Schriever, 1936). This property of the lateral line appeared to be associated with the ability of a fish to orient itself with respect to obstacles and sources of water currents. Although Reinhardt (1935) denied that fish were capable of localizing sound sources, Dijkgraaf (1934) and von Frisch and Dijkgraaf ( 1935) demonstrated clearly that fish could orient with respect to obstacles, nearby sources of water movements, and nearby low frequency sound sources. Some sort of cutaneous sensory system was postulated, and the lateral line system was considered a strong possibility in such orientations (Sand, 1937). The innervation of the lateral line system including head canals, indicates strongly that its function must be in some way related to that of

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the ear, i.e., in some form of vibration detection. Pearson (1936) traced out the course of the VIIIth nerve and showed that the peripheral sensory supply to the lateral line system was derived from the acoustic nerve VIII. In spite of this report, and the earlier descriptions of Herrick ( 1898), some recent textbooks and manuals of comparative anatomy still give the innervation of the lateral line and head canals as the facial (VII), glossopharyngeal ( IX), and vagus ( X ) nerves, based upon gross anatomical observations. This error has been pointed out most recently by van Bergeijk ( 1967a).

B. Mechanisms of Sound Detection 1. INNER EAR The morphological aspects of sound detection in fishes will be treated briefly here. The structure of the labyrinth, including the inner ear, has been reviewed in some detail by Grass6 (1958) and Moulton (1963), and will be covered by Lowenstein in another chapter in this volume. The pars superior consists of the semicircular canals and associated ampullae, and the pars inferior consists of the sacculus and lagena, each of which contain an otolith (Fig. 16). The earliest studies in which sound detection function in fishes was localized to the labyrinth were the extirpation experiments of Manning ( 1924) and von Frisch and Stetter (1932). Pearson (1936) described the central connections of nerves from the inner ear and postulated that the coarse fibers from the saccular root transmit sonic stimuli. Von Frisch ( 1938b) described the connection of the Weberian ossicles in Phorinus as transmitting vibrations from the swim bladder to the saccular otolith (sagitta) (Fig. 17). He also stated that the lagenar otolith can receive sonic stimuli by way of bone conduction. In Lebistes, which lacks a Weberian apparatus, Farkas (1938a,b) reported that the sagitta is the

Fig. 16. Simplified diagrams of inner ears of fishes: ( a ) the “typical” form and ( b ) the ostariophysine form. Redrawn from von Frisch (1936), after Tavolga (1965), with permission of the U. S. Naval Training Device Center.

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Fig. 17. Members of the order Ostariophysi are characterized by the presence of the Weberian apparatus. The swim bladder ( c ) serves as the primary transducer and vibrates (see arrows) in response to an impinging pressure wave; the vibration is transmitted to the largest of the Weberian ossicles, the tripus ( b ) ; the tripus is coupled to the fluids of the inner ear ( a ) through three additional ossicles (intercalariurn, scaphium, and claustrum). Redrawn from von Frisch (1938b), after Tavolga (1965), with permission of the U. S. Naval Training Device Center.

otolith that receives vibrations through the fenestra sacculi. The most definitive work was that of Dijkgraaf (1949, 1952a) who demonstrated that the auditory function of the inner ear resides in the sacculus and lagena, although the contribution of the lagena to sound detection is as yet not clear. Electrophysiological techniques were used by Zotterman (1943) to locate auditory reception in the macula sacculi. Similar results were obtained by Lowenstein and Roberts (1951) in elasmobranchs, but in addition they were able to detect response potentials from the utriculus. The electrical activity of single sensory nerve fibers of the acoustic nerve of the sculpin, Cottus scorpius, was detected and analyzed, and at least four types of neurons were identified on the basis of electrical activity in response to sound (Enger, 1963). The most recent neurophysiological studies on the inner ear of fishes have confirmed the preeminence of the sacculus as the acoustic detector. In the goldfish, saccular hair cells were found to be divided into a dorsal and a ventral group, with the kinocilia of the hair cells oriented at 180" in the two groups (Flock and Wersall, 1962). Two groups of afferent nerve fibers are present, one from the anterior and the other from the posterior sections of the sacculus, and the associated hair cells, including

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those of the lagena, were shown to respond to both vibratory and static bending ( Furukawa and Ishii, 1967a,b). Using neurophysiological techniques, some determinations of hearing capacities have been made in herring, Clupea, codfish, Gadus, and sculpin, Cottus, by Enger (1963, 1967a) and Enger and Andersen (1967). The connections between acoustic nerve fibers and Mauthner's neurons were reviewed by Moulton and Dixon (1967), together with extensive discussion and evidence for the relation of these connections to directional responses to sound in fishes. 2. SWIM BLADDER AND HEARING According to Griffin (1950, 1955) and Pumphrey (1950), a fish is essentially transparent to water-borne sound and its only acoustic discontinuity is the swim bladder (or other air chamber). Sound reception under water requires the presence of a transducer constructed of material very different in acoustic properties and density from the surrounding medium. Air bubbles are known to be excellent reflectors and resonators (Horton, 1959; Meyer, 1957) and certainly the swim bladder can serve efficiently as a transducer. Jones and Pearce (1958), N. B. Marshall (1951), and Midttun and Hoff (1962) have shown that fish swim bladders are effective sonic reflectors and that 50% or more of impinging sound energy is returned by the bladder, while a smaller percentage is reflected by the rest of the fish's body. Kleerekoper and Roggenkamp (1959) demonstrated that damage to the swim bladder in the catfish, Zctalurus,raised thresholds by 20 dB or more. It is quite probable, however, that some portions of the fish, such as the skull, may also serve as acoustic discontinuities and thus permit sound reception by bone conduction, although the swim bladder still appears to be the most obvious and efficient sonic transducer that the fish possesses. If the above is correct, then fishes with swim bladders should have better hearing than those without. Furthermore, those species in which the swim bladder is acoustically coupled to the inner ear should have the highest auditory sensitivity and broadest range. It appears that the Ostariophysi possess the lowest auditory thresholds and highest upper frequency limits. This is undoubtedly a function of the Weberian apparatus which couples the auditory signal received by the swim bladder to the inner ear in a manner analogous to the operation of the middle ear ossicles in mammals. Other air chambers can serve in similar fashion, as, for example, the branchial cavity in the labyrinthine fishes ( Schneider, 1941). Among nonostariophysines, there are a number of forms in which the

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swim bladder has anterior extensions which are either coupled directly to the perilymphatic fluid (as in many clupeids) or attached to the occipital region of the neurocranium (Froese, 1938; Grass&,1958; Tracy, 1920). Wohlfahrt ( 1936, 1938) described long, thin anterior extensions of the swim bladder in clupeids. These terminate in gas-filled capsules enclosed in bone and coupled to the perilymph through an elastic fenestra. Although auditory thresholds using psychophysical methods are not yet available for any clupeid fishes, Enger (1967a) obtained action potentials generated in the medulla in response to acoustic stimuli in the herring, Clupea harengus. The tentative audiogram so generated showed a rather flat frequency response over a range of 30-1200 Hz with a threshold of -20 to -25 dB pb, and a sharp increase to +35 dB pb at 4000 Hz. In contrast to the neurophysiological technique used by Enger (1967a), a number of workers have resorted to conditioning techniques in which the fish are trained to make some behavioral response in the presence of the test sound. By means of avoidance conditioning, Tavolga and Wodinsky (1963) showed that the squirrelfish, Holocentrus ascensionis, has a low threshold and broad frequency response spectrum. It is probable that this is related to the contiguity of the anterior end of the swim bladder to the skull, as described by E. M. Nelson (1955). Species with reduced or absent swim bladders should have poor hearing, but the evidence for this is sparse. Bull (1928) was unable to condition a blenny, Blennius, to respond to sound. In Gobius, Dijkgraaf (1949, 1952b) showed an upper frequency limit of only 800 Hz, and he postulated that most sound reception in this species took place through lateral line or cutaneous tactile senses. On this basis, sharks and other elasmobranchs should be virtually deaf, yet the studies of Kritzler and Wood ( 1961) and D. R. Nelson (1967a) and the electrophysiological work of Lowenstein and Roberts (1951) show this is not true. The validity of the statement that the fish is acoustically transparent needs to be reexamined, and the possibilities that the skull, vertebral column, and other portions of the body can act as acoustic discontinuities should be investigated. Furthermore the contribution of the lateral line to hearing needs clarification. 3. LATERAL LINEAND HEARING Fish possess two sensory modalities for the detection of underwater vibrations. In addition to the inner ear, they possess a series of integumentary sense organs collectively known as the lateral line system (Fig. 18). This system will be covered in detail by Flock in Chapter 8 of this volume. Most of the anatomical studies on the lateral line in fishes

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Trunk canal system

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Fig. 18. Lateral line system of the surfperch, Hyperprosopon. After Walker ( 1967). Reprinted from “Lateral Line Detectors” edited by Phyllis Cahn, copyright @ 1967 by Indiana University Press. Reprinted by permission.

have been summarized by Disler (1960), and the microscopic anatomy of the individual receptors was described by Dijkgraaf (1952a), Cahn and Shaw (1962), and many other authors (cf. Cahn, 1967) (Figs. 19 and 20). As reviewed by Dijkgraaf (1963a), some of the early literature

Fig. 19. Schematic of a neuromast unit of the lateral line system. Key: cu, cupula; ki, kinocilium; mc, mantle cell; ne, nerve ending; nf, nerve fiber; sen, sensory cell; st, stereocilium; and sup, supporting cell. After Iwai (1967). Reprinted from “Lateral Line Detectors” edited by Phyllis Cahn, copyright @ 1967 by Indiana University Press. Reprinted by permission.

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Fig. 20. Idealized cell of the acoustico-lateralis system. The position of the kinocilium determines the axis of sensitivity of the cell, and displacement in the direction of the arrow results in action potentials in connecting nerve fibers. After van Bergeijk (1967a), with permission of Academic Press.

on the function of the lateral line was contradictory, but strong indications were that it responds to minute water currents and to frequencies below 500 Hz (Kleerekoper and Roggenkamp, 1959). Elcctrophysiological studies by Suckling and Suckling (1950), Suckling (1962), Harris and van Bergeijk (1962), Jielof et al. ( 1952), and Kuiper (1956) have established the fact that the lateral line functions as a tactile receptor specialized for the detection of water displacements. Dijkgraaf ( 194713, 1967) developed the idea that the lateral line could function as a sort of low frequency sonar system to detect the presence of nearby obstacles. Acoustic energy is present in two forms: a pressure wave and a displacement. The pressure wave (far-field) is usually measured by a hydrophone, and its intensity drops off as the square of the distance from the source. Displacement (near-field) is detectable only close to the sound source since its intensity drops off as the cube of the distance. Harris and van Bergeijk (1962) demonstrated the importance of the near field in acoustic detection by fishes. They showed that the lateral line is strictly a near-field receptor, and operates only at low frequencies. An effective near field would exist at distances of about one-sixth of a wavelength, as calculated by van Bergeijk (1964).

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It is clear from the neurophysiological studies of Kuiper (1956), Jielof et al. (1952), Suckling and Suckling (1950, 1964), and others that the lateral line organ is a displacement detector, with a threshold in the order of magnitude of about 20 A (Harris and van Bergeijk, 1962). Based on additional behavioral studies, Dijkgraaf ( 1963a, 1964, 1967) concluded that these structures primarily respond to “current-like water disturbances.” He also made the point that the lateral line organs are not acoustic detectors, i.e., they do not respond to a propagated pressure wave. Many behavioral studies, however, have referred to the lateral line as responding to “sound.” As pointed out by van Bergeijk ( 1 9 6 7 ~ )Dijkgraaf‘s ~ original contention (1934) that the lateral line organ can only detect water motion is perfectly correct. However, the near-field effect is an inseparable component of acoustic energy, and the entire lateral line system acts as a near-field acoustic detector. The functional similarity of the inner ear and the lateral line can be illustrated by the following: (1) Both inner ear hair cells and lateral line organs have a fundamental structural similarity, even to the polarity of response and arrangement of kino- and stereocilia (Fig. 20); ( 2 ) both inner ear and lateral line are supplied by branches of the acoustic nerve; (3) both are essentially displacement detectors (van Bergeijk, 1967a). The lateral line system remained a near-field detector, while the inner ear, by virtue of the proximity of the swim bladder, became a far-field detector (van Bergeijk, 1967a). Both can be considered as acoustic sense organs. C. Hearing Capacities 1. PSYCHOPHYSICAL STUDIES

In the majority of studies on teleostean auditory capacities, the objective was to determine the upper frequency limits to which the animals could respond. Attempts have been made in only a few studies to measure absolute intensity thresholds ( Autrum and Poggendorf, 1951; Diesselhorst, 1938; Kritzler and Wood, 1961; Maliukina, 1960; Poggendorf, 1952; Stetter, 1929; von Boutteville, 1935). In most of these reports, only one or a few selected frequencies were actually tested. With the exception of the work of Griffin ( 1950), the intensity measurements were only approximations. Griffin’s determinations were based upon measurements taken with calibrated hydrophones, amplifiers, and decibel meters. For most of the ostariophysine fishes tested, the auditory thresholds were low, in the order of -40 to -60 dB pb, and the most sensitive frequency range was about 100-1500 Hz (Stetter, 1929; Diesselhorst, 1938; von Boutteville, 1935; Dorai Raj, 1960b; Autrum and Poggendorf, 1951;

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Kleerekoper and Roggenkamp, 1959). Upper frequency limits for these fishes were given as over 7000 Hz (Farkas, 1936; Kleerekoper and Chagnon, 1954; von Boutteville, 1935; von Frisch, 1938b). The first attempt to determine a complete audiogram for a fish was done with the bullhead, Zctalurus nebulosus, by Poggendorf ( 1952) (Fig. 21). In recent years, audiograms for two additional ostariophysine species have been reported: the goldfish, Carassius uurutus, by Enger (1966, 1967b), Jacobs and Tavolga ( 1967), and Weiss ( 1967, 1969); and the Mexican blind characin, Astyanux mexicanus, by Popper. ( 1970). A comparison of the three audiograms obtained for the goldfish shows some interesting results of different techniques. Enger (1967b) used a positive reward conditioning ( Fig. 22) , while Jacobs and Tavolga ( 1967) (Fig. 21) and Weiss (1967) (Fig. 23) used different forms of avoidance conditioning. Enger used a loudspeaker in the water and also in air; Jacobs and Tavolga used a loudspeaker in air, but in an enclosed chamber such that the backwave of the speaker was damped out; Weiss used a tank with two large speaker surfaces at the ends, with the speakers operating 180" out of phase. The quality of the stimulus in each of these experiments was somewhat different. The Enger underwater-loudspeaker

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Fig. 21. Comparison of audiograms for four species of teleost fishes, Goldfish, Carassius auratus, from Jacobs and Tavolga ( 1967); brown bullhead, lctalurus nebulosus, from Poggendorf ( 1952 ) ; blue-striped grunt, Haemulon sciurus, from Tavolga and Wodinsky ( 1965); and squirrelfish, Holocentrus ascensionis, from Tavolga and Wodinsky (1963).

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Fig. 22. Audiogram for the goldfish using air and underwater loudspeakers: ( 0 )loudspeaker in air and (0) loudspeaker in water. After Enger (1967b). Reprinted from “Lateral Line Detectors” edited by Phyllis Cahn, copyright @ 1967 by Indiana University Press. Reprinted by permission.

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Fig. 23. Audiogram for the goldfish obtained with an essentially near-field subject 5, and (0) subject 6. After stimulus: ( V ) subject 2, ( A ) subject 3, (0) Weiss ( 1967). Reprinted from “Lateral Line Detectors” edited by Phyllis Cahn, copyright @ 1967 by Indiana University Press. Reprinted by permission.

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system undoubtedly produced a complex of acoustic energy involving both pressure waves and displacement. The Enger air-loudspeaker system was even more complex acoustically, since the speaker was close enough to the water surface to induce near field, and the energy from the back of the speaker was not damped. The Jacobs-Tavolga system was based essentially upon the suggestions of Parvulescu (1964, 1967) and van Bergeijk (1967a), and was an attempt to attain a pure pressure field, although its “purity” could not be measured. The Weiss system produced a push-pull effect in the water and probably approached a pure near-field condition. Weiss (1967, 1969) described his apparatus as producing a “uniform sound field,” but he presented no data to validate this point. If by uniformity he meant equal sound pressure at all points, this would be a remarkable achievement, especially in a small tank. Although he made no measurements of displacement, he claimed to be able to separate inner ear from lateral line reception. By contrast, Cahn et al. (1969) obtained separate pressure and velocity (displacement) measures in a tank similar to Weiss’s, and they found variations in the pressure levels at digerent part of the tank over a range of about 10 dB. At lo00 Hz, Enger’s and Jacobs and Tavolga’s data were roughly in agreement, but Weiss’ thresholds were about 20 dB higher. All the audiograms were essentially flat over the 20&1000-Hz range, except Enger’s air-loudspeaker data, which showed a significant rise in threshold at 200 Hz. Above 1000 Hz, Enger obtained thresholds of about -10 dB pb at 5000 Hz, while the other two audiograms rose abruptly to about f 3 0 dB pb at 3000 Hz. A factor in threshold studies that must be considered is the level and spectrum of background noise in the experimental tank or chamber. Tavolga (1967b) demonstrated that masking noise effects in fishes are essentially similar to those in human hearing studies (Fletcher and Munson, 1937). In general, if the thresholds are 10 dB or more above the noise level in the region of the test frequency, the probability is high that the threshold values obtained are not masked and are unaffected by the ambient noise. The threshold data obtained by Jacobs and Tavolga (1967) gave values that were at least 10 dB higher than the noise level in the appropriate band, and at least 30 dB higher than the spectrum level (in noise per cycle). Unfortunately, Enger (196713) and Weiss (1967) gave no data on ambient noise levels. With so many differences in conditioning techniques, acoustic circumstances, and, above all, the lack of comparable noise level data, it is not possible at this time to bring all the above results into consonance. However, Wodinsky (1969) found that the high thresholds obtained by Weiss

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resulted from the omission of shock during the testing trials, and extinction of the response took place. It is possible, therefore, that Weiss’s data do not represent auditory thresholds at all. Such differences in technique may also explain why thresholds obtained by Weiss et al. (1969) on Ictalurus nebubsus were 10-20 dB higher than those reported earlier by Poggendorf ( 1952). Fishes without a Weberian apparatus have not been tested as extensively as the ostariophysines. However, the mormyrids and labyrinthine fishes possess air chambers directly coupled to the perilymphatic fluid and inner ear. The studies of Diesselhorst (1938), StipetiL (1939), and Schneider (1941) showed upper frequency limits of over 3000 Hz for mormyrids, with a threshold of -50 to -40 dB pb at 258 Hz (Diesselhorst, 1938). Schneider ( 1941) reported upper frequency limits of 4500 Hz for certain labyrinthines. Upper frequency limits in most other nonostariophysine species are considerably lower. Table I1 summarizes the majority of reports. Complete audiograms obtained for several marine teleosts were reported by Tavolga and Wodinsky (1963, 1965) and Wodinsky and Tavolga ( 1964) (Fig. 24). Based upon data on nine species from Bahamas waters, the most sensitive range was 200-600 Hz, with average thresholds of about 0 dB pb. The snappers (Lutjanidae), grunts (Pomadasyidae), sea basses, and groupers (Serranidae) tended to show thresholds of +lo to +20 dB pb in the 300-600-Hz range. Above this range, the threshold Table I1 Upper Frequency Limits

Genus and family

Gobius (Gobiidae) Corvina (Sciaenidae) Corvina (Sciaenidae) Sargus (Sparidae) Anguilla (Anguillidae) Lebistes (Poeciliidae) Lebistes (Poeciliidae) Mugil (Mugilidae)

A nguilla (Angaillidae) Mugil (Mngilidae) Corvina (Sciaenidae) Corvina (Sciaenidae) Mullus (Ilullidae) Gaidropsarus (Gadidae) Perm (Percidae)

Upper frequency limit (Hz)

Reference

800 1000 1500-2000 1250 600 435 (640 in young) 2068 1600-2500

Dijkgraaf (1952b) Dijkgraaf (195213) Maliukina (1960) Dijkgraaf (1952b) Diesselhorst (1938) Farkas (1935) Farkas (1936) Maliukina (1960)

Threshold (re 1 pb) -20 to 0 dB a t 250 HZ -50 dB a t 640 Hz -45 dB a t 320 Hz -50 dB a t 500-600 Hz Below -30 dB at 450-900 Hz -30 dB a t 750 Hz -14 dB at 100 Hz

Diesselhorst (1938) Maliitkina (1960) Maliukina (1960) Maliukina (1960) Maliukina (1960) Maliukina (1960) Wolff (1967)

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Fig. 24. Comparison of the audiograms of the grunt, Haemulon, the squirrelfish, Holocentrus, and the human. All curves are plotted against the extreme lefthand ordinate in terms of acoustic power (W/cmz). The equivalent acoustic pressures in air (against which the human audiogram is plotted) are on the right ordinate. After Wodinsky and Tavolga (1964), with permission of Pergamon Press.

curve rose sharply to $40 or +SO dB pb at 1500-2000 Hz. At frequencies below 300 Hz, these species exhibited high thresholds at first, but after additional training and testing, the thresholds were 0 to +lo dB pb at 100 Hz. Cohen and Winn (1967) determined the audiogram for the midshipman, Porichth ys notatus, electrophysiologically, and they based their thresholds on a 20 p V saccular microphonic response. The lowest thresholds were about +7 dB pb at 30 Hz. The curve rose slowly to about +22 dB pb at 120 Hz, then showed a sharp dip to +11 dB pb at 150 Hz. Above 150 Hz, the audiogram rose steeply to +40 dB pb at 240 Hz. Significantly, the dip at 150 Hz corresponded roughly to the average fundamental of the sounds normally produced by this species. Some species showed higher sensitivities. The squirrelfishes, Holocentrus, and a few others were found to have thresholds of -20 dB pb at 600 Hz and could respond to frequencies as high as 2000 Hz. The only data that are presently available on the hearing of pelagic species were reported by Iversen ( 1967) (Fig. 25). The yellowfin tuna, Thunnus albacares, possessed lowest thresholds of -13 dB pb at 300 Hz and -17 dB pb at 500 Hz. The audiogram rose steeply to about +20 dB pb at 1000 Hz and showed a more gradual rise toward the low end to about +20 dB pb at 100 Hz. These pelagic, fast-swimming fish are difficult to maintain under any aquarium conditions. This study is particularly note-

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Fig. 25. Audiogram of the yellowfin tuna, Thunnus albacares: ( 0) 50 cm and ( A ) 60 cm. The lower dotted line indicates the background noise plotted as total noise in a narrow band. After Iversen (1967), with permission of Pergamon Press.

worthy since the fish not only kept in good health for considerable periods of time but were also trained and tested repeatedly. It should be noted that Iversen provided data on ambient noise in his experimental tanks, and it seems clear that his threshold data were unmasked values, i.e., unaffected by the ambient noise. The shape of the audiogram for the Atlantic cod, Gadus nwrhua, is significantly different from those of other marine fishes tested. Buerkle ( 1967) showed that at frequencies below 200 Hz, the thresholds (determined by conditioned cardiac rhythm changes) were in the order of 0 to -10 dB pb. The curve rose steeply to about +20 dB pb at 400 Hz, and this frequency probably marks the upper limit of hearing in the species. It is also probable that the lateral line is the primary receptor. Buerkle (1968) also demonstrated the effect of masking noise on these thresholds. The flat portion of the audiogram (35-141 Hz) was most consistently affected by the masking noise, and the signal-to-noise ratio in this range was about 20 dB, using the spectrum level (noise per cycle) as the reference. In a report of preliminary data, two species of pollack (Pollachius pollachius and P . virens) were found to have a hearing range similar to that of the cod. The lowest threshold was at about -9 dB pb at 300 Hz, and the audiogram rose sharply above 450 Hz (Parrish et al., 1968). In a series of studies on fishes of the family Percidae, Wolff (1967,

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1968) used avoidance conditioning to demonstrate auditory thresholds. In the common perch, Perca fluviatilis, the lowest threshold was about -14 dB pb at 100 Hz, and the curve rose sharply to f35 dB pb at 50 Hz, and almost +45 dB pb at 200 Hz. In the pike perch, Lucioperca Sandra, thresholds were about 0 to $5 dB pb from 50 to 200 Hz, rising to $60 dB pb at 800 Hz. The audiogram for the stone perch, Acerina cernuu, was similar in shape to that of the pike perch, but consistently about 10 dB higher, It is possible, as in the case of the cod, that these percids may be primarily sensitive to displacements mediated by the lateral line. Deserving special mention is the attempt by Kritzler and Wood (1961) to determine a complete audiogram in the bull shark, Curcharhinus leucm. Their data, based upon positive reward conditioning, ranged in threshold values from +lo dB pb at 100 Hz, to a low level of about -15 dB pb 400-600 Hz, to over +lo dB pb at 1400 Hz. Considering the fact that the shark has no swim bladder and therefore receives all sounds either through direct conduction to the inner ear or by way of the lateral line system, these low thresholds are quite remarkable. This may serve to indicate that an air chamber need not function as the main transducer in sound reception, and the acoustical difference between the water medium and the bone or cartilage of the neurocranium may be sufficient to permit detection of frequencies up to 1000 Hz. The above report by Kritzler and Wood (1961) was the first behavioral study of hearing in any elasmobranch fish, although the ability of sharks to detect and respond to acoustic signals has long been known (Parker, 1910a). Interest in the hearing of sharks has, of course, been spurred by practical aspects of dealing with attacks of these animals on human beings. Many of the problems in experimental work on the hearing of sharks have been summarized by Wisby et al. (1964), and Backus (1963). In a preliminary report, D. R. Nelson and Gruber (1963) reported that they were able to attract sharks in open sea conditions to a sound source playing back recordings of low frequency pulses, similar to those produced by a struggling, wounded fish. Davies et a2. (1963) conditioned four species of sharks to respond to sounds and obtained data on response to both pure tones and octaves of broad-band noise. The results gave a fairly flat audiogram curve ( + l o dB pb) from 50 to 7000 Hz. The pressure levels ranged from +5 to +2S dB pb, and the lowest sensitivities of the animals were within a few decibels of the background noise levels at the tested frequencies. The authors concluded that the sharks are capable of determining the location of the sound source but are not capable of any significant frequency discrimination. These results should be considered as highly preliminary

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and probably do not represent true thresholds or audiograms, since it is quite probable that under conditions of low ambient noise the shape and level of the sensitivity curves would be very different. In other studies on the hearing of sharks, Wisby et al. (1964) and D. R. Nelson (1967b) obtained heart-rate responses to sound pressures of about +30 to +40 dB pb in the lemon shark, Negaprion brevirostris, at frequencies up to 1000 Hz. Dijkgraaf (196313) obtained similar values at 180 Hz in the dogfish, Scyliorhinus caniculu, using classic conditioning. Neither of these studies showed thresholds as low as those reported by Kritzler and Wood (1961). Nelson (1967a) also found thresholds in the lemon shark, Negaprion breuirostris, that were significantly different from the values reported by Kritzler and Wood (1961). His data gave values of from 0 to -10 dB pb from 10 to 320 Hz, and a sharp rise to about +30 dB pb at 640 Hz (Fig. 26). It is probable that differences in the conditioning techniques and, especially, in the acoustic conditions may account for the differences in threshold values obtained by various investigators.

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Frequency Fig. 26. Audiogram of the lemon shark, Negaprion breuirostris, plotted together with the mean filtered ambient noise: (-) mean hearing thresholds and ( - - - ) mean filtered ambient noise. After Nelson ( 1967a).

2. DISCRIMINATORY CAPACITIES A further step in the psychophysical study of hearing in fish is the investigation of the ability to discriminate sounds of different intensities and frequencies. It is clear that in contrast to mammalian hearing the

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frequency range of most fish is significantly narrower. Although in some species thresholds can be obtained at frequencies up to 3 or even 5 kHz, the intensities of the test sounds are in the 20-30 dB pb range, and such acoustic intensities are probably much higher than sounds normally encountered in the fish‘s environment. The usable frequency range in most fish, therefore, does not extend beyond about 2 kHz (higher in ostariophysines), and the dynamic range covers a span of about 60 dB. By contrast, human hearing has a usable range of at least 10 kHz and over 100 dB. Within the narrow frequency and dynamic range of fish, the ability for discrimination has been found to be remarkably good. The first reports on intensity discrimination were those of Wohlfahrt (1939), who concluded that this ability was completely lacking in fish, and Dijkgraaf and Verheijen (1950), who were able to show that the minnow, Phoxinus laevis, was capable of distinguishing between tones of equal frequency but different intensity. In another ostariophysine, the goldfish, Curussius auratus, Jacobs and Tavolga ( 1967) demonstrated that the “just-noticeable-difference” ( i n d ) was from 3 to 6 dB. This compares to a ind of about 0.5 dB for human hearing ( Licklider, 1951) . The ability of fish to discriminate between different frequencies was first studied by Stetter (1929), who found that Phoxinus was capable of discriminating between two tones that were about a minor third apart, i.e., about a 20% difference in frequency. For the same species, Wohlfahrt ( 1939) obtained a ind of about 6% ( a half tone). This discriminatory capacity was found to be somewhat temperature dependent (Dudok van Heel, 1956). Much of the above work was done with poorly controlled intensities and crude sound-producing devices (flutes, tuning forks, etc. ) . Using more accurate sound generators, Dijkgraaf and Verheijen ( 1950) reported that Phoxinus was capable of discriminating tones with a 3% (one-quarter tone) difference in frequency. Using more accurately calibrated equipment, standard psychophysical techniques, more animals, and a different conditioning procedure, Jacobs and Tavolga (1968) were able to confirm these data for the goldfish. The mean ind at 200 Hz was 9.4 Hz (4.7%),at 500 Hz the ind was 17.4 Hz (3.5%),and at 1000 Hz the ind was 50.1 Hz (5.0%).For comparison human subjects possess a ind of 0.448%at 100 Hz and 0.243%at 1000 Hz. It is significant that species of fish other than Ostariophyi have a much larger ind for frequency. In two species of mormyrids, Stipetih (1939) reported a ind of 12-25%, and Dijkgraaf (1952b) stated, that Gobius, Corvina, and Sargus were capable of discriminating a 9-15% difference in frequency. Even larger values of 50-100% were given for other nonostario-

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physines: Umbra (Westerfield, 1921), Anguilla (Diesselhorst, 1938), Macropodus ( Schneider, 1941), and the lemon shark, Negaprion (D. R. Nelson, 1967a). In addition to possessing lower absolute thresholds, the Ostariophysi also appear to have better discriminatory capacities, and the possibility appears that the Weberian apparatus may play a role in frequency analysis. The mechanism of frequency discrimination in fish was investigated by Enger (1963) by means of neurophysiological techniques. In the sculpin, Cottus, he demonstrated the presence of four types of neural units, some of which displayed a following response to the acoustic stimulus. He concluded that frequency discrimination in fishes takes place in part by the following response, and in part by a separation into low and high frequency sensitive units. This volley theory would not, however, explain the fine degree of discrimination demonstrated above (Jacobs and Tavolga, 1968). Furthermore, Enger used extremely high sound levels up to 50 dB pb, and van Bergeijk (1967a) pointed out that such intensities at close range would stimulate inertial receptors as well as acoustic receptors and produce, in effect, a vertigo. The inadequacies of a volley theory to explain human auditory discrimination led to the classic work of von B6k6sy (1960), where he demonstrated the applicability of a place theory. The place theory, however, rests on the morphological basis of a cochlea with a basilar membrane that is differentially responsive to different frequencies. Fish do not have a cochlea, or anything apparently analogous to a basilar membrane. The question of the mechanism of frequency analysis by the fish ear remains an intriguing problem. Much of the support for the place theory in human hearing is derived from psychophysical data on the effects of masking noise and the presence of a critical band (Fletcher and Munson, 1937; J. E. Hawkins and Stevens, 1950; Scharf, 1961,1966). Some evidence has been presented that a critical band for masking may exist in fish (Tavolga, 1967b), and van Bergeijk (1967a,b) pointed out that a basilar membrane as such is not essential for a place theory to apply. Any structure with some acoustic asymmetry would respond differentially to traveling waves of different frequencies. Even a bongo drum can generate sounds over a range of at least an octave, and van Bergeijk (1967b) presented a most plausible explanation for frequency analysis in the fish‘s ear. This “bongo drum hypothesis” depends upon the fact that the saccular otolith and its underlying macula behaves as a bounded membrane with sufficient acoustic asymmetry to resonate differentially at different frequencies. The problem of whether a fish can detect the direction of a sound source has been given little attention until recently. Kleerekoper and

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181.

Chagnon (1954) presented strong evidence that Semotilus can orient with respect to a sound source, especially if there are standing waves present. Reinhardt (1935) and von Frisch and Dijkgraaf (1935) came to the opposite conclusion, i.e., that fish could not localize a sound. Their data. however, showed that localization took place at close range to the sound source. Van Bergeijk (1964) reviewed the available data with respect ta the nature of the acoustic stimulus presented, and he emphasized that the distinction between the two forms of acoustic energy must be made: i.e., between far field (pressure) and near field (displacement). For localization to take place, a minimum of two receptors is necessary (Kuroki, 1967). Fishes have, at best, only a single pressure receptor: one median swim bladder coupled to the two inner ears. Displacement detectors in the form of lateral line organs, however, form a complex array of numerous units. Van Bergeijk's conclusion is, therefore, that fish are not able to localize except within the range of the near field of a sound source (about one-sixth of a wavelength). Through a combination of neuroanatomical, neurophysiological and behavioral techniques, Moulton and Dixon ( 1967) demonstrated that certain directional responses in fish consist of rapid tail flips. These are two-neuron reflexes involving a sensory and a Mauthner's neuron and are associated with a rapid escape response, although conditioning can alter this to an approach response. Moulton and Dixon proposed that these were directional resonses to a far-field stimulus, but the intensity of the test sounds was high and the loudspeaker was close to the animal in their experimental conditions. It is probable that a substantial near field was generated (cf. comments by Tavolga and van Bergeijk and p. 232 in Moulton and Dixon, 1967). Experiments and observations on the movements of carp in a large tank, monitored by means of a matrix of photoconductive cells, showed that orientation in a sound field takes place by a sort of klinotaxis. In response to nodal and antinodal sound pressure variations, the turning angle of the fish is modified, and it is postulated that this response is mediated by Mauthner cells ( Kleerekoper and Malar, 1968). According to the analysis by van Bergeijk ( 1964), elasmobranchs, lacking a swim bladder, should not be able to detect far-field energy. In field observations, however, D. R. Nelson and Gruber (1963) and Wisby and Nelson (1964) were able to attract sharks with low frequency signals (20-60 Hz). The range over which this attraction took place (about 200 meters) brings up the possibility that the animals were reacting to a far-field pressure wave. However, sensitivity to displacement energy in the lemon shark, Negaprion brevirostris, was found to be below 10 A in some cases (Banner, 1967).

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D. Evolution of Hearing Probably one of the most primitive interactions between an organism and its environment is one that is dependent upon physical contact. On the multicellular level of organization, the specialization of contact receptor cells appears to be one of the earliest to evolve. It is clear that the specialization of an organ for reception of sound should be traceable from primitive touch receptors with displacement-sensitive hairs. The classic approach to the study of this evolutionary sequence is exemplified in the review by Pumphrey ( 1950). The essentials of the sequence are: free neuromasts that are groups of hair cells; the addition of a gelatinous cupula and the organization of the neuromasts into more or less enclosed lateral line organs; the development of otoconia and, later, otoliths that mass-load the hair cells; and the enclosure of the hair cell groups within the labyrinth. The functional sequence is : simple mechanoreceptors that develop into more sensitive displacement detectors; enclosed mechanoreceptors become pressure sensitive as a result of the intervention of an air-filled swim bladder; and in terrestrial vertebrates, the middle ear takes on the coupling function of the swim bladder. According to Pumphrey (1950), the response of the inner ear to gravity is “a byproduct of the improvement of hearing.” Based upon a reexamination of the concepts of “sound and “hearing,” van Bergeijk (1966, 1967a) presented an intensive survey of the problem of the evolution of hearing in vertebrates and attempted to trace this evolution in physiological terms. The lateral line organ is essentially a displacement detector and, as a result, a hydrodynamic motion detector. The organization of the lateral line system, however, provides new inputs into the acoustic centers of the central nervous system, and the lateral line system becomes a near-field hearing organ, with the capabilities of localizing sound sources (van Bergeijk, 1964). By contrast with Pumphrey, van Bergeijk (1967a) proposed that the inner ear labyrinth arose as an inertial receptor organ. Only the later development of a swim bladder ( a hydrostatic organ) gave the labyrinth the property of hearing. The acoustic discontinuity of a swim bladder in an aquatic organism enables it to respond to pressure waves, i.e., far-field sound, and the bladder therefore generates a local near field that can excite the inner ear, Specializations such as the Weberian apparatus improve the coupling of the swim bladder and the inner ear and enhance far-field hearing. Van Bergeijk carried this evolutionary sequence into the development of hearing in terrestrial vertebrates. His entire argument was brilliantly presented and thoroughly documented, and it deserves recognition as a milestone in the field of biology.

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IV. ACOUSTIC COMMUNICATION IN FISH

The channels available for animal interactions are: photic (visual), mechanical ( including tactual and acoustical), thermal, chemical (gustatory and olfactory), and electrical (limited to certain fishes). Information transmitted along any of these channels can be broadly classified as: long range vs. short range and directional vs. nondirectional. Each of these channels has certain limitations in an aquatic medium as compared with a terrestrial environment. The photic channel is severely limited in water (Dietrich, 1963), especially in seawater in regions of high planktonic concentration. The probability is that the range for effective vision in the marine environment is generally less than 1 meter, and in areas of high turbidity this effective range may be reduced to only a few centimeters. The chemical channel is potentially an effective one in water, because of the large range of substances that are easily suspended or dissolved in water. This channel, however, is slow and nondirectional, and the source of the stimulus can be located only by means of some kinesislike movements in which the animal simply moves about until it finds areas of progressively higher stimulus concentrations. Although useful in a terrestrial environment, the thermal channel is virtually unavailable to aquatic animals, especially to ectothermic forms such as fishes. Water absorbs heat rapidly, and a thermal gradient attenuates much too fast for any effective reception as a stimulus in an interaction. Both ac and dc electrical fields are readily set up in water, especially in saltwater, and many species of fishes, in addition to the well-known electrical forms, are now known to be able to detect electrical potentials produced by other organisms. How widely this energy channel is used in interaction among fishes still remains to be studied. Aside from the short-range, direct contact function of tactile receptors, the mechanical channel offers several advantages for interactions and communication among fishes. Acoustic energy under water is effective as pressure and as displacement. Both the inner ear and the lateral line are essentially displacement sensitive, but the inner ear receives nearfield displacements from the nearby swim bladder. The swim bladder acts as a transducer for pressure waves and transforms them into local near-field effects (Harris and van Bergeijk, 1962; van Bergeijk, 1964). Pure pressure waves are efficiently propagated in water and this form of acoustic energy is probably the most rapid and effective channel for long-range interactions. At short range, directional orientation to a sound

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source by fishes can take place in the near field, probably by means of the lateral line. The majority of sounds produced by swim bladder mechanisms in fishes range from 50 to 100 Hz, and this would provide an effective near-field range of from about 50 to 3 meters, respectively. The acoustic channel, therefore, is one of long effective range and high information content for interactions among aquatic animals, especially fishes. It is clear that fishes interact with each other in a variety of ways, using various sensory modalities, and the interactions occur in many different behavioral contexts. Some of these interactions have been called “communication,” but the exact definition of animal communication is not always clear. Some authors, for example, have favored a broad definition that could conceivably include all kinds of interactions, while others restricted the use of the term “communication” to intraspecies interactions (Frings and Frings, 1964). Attempts have been made to find factors in common between human communication and that of other animals (Marler, 1961; Sebeok, 1965). Many of the approaches to animal communication involve cross-phyletic comparisons with little regard for the differences in phyletic position and level of organization of the organisms compared. The concept of levels of organization is basic to biological science, and this concept is particularly applicable to behavior. Schneirla (1953) postulated a hierarchical arrangement of levels of behavioral integration with each level defined by qualitatively different organization and development of integrative systems. Tavolga (1969, 1970) applied the concept of levels of organization to the definition of communication. Communication is not a single phenomenon; rather, there are many kinds of communication. There are many kinds of interactions that are characteristic of different levels of organization, and only some of these levels involve communication in the strict sense. Interactions among fishes occur at various levels of complexity and organization. The understanding of these interactions must depend upon an appreciation of the level involved in each case and the developmental history of the behavior. Levels of interaction among organisms were defined by Tavolga (1969, 1970) as follows: Vegetative: Interactions by virtue of physical presence alone, as well as through growth or tropism. Tonic: Interactions resulting from the more or less continuous processes fundamental to species-typical development and function, e.g., homeostasis. Phasic: Interactions that result from discontinuous, more or less

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regular stages or events in the development of an organism. This would include some primitive types of sex discriminatory and food discriminatory behavior. Signal: Interactions in which receiver and/ or emitter utilizes some specialized structures. Specialized forms of behavior would also be involved. Symbolic and Language: Interactions based on high levels of nervous integration, as in primates, including man. The term “communication,” therefore, could include any one or more levels in the above heirarchy of interactions. Restriction of the scope of the term now becomes practical and meaningful. Signal, symbolic, and language levels would comprise communication, while the vegetative, tonic, and phasic levels would include primitive forms of interactions (Tavolga, 1969). For communication to take place any one of the following conditions should be met: (1) The emitter must possess a specialized stimulus-producing mechanism (chemical, morphological, or behavioral); (2) the stimulus must occupy a narrow portion of the available spectrum of the channel (frequency range, duration, patterning, chemical specificity); and ( 3 ) the receiver must possess specialized receptors and respond in a specific manner. Most fishes can react with each other on the tonic level. Excretory wastes, normal mucous secretions, carbon dioxide output, and other metabolic byproducts are readily diffused through water and can affect other animals in some physiological fashion. Body shapes, basic color patterns, normal locomotion, and other manifestations of homeostasis and maintenance behavior can result in both intra- and interspecies interactions. Phasic level interactions are also common in fishes. The reaction to Schreckstoff is an example, even though it can sometimes be speciesspecific. Many predator-prey interactions are on this level, as well as much of aggressive and territorial behavior. Schooling is primarily a phasic level interaction, and some aspects of reproductive behavior are also phasic. Although there is little evidence on signal level interactions in fishes, indications are that the exchange of specific signals may be important in reproductive behavior, especially during the prespawning, and parental care stages. The courtship stage is probably most crucial, since species and sex discrimination takes place at this time, and a continuing series of attracting interactions is necessary to hold the pair in proximity until the proper physiological state for spawning is reached. Such interactions can also stimulate the members of the pair to develop into spawning con-

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dition. Specialized reproductive activities, therefore, are the main interaction types among fishes that fall into the category of communication as defined here. Many of the sounds described and cataloged by Fish (1954, 1964) and her co-workers were elicited from captive animals under various unspecified conditions of duress, including administration of electric shock. It is not known what behavioral significance these sounds could have. Moreover, the electric shock method may in some cases produce a neuromuscular response rather than a true behavior. Any animal with a swim bladder is potentially capable of sound production if the body wall muscles are abruptly stimulated to contract. Interpretations of biological significance, therefore, can only be made on the basis of field recordings or of relatively unrestrained animals under experimental conditions. Some stridulatory sounds, such as those of grunts (Pomadasyidae) and other forms that grind their pharyngeal teeth, have been recorded only in air or from specimens grasped under water in an aquarium ( Burkenroad, 1930). The fin ray stridulations of marine catfish are produced when the animal is captured and pulled out of water (Tavolga, 1960). Such sounds are very unlikely to have any biological significance. Many of these stridulatory sounds have most of their acoustic energy at frequencies above the hearing range of most fishes and thus should be considered unlikely to have any behavioral significance. The gnashing sounds of feeding should be within the hearing range of fishes, but these sounds have not yet been shown to have any communicative value. It is quite possible that individuals hearing other fish eating may soon learn to come for food to the source of the sound. Hydrodynamic sounds may also play a role in the life of both a predator and a prey species. Sharks have long been known to approach the vicinity of a wounded fish, and D. R. Nelson and Gruber (1963) proposed that much of this attraction is the result of low frequency acoustic stimuli. Young lemon sharks, Negaprion brevirostris, will not only approach but will even attack a hydrophone that is emitting pulses of broadband noise, and similar hydrodynamic sounds were found to attract sharks (Banner, 1968). A detailed description of field studies on the effects of sounds on free-ranging sharks shows dramatic results (Myrberg et al., 1969), but it is not yet clear what the specifications and parameters of an adequately attractive stimulus are. Not only sharks but also other predatory fishes have been shown to be attracted to the source of low frequency sound pulses, especially those hydrodynamic disturbances associated with active predation ( Richard, 1968). Such interactions are probably on the phasic level, sirice the stimuli

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produced by the prey animals result from simple locomotor behavior, and the responses of the predators are nonspecific approaches. A number of sounds are produced in some stressful situation. Groupers, squirrelfish, jacks, and a variety of other species have been described as doing so (Fish, 1954; Moulton, 1958; Tavolga, 1960). Approach of foreign objects, a poke of a stick, etc., are examples of such stimuli. Whether the sound is indicative of alarm, fright, anger, annoyance, or other emotional state is not known. When the approach of another fish stimulates sound production, then there is a likelihood of the sound functioning as a warning call from a territory holder toward an intruder, as in groupers ( Moulton, 1958; Tavolga, 1960), squirrelfish ( Winn et al., 1964), toadfish (Tavolga, 1960; Gray and Winn, 1961), pinfish (Caldwell and Caldwell, 1967), triggerfish (Salmon et al., 1968), and several other forms listed by Winn (1964). Most of these interactions are probably tonic or phasic in level, but the data on toadfish (Winn, 19f37) indicate a more specific signal level communication. Since ancient times, the relation of certain kinds of fish sounds to spawning behavior has been known. This is especially true of members of the croaker and drumfish family (Sciaenidae). In many parts of the world, including the Florida coast, fishermen commonly listen for the deep rattling sound of drumfish during their spring spawning migration. Schneider and Hasler (1960) described the spawning sounds of the freshwater drum, Aplodinotus. The European sciaenid Corvinu has been reported by Dijkgraaf (1947a) and by Protasov and Aronov (1960) as producing sounds in connection with spawning behavior. The codfish, Gadus callarias, and the haddock, Melanogrammus aeglefinus, have been reported to produce low frequency sounds during the spawning season ( Brawn, 1961; Hawkins and Chapman, 1966). The boat-whistle sounds of the toadfish, Opsanus, were thought to be related to spawning or to territorial behavior (Fish, 1954; Fish and Mowbray, 1959; Tavolga, 1958c, 1960). Gray and Winn (1961) and Winn (1964, 1967) have presented data showing that the sounds appear to be emitted by males only and serve to attract females to nesting sites. Winn ( 1967) also demonstrated the interactions between sounding territorial toadfish. Positive evidence of the function of fish sounds in reproductive behavior have come from studies on freshwater minnows (Delco, 1960; Winn and Stout, 1960; Stout, 1963), on a characid (K. Nelson, 1965), on tidal zone species of gobies and blennies (Tavolga, 1956, 1958a,b), and on certain cichlid fishes (Myrberg et al., 1965). Experiments involving playback of sounds to the fish were used to establish the fact that sounds produced by courting males could attract females. It is probable

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that many of the instances of sounds used in reproductive behavior represent signal level communication, but needed data on specificity of the signal are often lacking. Although no sex differences appear to exist in the morphology of the sonic mechanisms of the Batrachoididae, only male toadfish, Opsanus tau, are known to emit boat-whistle sounds (Gray and Winn, 1981; Winn, 1964), and only males of the midshipman, Porichthys notatus, produce any sounds at all ( Cohen and Winn, 1967). Some species appear to produce sounds when schooling or aggregating. Aggregations of sea robins produce a characteristic staccato call (Moulton, 1956), and nighttime schools of marine catfish form large choruses ( Tavolga, 1960). In some long-term observations of local populations, Breder (1968) found that the sea catfish, Galeichthys felis, aggregate and produce choruses of the “percolator” sounds from April to October, with a lull in July and August. These choruses usually start after 5 P.M. and cease before 11 P.M. Optimum water temperatures for chorus formation were from 74” to 89°F) and increased chorusing was noted during new moon periods. Breder (1968) also observed that the boat-whistle sounds of the toadfish, Opsanus beta, were normally heard in March, April, August, September, and October, usually most frequently when the Gabichthys choruses were most vigorous. The repetition rate of the toadfish calls was found to be temperature dependent, averaging 0.93lmin at 74°F and 1.92lmin at 83°F. Toadfish calls of this Florida west coast species disappeared at water temperatures below 73°F and above 91°F. As in the catfish (above), many species seem to show a daily rhythm in their sonic behavior. Winn et al. (1964) demonstrated this cyclic activity in the squirrelfish. This species, as well as many others, tend to show dawn and dusk peaks of sonic activity, possibly correlated with feeding or territorial movements. Cummings et al. (1964) found a cyclic occurrence, with dawn and dusk peaks, for many sounds of biological origin as monitored by a shore-based hydrophone system. Since so many of the sounds produced by different species are similar, Winn (1964) proposed that the coding of information may be through temporal patterning. With the exception of a few species, like the toadfish, that produce harmonic tones, most fish sounds are short pulses with a fundamental near 100 Hz. There are, however, distinct differences among species in the grouping and repetition rates of these pulses, It is conceivable, according to Winn, that species discrimination and the communication of information as to emotional state ( i.e., alarm, territorial

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defense, and prespawning behavior) is by way of temporal coding. This type of coding would be characteristic of the signal level of communication. The presence of echolocation abilities among fishes have yet to be demonstrated. Griffin (1955) described a single instance in which a deep-sea species (no identified) was recorded and echoes of its cries could be detected bouncing back from the ocean floor. On this basis, Griffin proposed that fishes could conveivably utilize echoes of their own sounds for orientation. These observations, however, are in need of confirmation. V. PROBLEMS AND PROSPECTS FOR THE FUTURE The study of underwater bioacoustics is an interdisciplinary field and has captured the interests of physicists, acousticians, engineers, psychologists, physiologists, and scientists in many other specialities. As noted earlier, it is also a rapidly expanding field and as a by-product of the expansion, numerous new areas of research are unfolding. Some of these are basic and some applied, and it was pointed out by Galler (1967) that this field has served as a good example of the interaction between basic and applied research. Ever since the development of ASDIC and Sonar, the effectiveness of echo-ranging equipment in detecting marine life has shown a steady increase. Although not directly related to sound production and detection by fishes, the utilization of these techniques for detection of fishes has moved to increasing degrees of precision (Hester, 1967; Cushing, 1967; Weston, 1967) and deserves mention here. Sonar has already proved its value in assisting the commercial fisherman, and it promises to be an excellent tool for the study of fish locomotion and the behavior of fish schools. The utilization of sound to guide fish has only recently been applied commercially. The bibliography compiled by Moulton and Backus ( 1955) showed that these techniques for guiding or attracting fishes have been utilized by fishermen in many parts of the world. Some of the techniques are primitive and of ancient origin (Busnel, 1959; Wolff, 1966). Recent attempts have been made, using modern electronic equipment, to attract fishes by playback of their feeding sounds, and some success in this area has been reported ( Hashimoto and Maniwa, 1967). Prelirninary studies on guiding migrating salmon by means of acoustic stimuli have also shown promise for the future (Vanderwalker, 1967). The recent observations

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by Richard (1968) demonstrated the feasibility of attracting a variety of predatory fishes by means of low frequency sound. For the successful development of these techniques, it is clear that more basic information is still required on the hearing range of many of the commercially important species, as well as more information on the behavioral significance of sounds produced by these fishes. Although the technique of listening for sounds produced by fishes is known as an art among fishermen in several areas of the world (Moulton, 1963), the use of this method in major commercial fisheries has yet to be tested. Some preliminary studies have been attempted, using a combination of sonobuoy and telemetry equipment ( Hashimoto et al., 1960). Considering the technology now available, it should be feasible to detect and identify fish sounds electronically, although much needed information on identifying characteristics of commercially desirable species is still lacking. It is no longer sufficient to present another instance of a fish making sounds, and such information needs to be correlated with the behavioral context of the sound. Long-term recordings and observations, like those supplied by the acoustic-video installation of the Institute of Marine Science at Bimini, Bahamas (Kronengold et al., 1964), will be increasingly necessary. Bioacoustic observations by small submersibles hold much promise for the future of the field (Backus et al., 1968))and some scuba divers are already becoming equipped with listening devices. In this connection, the identification of many hitherto unspecified field contacts will become possible. Behavioral studies, however, will have to be supplemented by laboratory observations and experiments where environmental conditions can be controlled. A problem that has troubled both field and laboratory investigators has been the exact specification and description of the acoustic stimulus. Watkins (1967) pointed out the pitfalls of analyzing equipment, but, further, it is extremely difFicult to separate the two forms of energy that usually exist together in an underwater sound field: pressure and displacement. Most hydrophones are basically pressure transducers and will detect pressure changes produced by a near field as well as those of a far field. The needs of biologists for a small, but sensitive, displacement transducer have not yet been satisfied (Tavolga, 1967cj, and this is another area in which an interdisciplinary approach to the problem is necessary. The problem of frequency discrimination by fish is an intriguing one. If future evidence demonstrates the applicability of a place theory to fish hearing, it may necessitate a reexamination of the concept of the critical band.

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The mechanisms of sound production in fishes need further elucidation, especially in relation to the remarkable fast-acting properties of the sonic muscles. If these properties, as well as those of common innervation and embryonic origin, are found to be as widely distributed among the diverse families of fishes as is presently apparent, then questions begin to arise as to the evolutionary origin of sound production. Data are now being accumulated at a rapid rate on the behavioral significance of fish sounds. Acoustic interactions and communication among fishes are evidently more common than was thought only about 20 years ago. Both laboratory and field investigations are now establishing the behavioral contexts for such interactions in many species. One area of this study that has yet to be examined is the ontogeny of these interactions. A strong trend in modern animal behavioral study is toward an understanding of the development of behavior. It is no longer appropriate to simply label a behavior as innate or learned, but it is necessary to investigate the physiological, experiential, genetic, and other antecedents of the behavior as it develops in the individual (Schneirla, 1965). One of the few ontogenetic behavioral studies in fishes has been on the development of schooling. Shaw (1960, 1961) showed the possible relationship of the optomotor response in larval fishes to aggregation and schooling. Investigations of the development of sonic behavior in fishes are clearly indicated and desirable. The future of the field of fish bioacoustics is exciting, and areas for research are available to workers from many disciplines. Many basic problems still exist, but at the same time the application of the basic information is feasible and has a great potential toward improving our understanding of aquatic resources in general. ACKNOWLEDGMENTS The author’s research in this field has been supported in part by grants GB-1574 and GB-4364 from the Psychobiology Program of the National Science Foundation. Support was also received through Contract 552 (06) between the Office of Naval Research and The American Museum of Natural History. This report, in part, serves as a final report for this contract. Some of the review work was done in connection with Contract N61339-1212 between the U. S. Naval Training Device Center, Orlando, Florida, and The American Museum of Natural History, and many of the figures used here are reproduced with the permission of the U. S. Naval Training Device Center. The author would like to add a special personal acknowledgment to the late Dr. Willem A. van Bergeijk. It was through his inquiring and incisive thinking that the author was led to explore many avenues of this field. His untimely death, as he was approaching the peak of a brilliant career, was a loss not only to the immediate field but to science in general. This opportunity is taken to dedicate this chapter to a friend and colleague.

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I. STRUCTURE

The labyrinth is considered to represent the final stage in the process of submergence of neuromast organs of the lateral line type, and it has become completely or, at any rate, almost completely, separated from the outside medium and enclosed in the brain case. The “duct’us endolymphaticus” maintains a tenuous connection with the outside world in the elasmobranchs only. The labyrinth originates ontogenetically from a common placode with the lateral line. Wilson and Mattocks (1897) described the early states in the formation of a common rudiment ( Anlage) in the early salmon embryo. On about the twelfth day, the “embryionic shield thickens into a neural keel. Immediately at each side of this is left a comparatively thick streak of ectoderm which extends through the anterior two thirds of the embryo. It is the lateral sensory rudiment. Toward the end of the thirteenth day the middle portion of the rudiment begins to be invaginated to form the auditory sac. The auditory sac comes thus to lie between a preauditory and a postauditory rod of cells which give rise to the infra- and supraorbital canals of the head and to the lateral line of the trunk, respectively. 207

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A

PC

cs

A C

L

Fig. 1. The four chief morphological types of labyrinth in the agnathous and gnathostome fishes. ( A ) Myxine, after Lowenstein and Thornhill, 1971; ( B ) Lampetru, after Lowenstein et al., 1968; ( C ) elasmobranch, after Retzius, 1881; and ( D ) cyprinid, after von Frisch, 1936. AC, anterior crista; B, basal arm of crista; CC, crus commune; CS, ciliated sac; ED, endolymphatic duct [omitted in ( B ) and ( D ) ] ; H, horizontal arm of crista; HC, crista of horizontal canal; MC, macula communis; ML, macula lagenae; MN, macula neglecta; MS, macula sacculi; MSL, macula sacculi and macula lagenae; MU, macula utriculi; PC, posterior crista; and V, vertical arm of crista.

The typical subdivision of the laybrinth into semicircular canals and otolith organs is phylogenetically fundamental, and it is found in the labyrinth of the cyclostomes and, according to Stensi6 (1927), in the fossil ostracoderm Kiaermpis. Figure 1shows the four chief morphological types of labyrinths occurring in the fishes, and apart from the absence of the horizontal semicircular canal in the cyclostomes and the persistence of an external opening of the ductus endolymphaticus in the elasmobranchs, the chief variation concerns the relative proportions and positioning of the otolith organs, utriculus, sacculus, and lagena. The complete absence of a cochlea or any cochlear rudiment differentiates the fish labyrinth from the inner ear of all other vertebrates.

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A. Semicircular Canals The semicircular canals are curved membranous tubes filled with labyrinthine fluid. During their development they are folded off the general labyrinthine cavity with which they remain continuous at both ends. At one end the canals widen into a spherical ampulla which harbors the sensory apparatus. This consists of a crestlike ridge, the crista ampullaris, which juts out into the ampullary lumen. The crista carries the sensory epithelium made up of sensory cells and supporting cells. Ciliary hair processes project from the top of the sensory cells into the lumen of the ampulla. They are ensheathed in a jellylike cupula which reaches from the top of the crista to the opposite wall of the ampulla. The cupula fits into the ampulla like the blade of a turbine and is therefore deflected by movements of the endolymph within the canal. Such movements relative to the wall of the canal take place whenever the canal is subjected to angular acceleration by movements of the head in space. In the gnathostomes the three semicircular canals are arranged in three planes of space approximately at right angles to each other. Two lie in a vertical and one in a horizontal plane when the head occupies its normal position in space. B. Otolith Organs

Fundamentally all otolith organs are designed on the statocyst principle. There is a sensory epithelium consisting of sensory cells with an apical system of hair processes and of supporting cells. Above this epithelium lies a mass of crystalline matter, usually composed of calcium salts. This is specifically heavier than the surrounding endolymph, and the otolithic mass will therefore “seek the lowest possible level. In doing so it will slide along the sensory epithelium whenever this slants from the truly horizontal level. Two types of otoliths are known. The first is solid and of a constant shape characteristic of the species and in fact of the specific otolith organ within the labyrinth. Thus, for example, in the minnow, Phoxinus laevis, the lapillus in the utriculus is near spherical, the sagitta in the sacculus is arrow-shaped with winglike ridges, and the asteriscus in the lagena is flat and star-shaped with a horseshoelike ridge on one side ( Wohlfahrt, 1932). They show annual growth rings by means of which the age of a bony fish can be fairly accurately assessed. The second type of otolith is made up of a pastelike mass of crystalline “statoconia” in a ground substance. The over-all size and shape of such statolithic masses can be fairly constant, but they appear to be able to

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show plastic flow. The solid otoliths are anchored to the membranous wall of the labyrinth by systems of connective tissue strands functioning like guy ropes. Such structures are sparingly developed in connection with pastelike otolithic masses, In the latter case the otolithic mass of one otolith organ can be continuous with that of another. In the elasmobranch labyrinth such continuity has been observed between otolithic masses in sacculus and lagena ( 0. Lowenstein, unpublished observations ) . The general shape and layout of the labyrinth in the various classes of chordate animals has been well described in the classic work of Retzius (1881), and his findings are remarkably accurate considering the restricted range of techniques at his disposal. A phylogenetic line of change leads from the myxinoids with two ampullae at either end of a dorsal canal-like space and two ampullae at the end of two vertical canals and a so-called macula communis via the petromyzonts with a macula already spatially subdivided into what may be interpreted as homologs of the maculae of the gnathostome labyrinth (Lowenstein et al., 1968; Lowenstein and Thornhill, 1971 ) to the gnathostome labyrinth with three semicircular canals and three well-separated otolith organs, viz., the utriculus, sacculus, and lagena, each equipped with a sensory macula carrying an otolith. The labyrinth of Myxine (Fig. 1A) lies fully enclosed in a cartilaginous capsule. On the floor of the membranous labyrinth lies a macula consisting of sensory hair cells and supporting cells. The hair processes of the sensory cells project into a jellylike “otolith membrane” which is loaded with numerous large spherites of carbonate of lime. They form a continuous otolithic mass. The macula itself lies in a near vertical plane with its anterior and posterior margins slightly curved toward the horizontal. As will be seen below, there is ultrastructural evidence that this macula communis contains three areas corresponding to the three maculae of the gnathostome labyrinth. There are two roughly spherical ampullary spaces above the macula each with a ring-shaped crista carrying an epithelium of sensory hair cells. Their orientation within each crista makes it plausible that the ampullae correspond basically to the anterior and posterior vertical ampullae of the gnathostomes. However, it can be shown that they are capable of signalling horizontal angular accelerations, i.e., of playing the role later taken over by the horizontal canal of the gnathostome labyrinth ( Lowenstein, 1970; Lowenstein and Thornhill, 1971). The upper cavity of the labyrinth of Myxine represents a fairly wide open space without any special compartmentalization that could properly be described as tubular vertical canals. The VIIIth nerve is subdivided into branches supplying the two ampullae and the various regions of the macula.

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The labyrinth of the lamprey, Lampetra fluuiatilk, has been well described by de Burlet and Versteegh (1930) and recently by Lowenstein et al. ( 1968). It is significantly more complex than the labyrinth of Mgxine (Fig. 1B ) . A unique feature which does not exist in the labyrinth of any other chordate are two large ciliary sacs forming the center of the cavity of the membranous labyrinth. Both the anterior and the posterior sac are further subdivided into upper and lower recesses. They are lined with an epithelium of large ciliated cells whose long and powerful cilia beat incessantly and create four vortices of endolymph currents of constant direction. Unfortunately, the functional significance of this imposing intralabyrinthine structure is still completely obscure. Around the ciliary spaces are grouped the various sensory epithelia. The macula communis still carries a continuous otolithic mass of calcareous crystals. However, its morphological and functional division into discrete areas is well pronounced and emphasized by the topographic arrangement of the sensory cells with these. A horizontal anterior macula area, a medium vertical macula area, and a posterior horizontal macula area may be held to correspond to the macula utriculi, macula sacculi, and macula lagenae of the gnathostome labyrinth. There is also an isolated dorsal patch of sensory epithelium corresponding to the macula neglecta. There are two ampullae each with a trifid crista and canallike tubes folded off the dorsal labyrinth cavity. These obviously correspond to the anterior and posterior vertical canals of the gnathostome labyrinth. They meet dorsally and their spaces are separated by a valvelike flange of tissue. There is no crus commune, nor is there a trace of a horizontal canal. As in Myxine, it can be shown that the cristae of the two vertical ampullae are capable of monitoring horizontal angular accelerations ( Lowenstein, 1970). From the elasmobranchs onward the labyrinth carries the full complement of sensory end organs. Shape, length, diameter, and mutual angle of the semicircular canals and otolith organs may vary widely, and this variation may be of important functional significance. C. The Sensory Hair Cell

Recent ultrastructural research has shown that the hair cells of the end organs of the acousticolateralis system are homologous. They are always surrounded by a framework of supporting cells, the cell bodies of which usually lie below the base of the sensory cells to make contact with the basal membrane of the sensory epithelium. In the fishes, in contrast to birds and mammals, the sensory hair cells are all of one morphological

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type (Fig. 2 ) . They are cylindrical cells with a large nucleus. At their apical end the cytoplasm contains an electron-dense so-called cuticular plate. This receives the simple elongated roots of numerous so-called stereocilia, hairlike processes with a uniform inner structure of thin longi-

Fig. 2. Schematic drawing to represent the general structure of the sensory epithelia in the labyrinth of the ray. Two sensory cells are shown. One has a hair process composed of “large” diameter and the other a hair process composed of “small” diameter stereocilia. Nerve endings make contact with the base of the sensory cells. The supporting cells bear on their free apical surfaces numerous microvilli and a short ciliary rod. BM, basement membrane; CR, ciliary rod; CU, cuticle; GA, Golgi apparatus; K, kinocilium; M, mitochondrion; MS, myelin sheath; MR, membrana reticularis; MV, microvillus; MVB, multivesicular body; N, nucleus; NE, nerve endings; SE, sensory cell; STL, ‘large’’-diameter sterocilia; STS, ‘‘small’’-diameter sterocilia; SU, supporting cell; and V, vesicle. From Lowenstein et al. (1964).

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213

tudinal fibrillae. Their number varies between a few tens and over a hundred and their length is usuaily graded, their tips forming a slope away from the single so-called kinocilium. This kinocilium is rooted in the cytoplasm outside the cuticular plate by means of a typical basal body or centriole. The kinocilium is, in fact, a true cilium, characterized by nine peripheral and two central double longitudinal filaments. The kinocilium is often much longer than the longest stereocilia. The centriole is composed of nine triplet tubules in continuity with the double filaments of the ciliary shaft. On the side facing away from the cuticular plate and from the stereocilia, one of the triplet tubules has a lateral excrescense of electron-dense material, the so-called basal foot. The arrangement of hair processes is thus strictly polarized, and it has been shown that this polarization is of fundamental functional significance ( Lowenstein et al., 1964). The hair cells of the cristae and maculae have been found to be oriented within the sensory epithelia in a constant characteristic pattern. It is assumed that the mechanical deformation of the hair bundle, or at least the shearing of the kinocilial base in the direction of the basal foot, has an excitatory effect and deformation in the opposite direction an inhibitory effect on the activity of the hair cell and its associated sensory neuron. The innervation of the hair cell is complex (Fig. 3 ) . At its base there are at least two types of nerve endings. Sensory neurons, the pericaria of which lie in the course of the branches of the VIIIth nerve innervating a given end organ, send their dendrites to the base of the sensory cells, where they synapse with the cell body or, more accurately, where the cell body of the sensory cell synapses with them. Opposite the nerve endings which may lie deeply in an indentation of the base of the sensory cell are found in the cytoplasm of the sensory so-called synaptic bars surrounded by a halo of synaptic vesicles. This synapse has the appearance of a typical chemically transmitting synaptic structure. The nerve ending opposite is relatively clear of vesicular inclusions but contains

_-

Fig. 3. Schematic drawing of the innervation of a hair cell (Type 11). Modified after Wersall et al. ( 1967).

214

0.LOWENSTEIN

numerous mitochondria. Conduction is obviously directed from hair cell to afferent nerve ending. Besides these postsynaptic nerve endings the hair cell is innervated by apparently efferent nerve endings. On the hair cell side of these one finds a flattened space or synaptic sac surrounded by a membrane located close to the plasma membrane of the cell. The presynaptic nerve ending, the terminal branch of a relatively thin efferent nerve, is filled with synaptic vesicles. Conduction is clearly from nerve ending to hair cell and may serve an inhibitory function.

11. FUNCTION

The labyrinth has four chief functions: It is an organ which is ( 1 ) concerned with the maintenance and regulation of muscle tone, ( 2 ) a receptor for angular accelerations, ( 3 ) a gravity receptor, and (4) a sound receptor. This follows from the fact that bilateral total labyrinth extirpation is followed by temporary or lasting loss in muscle tone, by severe disturbances of equilibrium, and by deafness. A. Equilibrium

In the fishes the effector organs showing equilibrium reflexes are the eyes, the fins, and to a lesser degree, the trunk musculature. The reflexes are controlled by the receptors of the semicircular canals and of the otolith organs, and they occur as compensatory rotary and vertical eye movements and postures during active and passive changes of position from the “normal.” Rotation of the fish in a horizontal plane evokes horizontal rostro-caudal eye deviations and nystagmus, easily observable when watching a fish executing right and left turns. These reflex movements and postures of the eyes facilitate the fixation of a certain visual field during spatial displacement of the head. When the compensatory eye deviation has reached its anatomical maximum, the eye is brought back with a jerk to its normal position in the orbit. Compensatory deviation and jerklike return together are known as the nystagmus representing its slow and quick phase, respectively. The slow phase runs counter to and the quick phase in the same direction as the rotary displacement of the head. The slow phase is controlled by the labyrinth (and optokinetically through the eye itself), whereas the quick phase is of central nervous origin. Figure 4 shows the reflex response of the fins of the minnow, Phoxinus phoxinus, to passive sideways tilting toward the right. The posture of the

7.

THE LABYRINTH

215

Fig. 4. Compensatory fin posture and vertical eye deviation in a fish tilted on to its right side. From Lowenstein ( 1936).

fins automatically leads to a righting of the fish as soon as a forward movement takes place (righting reflex) ( Lowenstein, 1932). Reflex bending of the trunk to right and left occurs in response to left and right turns, respectively. 1. THE SEMICIRCULAR CANALS

Since in life the cupula has about the same refractive index as the endolymph and shrinks drastically on fixation, its size was grossly underestimated until Steinhausen (1931, 1933, 1935) and Dohlman (1935) showed that it extends to the opposite wall of the ampullary dome, and during its deflections glides in swing-door fashion along it with a minimum of endolymph leakage. It was thus established that cupula and endolymph form a rigidly coupled system and that the elastic cupula is to be defined as a highly damped torsion pendulum with a period, in the case of the pike, Esox lucius, in the neighborhood of 20 sec. Deflections of the cupula under the impact of an angular acceleration in the appropriate plane of space represent the stimulus to which the animal reacts by dynamic effector reflexes in the form of compensatory movements of eyes and limbs. In the elucidation of the mode of function of the semicircular canals as such, and the interaction of the six canals in the maintenance of dynamic equilibrium, elimination experiments and experiments with artificial mechanical and caloric stimulation have served to establish a fair amount of information (Maxwell, 1923; Lowenstein, 1936). There remained, however, a hard core of controversial problems, the solution of which had to await the results of oscillographic analysis of the responses from isolated labyrinthine end organs. Fortunately, the elasmobranch labyrinth was found to be eminently suitable for this type of experiment. The various branches of the VIIIth nerve are relatively easily accessible through the cartilaginous skull, and, what is more, the organ survives for considerable periods of time in the isolated brain case. A thorough qualitative and quantitative analysis of the stimulus-response relationships governing the function of the semicircular canals could thus be carried out in the labyrinth of the dogfish (Lowenstein and Sand,

216

0. LOWENSTEIN

1936) and the ray, Raja clavata (Lowenstein and Sand, 1940a,b; Groen et al., 1952; Lowenstein, 1954). It was found that there is a spontaneous discharge of sensory impulses from a considerable proportion of the sense endings from each ampulla when the labyrinth is at rest (resting discharge). During angular displacement in the appropriate direction the discharge frequency of impulses is increased or inhibited as the case may be (Fig. 5 ) . The horizontal canal responds to rotation about the vertical primary axis of the fish, but it is unaffected by rotation about its longitudinal and transverse axes. The impulse discharge is augmented by ipsilateral ( ampulla-trailing ) and inhibited by a contralateral ( ampullaleading) rotary displacement. The anterior and posterior vertical canals respond to rotation about all three primary axes. During rotation about the horizontal longitudinal axis, the four vertical canals function in pairs with anterior and posterior canals of one side being arrayed against those opposite, and the discharge

Fig. 5. Impulse discharge from a single end organ in the left horizontal semicircular canal of the ray, Raja clauatu, in response to acceleration to the left ( A ) and to the right ( B ) . The lower strip of each part is the continuation of the upper. Time at the top of records is 0.05 sec. Acceleration is recorded by the signal line at the bottom. Successive gaps mark the revolution of the turntable through 12'. From Lowenstein and Sand (1940b).

7.

THE LABYRINTH

217

activity is augmented in the canals undergoing ampulla-leading and inhibited in those undergoing ampulla-trailing displacement. It is reasonable to assume that the resting discharge recorded in the afferent nerve fibers derives from constant excitatory activity at the synapse between hair cell and afferent nerve endings. This may result from constant percolation of transmitter substance across the synapse. Excitation may then result from an increase in transit of transmitter substance, inhibition from the cessation of its flow. The modulations in the amount of transmitter substance discharge must then in their turn be governed by the state and direction of deformation of the hair processes. However, the intermediate links in the chain of events between mechanical deformation and electric activity in the afferent nerves are not yet clearly understood. It is now certain that the polarization of the hair cells as described above is an important factor in the mode of functioning of the semicircular canal. Their orientation on the cristae is uniform. On the crista of the horizontal canal the kinocilia in the individual hair bundles point away from the canal end of the ampulla toward the ampulloutricular opening. Thus ampulla-trailing acceleration will lead to a deflection of the hair bundle in order to make the kinocilium bend toward the side of its basal foot. Such a deformation is excitatory. On the cristae of the vertical canal the kinocilia of the individual hair bundles uniformly point toward the canal end of the ampulla and away from the ampulloutricular opening. The hair bundles undergo excitatory deflection in this case during ampulla-leading acceleration. Postrotatory aftereffects in the form of poststimulatory depression and postinhibitory augmentation of the discharge activity are clearly correlated with well-known effector responses such as the postrotatory eye nystagmus. A functional correlation between the discharge activity in the six semicircular canals and the reflex responses of the six eye muscles was established, and it satisfactorily fulfilled all aspects of Sherringtonian muscle antagonism with respect to the labyrinthine eye reflexes (Lowenstein and Sand, 1940a). The existence of a resting discharge in the end organs of the semicircular canals is functionally highly significant. Above all it serves here as the background for bidirectionality, i.e., for the sensitivity of a given semicircular canal to rotations in opposite direction which lead to an increase or decrease in the impulse discharge rates. Apart from this, the steady influx of spontaneous activity from the sense organ into the central nervous system must contribute to the maintenance of muscle tonus. This was directly demonstrated in the pike, Esox lucius, where operative interruption of the nerve supply to one horizontal ampulla led to a pro-

218

0. LOWENSTEIN

tracted asymmetry in the tonus of the horizontal eye muscles. This asymmetry could be augmented by ipsilateral and diminished by contralateral horizontal rotation, a fact furnishing further evidence for the basic bidirectionality of the semicircular canal mechanism ( Lowenstein, 1937). 2. THE OTOLITHORGANS The prototype for the otolith organ is fully elaborated in the invertebrate statocyst, and gravity perception is one of the fundamental factors in animal orientation. It is interesting, however, that an auditory function was originally attributed to all so-called otocysts. Eimer (1878) and Romanes (1885) were the first to apply the newly discovered equilibrium function of the vertebrate ear to the lithocysts of medusae, attributing to them, in addition to their assumed auditory function, the regulation of locomotory movements. In the vertebrates the detailed study of the mode and function of the otolith organs has all along been handicapped by their inaccessibility to separate elimination or stimulation. The operative interruption of the nerve supply of individual otolith organs is generally almost impossible because of the shortness of the nerve branches, and the removal of the otoliths usually involves wide openings in the vestibular space with considerable endolymph loss and spread of the damage to all other parts of the labyrinth. It is therefore not surprising that for a long time the functional analysis of the otolith organs was mainly based on the interpretation of the spatial arrangement and the anatomic relationships of the otoliths and their sensory epithelia, supplemented by rather contradictory results of experiments with artificial mechanical and other stimulations carried out in elasmobranchs by Kubo (1906) and Maxwell (1923) and in the pike by Ulrich (1935). When it became clear, however, that fish react to sound over a considerable range of frequencies (see below), and that their power of sound perception and pitch discrimination is chiefly localized in the labyrinth, the conclusion became inescapable that otolith organs must be capable of gravity and sound perception alike. Apart from the fact that both sensory functions imply sensitivity to linear acceleration, they are so different in their biological significance that it was hard to believe that any single one of the three otolith organs, namely, utriculus, sacculus, and lagena, could combine both functions. The results of the search for a fundamental division of labor among the three otolith organs in fishes can be summarized as follows. In the elasmobranch Mustelus canis (Parker, 1909; Maxwell, 1923), in the bony fish, Pseudopleuronectes americanus ( Lyon, 1899), and in Carassius auratus ( Manning, 1924), Cynoscion regalis

7.

THE LABYRINTH

219

(Parker, 1908), and Gobius jozo (Werner, 1929), and finally in Phoxinus phoxinus ( Lowenstein, 1932; von Frisch and Stetter, 1932), separate functional elimination of parts of the otolith system showed that the utriculus is able to control the whole range of postural responses to positional changes. These responses consist of lasting eye deviations and fin postures, the compensatory significance of which is the same as that of the dynamic effector responses to angular accelerations. Bilateral elimination of the sacculus and lagena complex leaves the fish with its gravity responses practically unimpaired, whereas the bilateral elimination of the utriculus abolishes the whole range of known postural reflexes. The anatomical subdivision of the labyrinth into a pars superior (utriculus and semicircular canals) and a pars inferior (sacculus and lagena) is therefore physiologically significant. Often the separation of these two parts is considerable, e.g., in the minnow, Plzoxinus, only a narrow canal, the canalis uticulo-saccularis joins them, while in Gobius jozo the two parts are completely separated. Corresponding results were obtained in all other vertebrate classes (Lowenstein, 1936), although there remained a suspicion that in some cases the lagena may participate in the control of equilibrium [Schoen and von Holst (1950) in the fish, Gymnocymbus ternetzii; MacNaughton and McNally (1946) in the frog]. The extension of the oscillographic analysis of impulse responses from the semicircular canals to the less accessible otolith organs carried out by Lowenstein and Roberts (1949) in the elasmobranch Raja clavutu showed quite clearly that the utriculus macula does in fact respond to positional changes in all directions and that it is thus potentially capable of controlling the whole range of postural responses. Figure 6 shows a single-unit response of the utriculus to a full-circle tilt about the horizontal longitudinal axis ( lateral tilt), The discharge frequency shows a clear maximum near a position in which the labyrinth under observation lies uppermost (side up), and a minimum near the spatially opposite position (side down). A similar picture would be obtained during foreand-aft tilting about the horizontal transverse axis of the fish. Besides units which have static discharge rates strictly corresponding with certain spatial positions of the labyrinth (position receptors), there are other end organs in the macula which react by a change in their discharge rate to the change of position as such, irrespective of the direction of the change, returning to a basic discharge rate whenever the head has come to rest in a new position (out-of-position receptors). It was found that the posterior third of the sacculus macula closely adjoining the lagena also responds to positional changes and that it does so in much the same way as the utriculus. The two structures therefore overlap in range, which means that one of them could be considered to

220

0. LOWENSTEIN

Fig. 6. Continuous record of the response from two end organs of the utriculus of the ray, Raja clauata, to a full-circle lateral tilt. Time marker at top of record 24/sec. Rotation signal at the bottom: 1 gap/3’. Constant speed of tilting approximately 10°/sec. The maximum discharge frequency lies near the side-up and the minimum near the side-down position. From Lowenstein and Roberts (1949).

be dispensable from the point of view of equilibrium control. The lagena, on the other hand, shows a distinctly different mode of reaction, responding to both lateral and fore-and-aft tilting, but showing a sharp maximum of the impulse discharge rate in or near the “normal” position. The lagena may therefore be considered to signal by a steep increase in activity the return of the head to the normal spatial position. The term “into-level

7.

221

THE LABYRINTH

receptor” used for the lagena by MacNaughton and McNally (1916) appears thus to be very appropriate. Thus it appears that in the elasmobranch labyrinth sensitivity to gravitational stimuli is found in all three otolith organs. How far this is utilized in the elicitation of equilibrium responses is another question which cannot be decided by electrophysiological experiments. We shall return to the discussion of this question when dealing with the auditory function of the fish labyrinth. The ultrastructural investigation of the maculae of the labyrinth of the ray revealed the picture of orientation of the hair cells (Fig. 7). A similar topographic analysis by Flock (1964) in the bony fish, Lota vulgaris, resulted in a similar picture (Fig. 8 ) .

*x

Moculo neglecta

Fig. 7. Diagrammatic representation of the polarity of sensory hair bundles found in the cristae and maculae of the left labyrinth of the ray. Part of the dorsal wall of the sacculus above the macula neglecta and of the posterior wall of the lagena has been cut away to show their two sensory areas. In this schematic rendering of the sensory hairs the orientation of the hair bundle is symbolized by an arrow, the arrowhead indicating the position of the kinocilium. After Lowenstein et al. (1964).

Here, as in the semicircular canals, the directional arrangement of the unilaterally polarized hair cells is of fundamental functional significance. The population of hair cells in each macula represents a pattern of response directionalities, and the maculae do not therefore respond to uniform otolithic shearing forces tangentially to their whole surface. They are subdivided into different regions of often diametrically opposed hair cell orientation. Excitatory or inhibitory responses occur simultaneously or in succession as the otolithic mass flows along the macula surface under the influence of a gravitational or inertial stimulus.

222

0.LOWENSTEIN

Medial

Fig. 8. Schematic illustration of the pattern of morphological polarization of the sensory cells in the labyrinth of Lota uulgaris. From Wersall et al. (1967).

It was shown that the hair cells in the elasmobranch utriculus give excitatory responses to tilts around all axes, i.e., to diagonal, to fore-andaft, and to lateral tilts. In accordance with this, and with our basic hypothesis, we find in the utriculus macula hair cells whose kinocilia point outward interspersed with others pointing inward. However, the longitudinal axis of the utriculus itself runs at an angle to the longitudinal axis of the skull pointing forward outward, and the functional axis of the hair cells appears to run radially perpendicular to the periphery of the oval-shaped utriculus macula. It is, therefore, clear that the arrangement of the hair cells in the elasmobranch utriculus resembles that described for the utriculus macula of Lota by Flock (1964). They differ insofar as in Lota oppositely directed hair cells are separately assembled into a marginal inward looking and a central outward radiating field, whereas in an elasmobranch they are interspersed. In both cases the hair cell directions fully account for the response picture described for the elasmobranchs by Lowenstein and Roberts ( 1949). The preponderance of responses to side-up tilting over those to side-down tilting reported by these authors is not borne out by the configuration of the hair cell map, but was in all probability a consequence of selection of recording sites enforced by anatomical circumstances. Characteristic response pictures must therefore be expected to be obtainable from single units in the utriculus on tilts

7.

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223

about all possible axes. Fundamentally, therefore, it is perfectly feasible to postulate that the utriculus alone is capable of controlling all postural responses ( Lowenstein, 1932, 1936). However, the evidence from the work on elasmobranchs shows that in these animals at least the lagena has an important graviceptor role. During tilts its responses run counter to those of the utriculus insofar as the majority of units are found to have maximum discharge rates in or near the “normal” position of the skull in space. It is quite likely that further scrutiny might disclose more units the maximum activity of which occurs in the upside-down position. This guess is based on the presence in the near vertically extended macula lagenae of hair cells pointing dorsally interspersed with others pointing ventrally. It is also likely that the lagena may respond more sensitively to fore-and-aft than to lateral tilts. The macula sacculi lies in a near dorsoventral plane on the ventromedial aspect of the recessus sacculi. The hair cell map is simple. Two populations of hair cells are divided by a longitudinal line. Above it they point upward, below it downward. There is very little overlap along the dividing line. A scrutiny of the gravity responses from the posterior part of the sacculus shows similarities with those obtained from the utriculus. It might be expected that further electrophysiological mapping might show a preponderance of responses to lateral tilting since it is difficult to point to a topographically suitable substrate for a response to pure foreand-aft tilts. A further extrapolation from topography would be to expect good maxima nearer the normal and upside-down position during fullcircle lateral tilts, in contrast to the lagena in which these maxima may preponderate in the course of fore-and-aft tilting. The survey of the situation in the elasmobranch labyrinth based on published data may now be followed by a resume of results of work on the cyclostome labyrinth in Myxine and Lampetra carried out during the past few years by the author and collaborators. The labyrinth of the lamprey, Lampetra fluviatilis ( Lowenstein et al., 1968), contains an otolith-covered sensory epithelium clearly divisible into areas which may or may not be true homologs of the maculae of the gnathostome labyrinth (Fig. 9 ) . The interesting feature of these maculae is the continuity of the sensory epithelia and also of the overlying otolithic mass. The epithelium of the vertical macula is connected with that of the anterior and posterior horizontal maculae by upward twisting regions in which the hair cell pattern is transitional. In the vertical macula, the hair cells are arranged in two populations divided by a longitudinal line above and below which they point upward and downward, respectively, There is little overlap. This hair cell arrangement is identical with that of the gnathostome sacculus. The arrangement of the hair cells in

224

0. LOWENSTEIN Dorsal

Anterior horizontal rnacula Median

Lateral

Posterior

Ventral

Fig. 9. Diagram to illustrate the general orientation of the sensory cells of the macular areas of the right labyrinth of the lamprey. The arrowheads indicate the position of the kinocilium. From Lowenstein et al. ( 1968).

the anterior and the posterior horizontal maculae is strikingly symmetrical. In the outer halves of both horizontal maculae the hair cells point toward the lateral periphery. In the medial half of the anterior horizontal macula they point forward and in the medial half of the posterior horizontal macula backward. There are groups of diagonally directed hair cells. In the anterior horizontal macula they are found chiefly along the border between the forward and outward pointing populations and point diagonally forward outward. In the posterior horizontal macula the diagonally directed cells point backward outward and lie along the border between the backward and the outward pointing populations. The hair cell maps of the anterior and posterior horizontal maculae show therefore a remarkable degree of symmetry in opposition. We have evidence that embryologically the vertical macula develops first and then spreads upward to give rise to the horizontal maculae ( R. A. Thornhill, unpublished observations). The hair cell orientation in the adult maculae can be well understood in the light of this embryonic history. The anterior horizontal macula, the largest of the macular areas, appears to be the obvious homolog of the gnathostome macula utriculi. Insofar as the other areas are concerned it is felt that the earlier attempts

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225

to homologize the anterior transitional area with the sacculus, the vertical area with the lagena, and the posterior area with the macula neglecta (de Burlet and Versteegh, 1930) may well have to be revised in the light of the ultrastructural findings concerning the pattern of hair cell orientation. It is tempting to suggest that the vertical macula, including perhaps the transitional area between it and the anterior horizontal macula, is the homolog of the sacculus and the posterior horizontal macula that of the lagena. It is true, the gnathostome lagena lies usually in a near-vertical plane at about right angles to that of the sacculus. However, the spatial dispositions of maculae are subject to great variation in the gnathostomes. For instance, in some elasmobranch (dogfish) labyrinths a portion of the usually horizontal utriculus macula lies in a near-vertical plane, whereas in others (Raja) both the usually vertical sacculus macula and the utriculus macula approach the horizontal. The macula neglecta of the lamprey may correspond to what de Burlet and Versteegh described as macula ductus dorsalis. The macula communis of Myxine is less complex at first sight (Lowenstein and Thornhill, 1971). It lies on the medial aspect of the labyrinth capsule and lies in a near vertical plane. Its anterior and posterior ends curve around becoming more horizontal in the process. There is no anatomically different middle region. However, the hair distribution corresponds closely to that on the three maculae in the lamprey, if one imagines these to be projected more or less into a single plane. What does this all signify in terms of function? The similarity between the horizontal maculae in the lamprey and the anterior and posterior ends of the macula communis in Myxine encourage the hypothesis that they may function in much the same way as has been demonstrated in the elasmobranch Raja clauata. The orientation of the hair cells on these two maculae can account for excitatory responses to fore-and-aft, lateral, and diagonal tilting in opposite directions. Whether or not the median vertical macula of the lamprey participates in gravity reception cannot be ascertained since the localization of the source of impulse responses in electrophysiological experiments on the isolated labyrinth ( Lowenstein, 1970) is not sufficiently accurate. There is, however, circumstantial evidence for the localization of vibration sensitivity in the vertical macula as well as in the so-called macula neglecta of the lamprey labyrinth. In both these sensory epithelia is found an aberrant type of hair cell, the hair bundle of which consists of an exceptionally tall and stiff kinocilium accompanied by a bundle of exceptionally short stereocilia. Clear-cut responses to vibration have been recorded from the eighth nerve of the lamprey. No such specialized hair cells are found in the macula communis of Myxine, and there is no evidence that the organ responds to vibration.

226

0. LOWENSTEIN

Furthermore, a macula neglecta is absent in Myxine. It appears therefore that the vertical macula may resemble the sacculus macula of the elasmobranchs by having a dual function. The specialized hair cells in the vertical macula are dispersed among the rest and not confined to any specific area of the macula. Early speculations on the relationships between the disposition of these maculae in space and the origin of the various postural reflexes of eyes and limbs await reappraisal in the light of such new evidence. Here is an important field for further research. One may, however, be justified in assuming that the utriculus is generally the chief receptor for gravitational stimuli ( Lowenstein, 1932, 1936). There are exceptions. There is evidence that in the herringlike bony fishes a part at least of the utriculus rather than the sacculus may be the organ concerned with hearing (Wohlfahrt, 1932, 1936) and it was shown that in the flatfish, Pleuronectes platessa and Platessa flesus, the chief gravitational responses derive from the sacculus with the possibility of participation by utriculus and/ or lagena ( Schone, 1964). Whereas von Frisch and Stetter (1932) believed that in the minnow, Phoxinus h v i s L., both sacculus and lagena may function as sound receptors, there is definite evidence that in the bony fish, Gymnocymbus tereutzii, and in the frogs, Rana sylvatica and Rana palusiris, the lagena participates in the control of postural equilibrium (Schoen, 1950; MacNaughton and McNally, 1946). It is interesting to note that removal of the lagena in these cases leads to instability in or near the normal position. The electrophysiological findings ( Lowenstein and Roberts, 1949) point to a similar functional range for the lagena in the elasmobranchs. In summary it may be said that the otolith organs respond to linear accelerations in general. They are therefore all potential gravity receptors. Besides this they may respond to linear translation, centrifugal stimuli, and rotating linear vectors during constant speed and accelerated rotations and finally to oscillatory linear accelerations in the form of vibrational and acoustic stimulation. Equilibrium in fishes is not controlled by the labyrinth alone. In the absence of highly developed proprioceptor mechanisms, visual orientation takes a considerable share in the control of posture and movement. This is supplemented by so-called dorsal-light reactions which manifest themselves in a tendency of the animal to turn its dorsal side in the direction of the incidence of illumination. The collaboration of eye and labyrinth in this field has been analyzed by von Holst and his collaborators by means of most ingenious experimental methods (von Holst, 1950). These investigations have made a significant contribution to the understanding of quantitative and qualitative aspects of otolith function.

7.

THE LABYRINTH

227

B. Hearing

The history of the problem of hearing in fishes has been reviewed in detail by von Frisch (1936), Kleerekoper and Chagnon (1954), and Enger (1968). It may suffice to recall here that from antiquity onward fish have been believed to be endowed with sound perception. This was believed to be localized in the internal ear and also in the lateral line. However, under the impact of the discovery of the equilibrium function of the labyrinth in the first half of the nineteenth century, and in view of the absence in the fish labyrinth of any structure corresponding with the cochlea of the higher vertebrates, the assumption gained ground that fish may after all be deaf, and that all earlier evidence for their power of sound perception ought to be considered with suspicion since it was probably based on a misinterpretation of circumstantial evidence. Even if sound perception could be convincingly demonstrated in fishes, it was argued, the term “hearing” should be used only if it could definitely be shown that the inner ear was the chief receptor organ concerned. After the publication of work by Zenneck (1903), Piper (1906), and Parker ( 1904, 1909), evidence in favor of hearing in fishes began to accumulate. A number of workers based their claims on observations of direct responses of various species of fish to sound stimuli. occurrence and repeatability of such responses was, however, often doubtfully documented, and no convincing picture emerged, chiefly because the various acoustic stimuli used were without biological significance for the experimental animal. Here, as so often, the method of conditioning helped to overcome the difficulty and yielded convincing evidence in favor of sound perception over a considerable range of frequencies in a number of families of fishes. 1. FREQUENCY RANGEAND PITCHDISCRIMINATION

Table I surveys the reliable data including, in a number of cases, the frequency range of responses. The Ostariophysi show a significantly better performance insofar as threshold, range, and pitch discrimination are concerned, and this fact is held to be connected with the presence of a swim bladder and its linkup with the labyrinth by the chain of Weberian ossicles (von Frisch, 1936; Poggendorf, 1952; Kleerekoper and Roggenkamp, 1959). The evidence in favor of sound reception among teleosts is thus impressive and makes the assumption of a well-defined biological significance of hearing inescapable. Sound production exists in a great variety of types of fish and suggests the existence of acoustic communication among individuals. This

Table I A List of Teleosts in Which Hearing Has Been Reliably Demonstrated, with Data for Frequency Ranges Where Availableo Frequency range (in Hz) Family

Species

Low

High

B

Reference

Non-Ostariophysi Anaban tidae

Anabas scandens ( = A . testudineus)

Over 659

Betta splendens Colisa lalia Macro podus cupanus Macropodus opercularti Trichogaster leeri Trichogaster trichopterus Anguillidae Cottidae Cyprinodontidae

Embiotocidae Esocidae Gadidae Gobiidae

Labridae

Mormyridae

Anguilla anguilla Anguilla anguilla Cottus gobio Cottus scorpius Fundulus heteroclitus Lebistes reticulatus Lebistes reticulatus Cymatogaster aggregatus Umbra limi Umbra pygmaea Gadus aeglejinus Gadus callarias Gobius niger Gobius paganellus Periophthalmus koelreuteri Crenilabrus griseus Crenilabrus melops Crenilabrus pavo Gnathonemus sp.

26374699

36

488450

44

1200-2068

800 600-800 Up to 651

2 7 9 4 3136

Diesselhorst (1938) Schneider (1941) Schneider (1941) Schneider (1941) Schneider (1941) Schneider (1941) Schneider (1941) Bull (1928) Diesselhorst (1938) Stetter (1929) Froloff (1925) Parker (1904) Farkas (1935) Farkas (1936) Moorhouse (1933) Westerfield (1921) von Frisch and Stetter (1932) Froloff (1925) Froloff (1925) Dijkgraaf (1952) Dijkgraaf (1950) Diesselhorst (1938) Froloff (1925) Bull (1928) Froloff (1925) StipetiE (1939)

0

Percidae Sciaenidae

Sparidae

2069-3 100

Marcusenius isodori Acerina cernua Perca Jluviatilzs Corvina nigra Corvina nigra Corvina nigra Sargus anniilaris

1024 1000 1250

Diesselhorst (1938) Froloff (1925) Froloff (1925) Dijkgraaf (1950) Dijkgraaf (1952) Froloff (1925) Dijkgraaf (1952)

Ostariophysi

C haraoinidae

Cobitidae Cyprinidae

Gymnot.idae Siliiridae

a

Hemigrammus caudovittatus Hyphesscbrycon j a m m e u s Pyrrhulina rachoviana

6960

Nemacheilus barbat u!a Alburnus lucidus ( = A . alburniis) Carassius auralus

1740-3480

Carassius auralus Carassius carassius Idus melanotus ( = Leucisus idus) Idiis melanotus ( = Ikiiciscus idus) Leuciscus dobula IZutiliis rutil us Phoxinus phoxinus Phoxinus phoxinus Phoxinus phoxinics

3480

5524

25 16

Pimephales nolatiis 7’inca tinca h’lectrophorus elcclricus 11meiur us nebulos us Ameiurus nebulosus

From von Frisch (1‘336) and Kleerekoper and Chagnon (1954).

5000-7000 5000-6000

870-1035 Over 1300

von Boiitteville (1935) von Boutteville (1935) von Boutteville (1935) Stetter (1929) Zenneck (1903) Bigelow (1904); Manning (1924); Ilenker (1931) Stetter (1929) Froloff (1925) Ilenker (1931) Stetter (1929) Zenneck (1903) Zenneck (1903) von Frisch (1938) von Frisch and Stetter (1932) Stetter (1929); Denker (1931); Benjamins (1‘334); von Bontteville (1935); Hafen ( 1935) McDonald (1921) Froloff (1925, 1928) von Boutteville (1935) von Frisch (1938) Maier (1909); Haempel (1911); Parker and van Heusen (1917); Krausse (1918); von Frisch (1923); Stetter (1929)

i-

230

0.MWENSTEIN

aspect, however, lies outside the scope of the present chapter (but see Chapter 6 by Tavolga, this volume). Sound reception usually goes hand in hand with pitch discrimination. Table I1 summarizes the results in this field. Two different types of pitch discrimination have to be distinguished, namely, absolute discrimination between two frequencies when offered singly on different occasions and relative discrimination of frequencies of equal intensity offered in rapid succession in the form of a warbling note ( Wohlfahrt, 1939, 1950). In the latter case, discrimination reaches the astonishing lower limit of a quarter tone in the 400-800-Hz range. The absence in the fish labyrinth of a special morphologically recognizable frequency analyzer structure of the basilar membrane type adds additional physiological interest to these findings. Insofar as threshold sensitivities are concerned, relatively few reliable Table I1 Pitch Discrimination in Various Speciesa Range of tone distinction (in Hz)

Species

Umbra sp. Anguilla anguilla Gobius niger Corvina nigra Sargus annularis

Non-Ostariophysi 288 and 426 Less than one octaveb 4 up to l a toneb (9-15% frequency distinction) 9 up to l a toneb (9-15y0 frequency distinction) t up to 1: toneb (9-15y0 frequency distinction) octaveb

+

Marcusenius isodori (Mormyridae) One tone* Gnathonemus sp. (Mormyridae) Macropodus opercularis Varying between one tone and I f octavesb (Anabantidae) Phoxinus phoxinus Phoxinus phoxinus Phoxinus phoxinus

Ostariophysi A major third in 821-651 range; a minor third in 290-345 range tone in 987.7-1046.5 range (670 frequency distinction) t tone in 40k300 range

+

Reference

Westerfield (1921) Diesselhorst (1938) Dij kgraaf (1952) Dijkgraaf (1952) Dijkgraaf (1952) Diesselhorst (1938) Stipetid (1939) Schneider (1941)

Stetter (1929) Wohlfahrt (1939) Dijkgraaf and Verheijen (1950)

a From Kleerekoper and Chagnon (1952). b T h e frequency range in which distinction of tones was examined was not mentioned by these authors.

7.

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data are available. Poggendorf (1952) measured the absolute values of the threshold sound pressure in dynes per square centimeter in Ameiurus nebulosus for a range of frequencies and found a steep rise of thresholds above 1600 Hz. At the optimum frequency of 800 Hz, the absolute threshold is as low as that for the optimum frequency of 3200 Hz in man. At low frequencies Ameiurus is considerably more, and at high frequencies considerably less, sensitive than man. In cyprinids, too (Stetter, 1929; von Boutteville, 1935), the relative sensitivity to sounds in the middle frequency range was found to be of the same order of magnitude as in man. It is eminently clear that fish in which the labyrinth is associated with some kind of air space are very significantly more sensitive to sound than others (Enger, 1968). They also respond to frequencies far above 1000 Hz whereas many of the others are sensitive to low frequencies only. A specific physical link between air bladder and labyrinth such as the chain of Weberian ossicles in the cyprinids, characinids, and silurids (Fig. 10) appears to contribute to the superiority in sound reception of these fish over other ostariophysan forms (Table I ) . Poggendorf (1952) found that in the catfish the operative removal of one of the Weberian ossicles, the so-called malleus, reduced the sensitivity by 30-40 dB and the destruction of the air bladder of the catfish results in a loss of sensitivity by 13 dB at 330-750 Hz and 30 dB at 1500 Hz ( Kleerekoper and Roggenkamp, 1959). The highest sensitivity for sound lies generally well below 1000 Hz in nonostariophysan fish, often below 500 Hz or between 600 and 800 Hz. It is at present doubtful whether extrusions of the air bladder toward the labyrinth, as found in the Sparidae and Clupeidae, for instance, always confer higher sensitivity and upward extension of the audible frequency range (Dijkgraaf, 1952; Tavolga and Wodinsky, 1963, 1965; Enger, 1968). The best auditory performance is recorded for those nonostariophysan fish in which the labyrinth is associated with closely adjacent air-filled cavities such as the Mormyridae and the Anabantidae ( TabIe I). Apart from the study of unconditioned and conditioned behavioral responses, audiograms can be obtained by recording the impulse responses in the eighth nerve or the parts of the brain associated with it. This method is of crucial importance in the investigation of the localization of acoustic responses (vide infra),but it has yielded useful information on sensitivity and frequency limits. Thus Enger (1968), recording from auditory neurons in the medulla oblongata of the herring, Clupea harengus, found a threshold of probably less than -20 dB ref. 1 pbar for frequencies up to 1200 HZ with a steep rise in threshold above this frequency and a probable upper limit of responsiveness at 4000 Hz.

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Transverse

canal

Stapes Incus Malleus

Air bladder

Fig. 10. The coupling between swim bladder and the labyrinth by means of Weber’s ossicles in the ostariophysans. Weber’s ossicles, black. Redrawn from von Frisch ( 1936).

So far we have dealt with teleosts only. The elasmobranchs lack an air bladder and any other air space associated with the labyrinth, but it is clear from recent studies that they respond to sound both in the far and near field (Enger, 1968). Bull sharks, Curcharhinus leucas, were conditioned by Kritzler and Wood (1961) to respond to sound between 100 and 1500 Hz with a lowest threshold at 400-600 Hz (Nelson and Gruber, 1963). Nelson ( 1966) showed that sharks respond to low-frequency pulsed sound in the far field (15-25 meters) and that they locate a sound source in the open sea from as far as 200 meters. Thresholds are high (above 1 pbar). In experiments with the lemon shark, Neguprion breuirostris (Poey), Banner ( 1967) finds responses to sound between 20 and 1000 Hz stimulated in the near field with thresholds ranging between 20

7 . THE

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and 40 dB above 1 pbar according to frequency and method of transduction. The responses observed in these experiments were jaw movements and brief cessation of respiration either unconditioned or negatively reinforced by electric shocks. Lowenstein and Roberts ( 1951) recorded frequency-synchronized impulse responses from the isolated surviving labyrinth of the ray, Raja clavata, when the preparation was exposed to substrate-conducted mechanical vibrations. Such impulse responses showed a fixed phase relationship with the microphonic synchronous change in electric potential up to

Fig. 11. Record of a response from the sacculus in the isolated labyrinth of the thornback ray, Raia clavata, to a vibratory stimulus of 110 Hz. Eight traces are superimposed, and the relatively undistorted microphonic wave carries high-amplitude spike potentials. From Lowenstein and Roberts ( 1951).

234

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120 Hz (Fig. 11). At that time no reliable information on “hearing” in elasmobranchs was available. Nevertheless, the experiments established the fact that the labyrinth contained vibration-sensitive end organs. Hearing without the power of fairly accurate localization of the source of sound would appear to be of rather restricted value to an animal. However, experiments on Phoxinus by von Frisch and Dijkgraaf (1935), and on Plzoxinus and Ameiurus by Reinhardt ( 1935), both under natural conditions and in the laboratory, showed that only relatively high intensity sound offered at close range (10 and 20 cm) had a directive effect, and this effect was not impaired by the operative elimination of the labyrinth, the Weberian apparatus, and the lateral line organs. The authors therefore attributed this limited sound localization to the tactile sensitivity of the skin. Kleerekoper and Chagnon (1954), using low frequency vibrations in relatively small tanks, observed that Semotilus atromaculatus atromaculatus (Mitchill) orientated toward lines of highest intensity in a complex pattern of standing waves and intensity gradients from one or more sources. The authors make no statement as to the nature of the receptor organs involved in these reactions. 2. THE SEATOF ACOUSTICFUNCTION There is no reason to doubt that in fishes, as in other vertebrates, the inner ear is the chief organ concerned with hearing, and the work of von Frisch and his pupils has yielded convincing evidence that, at least in the Ostariophysi, the pars inferior of the labyrinth, viz., the sacculuslagena complex, is chiefly responsible for sound reception, whereas the equilibrium function of the labyrinth resides in the pars superior, viz., utriculus and semicircular canals ( Lowenstein, 1932). The fact that the sound-conducting structures, such as the Weberian apparatus and socalled acoustic windows in the cranium (von Frisch, 1938; Dijkgraaf, 1950), appear to be closely associated with the pars inferior in the Ostariophysi makes this assumption still more cogent. In other fish, such as the Clupeidae, the morphological situation is different. Here the swim bladder-labyrinth connection clearly aims at the utriculus, and the question arises ( d e Burlet, 1935; Wohlfahrt, 1936) whether potentially all otolith organs could take on an acoustic function, the decisive factor being the exposure to or insulation from vibratory stimuli, differing from case to case in accord with the morphological situation. That this may in fact be so is strongly suggested by the results of the electrophysiological experiments on the labyrinth of the elasmobranch Raia claavata (Lowenstein and Roberts, 1951) . In these experiments the propagated impulse discharges in response to low-frequency vibratory stimulation (up to 120

7.

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Hz) were recorded from nerve twigs from the anterior part of the sacculus macula, from the unloaded part of the utriculus macula (lacinia utriculi), and from the macula neglecta situated in the roof of the sacculus cavity. No such vibration responses were found in the lagena which proved to contain gravity receptors only. A cranial window, the fenestra of Scarpa, is anatomically related to the vicinity of the sacculus and the macula neglecta, and it is quite possible that the vibration sensitivity of part of the utriculus macula may not be utilized by the animal. It must not be overlooked that the participation of the lateral line in the elicitation of responses to sound is to be seriously considered in cases where the sound source is situated near enough to give rise to water displacement in the vicinity of the fish. The lateral line and its function is discussed elsewhere (Chapter 8 by Flock). However, it must be stated here that in a number of the experiments especially with elasmobranchs near-field effects could not be excluded. In the lower frequency range to which elasmobranchs are chiefly sensitive such near-field effects extend for a considerable distance from the source. At 100 Hz, for instance, or a wavelength of 15 meters the far- and near-field effects are equal at a distance of 2.4 meters from the source (Enger, 1968). It is therefore imperative, especially in the study of directional responses, to make clear the conditions of stimulation obtaining the experiments. Accepting the effect of a basic vibration sensitivity of the otolith organs in fishes, the great differences in the acuity of hearing and the power of pitch discrimination found to exist between the Ostariophysi and the rest may well result entirely from the elaboration of resonators and sound conductors associated with the labyrinth. The remarkable fact of a highly developed power of pitch discrimination in the absence of a morphologically obvious frequency analyzer still remains one of the problems of fish physiology. It is probable, however, that this may be based upon a synchronization between the stimulus frequency and the frequency of the impulse discharge. The fact that pitch discrimination is absent above 800 Hz would be compatible with this assumption on neurological grounds ( Lowenstein and Roberts, 1951). That there may after all be differential frequency sensitivity within the area of a given otolith organ has been claimed by Enger (1963). In an analysis of the responses of single “auditory” neurons in the brain of the sculpin, Cottus scorpius, Enger found four types of neurons classified by their resting activity which responded differently to sound. Type I had a regular resting activity but did not respond to sound, but Enger believes these to be static units. Type I1 units had no resting discharge and responded to sounds below 200 Hz. Type I11 units had an irregular

236

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resting activity and responded to a wide band of frequencies, and type IV units showed spontaneous bursts of activity and responded to sounds up to 300 Hz with frequency-synchronized patterns of response without adaptation. Enger believes that units I1 and I11 may well be connected with different parts of the otolith-bearing end organs mediating a placedetermined frequency discrimination, whereas type IV units subserve frequency-synchronized information transfer from sense organ to brain. Pitch discrimination has also been demonstrated for elasmobranchs by Nelson (1966) who claims that the lemon shark, Negaprion brevirostris, discriminates between about half an octave apart in the 40-60 Hz band. This refers to absolute pitch, the two tones having been offered at a time interval of about 1min. REFERENCES Banner, A. (1967). Evidence of sensitivity to acoustic displacemznts in the lemon shark, Negaprion breoirostris ( Poey ), In “Lateral Line Detectors” ( P. Cahn, ed. ), pp. 265-273. Indiana Univ. Press, Bloomington, Indiana. Benjamins, C. E. (1934). La fonction du Saccule. Rev. Laryngol. Otol. Rhinol. 55, 1233-1242. Bigelow, H. B. (1904). The sense of hearing in the goldfish Carassius auratus L. Am. Naturalist 38, 275-284. Bull, H. 0. (1928). Studies on the conditioned responses in fishes. 1. Marine B i d . Assoc. U . K . 15, 485-533. de Burlet, H. M. ( 1935). Vergleichend Anatomisches uber endolymphatische und perilymphatische Sinnesendstellen des Labyrinthes. Acta Oto-Laryngol. 22, 287305. de Burlet, H. M., and Versteegh, C. (1930). Uber den Bau und die Funktion der Petromyzonlabyrinthes. Acta Oto-Laryngol. Suppl. 13, 1-58. Denker, A. (1931). Uber das Horvermogen der Fische. Oto-Laryngol. 15, 247-260. Diesselhorst, G. (1938). Horversuche an Fischen ohne Weberschen Apparat. Z. Vergleich. Physiol. 25, 748-783. Dijkgraaf, S. ( 1950). Untersuchungen uber die Funktionen des Ohrlabyrinths bei Meeresfischen. Physiol. Camp. Oecol. 2, 81-106. Dijkgraaf, S. ( 1952). Ueber die Schallwahrnehniung bei Meeresfischen. Z. Vergleich. Physiol. 34, 104-122. Dijkgraaf, S . and Verheijen, F. J. (1950). Neue Versuche iiber das Tonunterscheidungsvermogen der Elritze. 2. Vergleich. Physiol. 32, 2A8-256. Dohlman, G. ( 1935). Some practical and theoretical points in labyrinthology. Proc. Roy. Sac. Med. 28, 1371-1380. Eimer, T. ( 1878 ). “Die Medusen.” Tubingen. Enger, P. S. (1963). Single unit activity in the peripheral auditory system of a teleost fish. Acta Physiol. Scand. 59, Suppl. 210. Enger, P. S. (1968). Hearing in fish. In “Hearing Mechanisms in Vertebrates” (A. V. S. de Reuck and J. Knight, eds.), pp. 4-17. Churchill, London. Farkas, B. (1935). Untersuchungen iiber das Horvermogen bei Fischen. Allattani Kozlem, 32, 1-20.

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Farkas, B. (1936). Zur Kenntnis des Horvermogens und des Gehororgans der Fische. Acta Oto-Laryngol. 23, 499-532. Flock, A. (1964). Structure of the macula utriculi with special reference to directional interplay of sensory responses as revealed by morphological polarization. J . Cell Biol. 22, 413-431. Froloff, J. P. (1925). Bedingte Reflexe bei Fischen, I. Pfliigers Arch. Ges. Physiol. 208, 261-271. Froloff, J. P. (1928). Bedingte Reflexe bei Fischen 11. Pflugers Arch. Ges. Physiol. 220, 339-349. Groen, J. J., Lowenstein, O., and Vendrik, A. J. H. ( 1952). The mechanical analysis of the responses from the end-organs of the horizontal semicircular canal in the isolated elasmobranch labyrinth. J. Physiol. ( London) 117, 329-346. Haempel, 0. (1911). Zur Frage des Horvermogens der Fische. Intern. Rev. Hydrobiol. 4, 315-326. Hafen, G. (1935). Zur Psychologie der Dressurversuche. 2. Vergleich. Physiol. 22, 192-220. Kleerekoper, H., and Chagnon, E. C. (1954). Hearing in fish, with special reference to Semotilis atromaculatus atromculatus ( Mitchill). J. Fisheries Res. Board Can. 11, 130-152. Kleerekoper, H., and Roggenkamp, P. A. (1959). An experimental study on the effect of the swimbladder on hearing sensitivity in Ameiurus nebulosus nebulosus (Lesueur). Can. J. 2001.37, 1-8. Krausse, A. (1918). Kritische Bemerkungen und neue Versuche iiber das Horverm'ogen der Fische. 2. Allgem. Physiol. 17, 263-286. Kritzler, H., and Wood, L. ( 1961 ). Provisional audiogram for the shark, Carcharhinus leucas. Science 133, 1480-1482. Kubo, I. (1906). Uber die vom N . acusticus ausgelosten Augenbewegungen. Arch. Gcs. Physiol. 115, 457482. Lowenstein, 0. ( 1932). Experimentelle Untersuchungen iiber den Gleichgewichtssinn der Elritze (Phorinus laevis L.). 2. Vergleich. Physiol. 17, 806-854. Lowenstein, 0. ( 1936). The equilibrium function of the vertebrate labyrinth. Biol. Rev. 11, 113-145. Lowenstein, 0. (1937). The tonic function of the horizontal semicircular canals in fishes. J . Exptl. Biol. 14, 473482. Lowenstein, 0. ( 1954). The effect of galvanic polarization on the impulse discharge from sense endings in the isolated labyrinth of the Thornback Ray (Raja clauata). J. Physiol. (London) 127, 104-117. Lowenstein, 0. (1970). The electrophysiological study of the responses of the isolated labyrinth of the lamprey ( Lampetra fluviatilis) to angular acceleration tilting and mechanical vibration. PTOC.Roy. SOC.B 174, 419-434. Lowenstein, O., and Roberts, T. D. M. (1949). The equilibrium function of the otolith organs of the Thornback Ray (Raja cluvata). J. Physiol. (London) 110, 392415. Lowenstein, O., and Roberts, T. D. M. (1951). The localization and analysis of the responses to vibration from the isolated elasmobranch labyrinth. A contribution to the problem of the evolution of hearing in vertebrates. J . Physiol. (London) 114, 471-489. Lowenstein, O., and Sand, A. (1936). The activity of the horizontal semicircular canal of the dogfish, Scyllium canicula. J. Exptl. Biol. 13, 416428.

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Lowenstein, O., and Sand, A. (1940a). The individual and integrated activity of the semicircular canals of the elasmobranch labyrinth. J. Physiol. (London) 99, 89101. Lowenstein, O., and Sand, A. (1940b). The mechanism of the semicircular canal. A study of the responses of single-fibre preparations to angular accelerations and rotations at constant speed. Proc. Roy. SOC. B129, 256-275. Lowenstein, O., and Thornhill, R. A. (1971). Submitted for publication. Lowenstein, O., Osborne, M. P., and Wersall, J. (1964). Structure and innervation of the sensory epithelia of the labyrinth in the Thornback Ray (Raja claaata). Proc. Roy. SOC. B160, 1-12. Lowenstein, O., Osborne, M. P., and Thornhill, R. A. (1968). The anatomy and ultrastructure of the labyrinth of the lamprey (Lampetra jluuiatilis, L.). Proc. Roy. SOC. B170, 113-134. Lyon, E. T. (1899). A contribution to the comparative physiology of compensatory motions. Am. J. Physwl. 3, 86-114. McDonald, H. E. ( 1921). Ability of Pimephales notatus to form associations with sound vibrations. J. Comp. Physiol. 2, 191-193. MacNaughton, I. P. J., and McNally, W. J. (1946). Some experiments which indicate that the frog’s lagena has an equilibrium function. 1. Laryngol. Otol. 61, 204-214. Maier, H. N. (1909). Neue Eeobachtungen iiber das Horvermogen der Fische. Arch. Hydrobid. 4, 393-397. Manning, F. B. (1924). Hearing in the goldfish in relation to the structure of its ear. J. Exptl. Zool. 41, 5-20. Maxwell, S. S. ( 1923). “Labyrinth and Equilibrium.” Lippincott, Philadelphia, Pennsylvania. Moorhouse, V. H. K. (1933). Reactions of fish to noise. Contrib. Can Biol. Fisheries 7 , 465-475. Nelson, D. R. (1966). Hearing and acoustic orientation in the lemon shark, Negaprion brezjirostris (Poey), and other large sharks. Dissertation Abstr. 27, 1. Nelson, D. R., and Gruber, S. H. (1963). Sharks: Attraction by low-frequency sounds. Science 142, 975-977. Parker, G. H. (1904). Hearing and allied senses in fishes. Bull. U. S. Fisheries Commun. 22, 45-64. Parker, G. H. (1908). Structure and function of the ear of the Squeteague. U . S. Bur. Fisheries Bull. 28, 1213-1224. Parker, G . H. (1909). Influence of the eyes, ears and other allied sense organs on the movements of the dogfish, Mustelus canis. U . S. Bur. Fisheries Bull. 29, 4%57. Parker, G. H. and van Heusen, A. P. (1917). The reception of mechanical stimuli by the skin, lateral line organs and ears in fishes, especially in Ameiurus. Am. J. Physiol. 44, 463489. Piper, H. ( 1906). Aktionsstrome vom Gehororgan der Fische bei Schallreizung. Zentr. Physiol. 20, 293-297. Poggendorf, D. ( 1952). Die absoluten Horschwellen des Zwergwelses ( Ameiurus nebulosus) und Beitrage zur Physik des Weberschen Apparates der Ostariophysen. 2. Vergleich. Physiol. 34, 222-257. Reinhardt, F. ( 1935). Uber die Richtungswahrnehmung bei Fischen, besonders bei der Elritze (Phoxinus Zuevis) und beim Zwergwels (Ameiurus nebulosus Raf.). 2. Vergleich. Physiol. 22, 570-604.

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Retzius, G. ( 1881). “Das Gehororgan der Wirbeltiere.” Stockholm. Romanes, G. H. ( 1885). “Jellyfish, Starfish and Sea-urchins.” Kegan Paul, London. Schneider, H. (1941). Die Bedeutung der Atemhohle der Labyrinthfische fur ihr Horvennogen. Z. Vergleich. Physiol. 29, 172c194. Schoen, L. ( 1950). Quantitative Untersuchungen uber die zentrale Kompensation nach einseitiger Utriculus-ausschaltung bei Fischen. Z. Vergleich. Physiol. 32, 121-150. Schoen, L., and von Holst, E. (1950). Das Zusammenspiel von Lagena und Utriculus bei der Lageorientierung der Knochenfische. Z. Vergleich. Physiol. 32, 552-571. Schone, H. ( 1964). Ober die Arbeitsweise der Statolithen-apparate bei Plattfischen. Biol. Jahresh. 4, 135-156. Steinhausen, W. (1931). Ober den Nachweis der Bewegung der Cupula in der intakten Bogengangsampulle des Labyrinthes bei der naturlichen rotatorischen und kalorischen Reizung. Arch. Ges. Physiol. 228, 322-328. Steinhausen, W. ( 1933). Ober die Funktion der Cupula in den Bogengangsampullen des Labyrinthes. Z. Hub-, Nasen- u. Ohrenheik 34, 201-211. Steinhausen, W. (1935). Ober die Cupula. 2. Huh-,Nasen- u. Ohrenheik. 39, 1962. Stensio, E. (1927). “The Downtonian and Devonian vertebrates of Spitzbergen I.” Oslo. Stetter, H. (1929). Untersuchungen uber den Gehorsinn der Fische besonders von Phoxinus luevis L. und Ameiurus nebulosus Raf. Z . Vergleich. Physiol. 9, 339447. Stipetik, E. (1939). Uber das Gehororgan der Mormyriden. Z. Vergkich. Physiol. 26, 740-752. Tavolga, W. N., and Wodinsky, J. (1963). Auditory capacities in fishes. Pure tone thresholds in nine species of marine teleosts. Bull. Am. Museum Nut. Hist. 126, 177-240. Tavolga, W. N., and Wodinsky, J. (1965). Auditory capacities in fishes: Threshold variability in the blue-striped grunt, Haemulon Sciurus. Animal Behauiour 13, 301-31 1. Ulrich, H. (1935). Die Funktion der Otolithen, gepriift durch direkte mechanische Beeinflussung des Utrikulus otolithen am lebenden Hecht. Arch. Ges. Physiol. 235, 545-553. von Boutteville, K. F. (1935). Untersuchungen iiber den Gehorsinn bei Characiniden und Gymnotiden und den Bau ihres Labyrinthes. Z. Vergleich. Physiol. 22, 162191. von Frisch, K. (1923). Ein Zwergwels, der kommt, wenn man ihm pfeift. Biol. Zentr. 43, 439446. von Frisch, K. (1936). Ober den Gehorsinn der Fische. Biol. Rev. 11, 210-246. von Frisch, K. (1938). Ueber die Bedeutung des Sacculus und der Lagena fur den Gehorsinn der Fische. Z. Vergleich. Physiol. 25, 703-747. von Frisch, K., and Dijkgraaf, S. (1935). Konnen Fische die Schallrichtung wahrnehmen? Z. Vergleich. Physiol. 22, 641-655. von Frisch, K., and Stetter, H. (1932). Untersuchungen iiber den Sitz des Gehorsinnes bei der Elritze. Z. Vergleich. Physiol. 17, 686-801. von Holst, E. (1950). Die Arbeitsweise des Statolithen-apparates bei Fischen. 2. Vergleich. Physiol. 32, 60-120. Werner, C. F. (1929). Experimente iiber die Funktion der Otolithen bei Knochenfischen. Z. Vergleich. Physiol. 10, 26-35. Wersall, J., Gleisner, L., and Lundquist, P.-G. (1967). Ultrastructure of the vestibular

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end organs. In “Myotatic, Kinesthetic and Vestibular Mechanisms” (A. V. S. de Reuck and J. Knight, eds.), pp. 105-120. Churchill, London. Westerfield, F. (1921). The ability of mud-minnows to form associations with sounds. 3. Comp. Psychol. 2, 187-190. Wilson, H. V., and Mattocks, J. E. (1897). The lateral sensory anlage in the salmon. Anat. Anz. 13, 658-660. Wohlfahrt, T. A. (1932). Anatomische Untersuchungen uber das Labyrinth der Elritze (Phoxinus laevis L. ). Z . Vergleich. Physiol. 17, 659-685. Wohlfahrt, T. A. (1936). Das Ohrlabyrinth der Sardine (Clupea pilchardus Walb.) und seine Beziehungen zur Schwimmblase und Seitenlinie. Z. Morphol. Oekol. Tiere 31, 371410. Wohlfahrt, T. A. ( 1939). Untersuchungen iiber das Tonunter-scheidungsvermogen der Elritze (Phoxinus laevis Agass.). 2. Vergleich. Physiol. 26, 570-604. Wohlfahrt, T. A. ( 1950). Wber die Beziehungen zwischen absolutem und relativem Tonunterscheidungsvermogen sowie iiber Intervallverschmelzung bei der Elritze ( Phoxinus laevis Agass. ). Z. Vergkich. Physiol. 32, 151-175. Zenneck, J. ( 1903). Reagieren die Fische auf Tone? Arch. Ges. Physiol. 95, 34-6.

8 THE LATERAL LINE ORGAN MECHANORECEPTORS" A H 3 FLOCK

.

I. Introduction . . . . . . 11. Structure of the Sense Organ . . . A. Methods of Ultrastructural Study . B. CanalOrgans. . . . . . C. Epidermal Organs . . . . 111. Sensory Excitation in the Hair Cell . A. Methods in Physiological Study . . B. Properties of the Receptor Potential . C. Origin of the Receptor Potential . IV. Transmission at the Sensory Synapse . V. Initiation of Nerve Impulses . . . VI. The Central Nervous System and Feedback VII. Conclusion . . . . . References . . . . . .

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241 242 243 244 247 248 249 251 253 255 258 259 261 262

I. INTRODUCTION

It is intended here to deal in particular with new techniques in the study of structure and function of lateral line organs. In morphological work conventional electron microscopy is complemented by the scanning microscope which opens a door to a new world of topographic beauty; intracellular recordings from single hair cells may provide a new insight into the mechanism by which hair cells transduce mechanical energy into electrical activity in sensory nerve fibers; and data processing by computer helps to analyze the message traveling in the nerve fibers to * This work was supported by grants from the Swedish Medical Research Council, Therese and Johan Anderssons Minnesfond, Elin and Sven Sjobergs Minnesfond, King Custaf V jubileumsfond and by the National Institutes of Health grant No. NB 03956-06. 241

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the central nervous system. This presentation is concerned with sensory transduction in the peripheral organ, for information about the biological significance of the organ system and behavioral aspects other sources are referred to. In 1966 the first conference on lateral line detectors was held. The proceedings from this conference contains a wealth of knowledge about this sensory system, and the reader is referred to this work as well as to a monograph by Dijkgraaf (1963) for extensive literature references.

11. STRUCTURE OF THE SENSE ORGAN

The mechanoreceptor organs of the lateral line system extract information of water motion from the external environment. Relevant modes of water motion include laminar flow (Gijrner, 1963) as well as surface wave eddies (Schwartz, 1967) and near-field displacement caused by vibrating sources (Harris and van Bergeijk, 1962). The adequate stimulus for an organ depends on the characteristics of the mechanical coupling between the organ and the environment. In each case, however, the adequate stimulus for the hair cell is the bending, or the angular displacement, of the sensory hairs that extend as a bundle from the apical end

Fig. 1. In the canal organs of Lota lotu the cupula rests on the sensory epithelium at the bottom of a bony half-cylinder.

8.

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of each receptor cell. This is effected by a gelatinous structure called the cupula which rests upon the sensory epithelium and to which the sensory hairs are attached. There are two types of lateral line mechanoreceptors: In canal organs the sensory area is situated at the bottom of a canal sunk down below the skin in a system of canals which is particularly well developed on the head (Jakubowski, 1967). Epidermal organs, as the name implies, are situated in the skin with the cupula extending into the outside water. A. Methods of Ultrastructural Study

It is relevant here to give a brief description of electron microscopic techniques. Canal organs in the teleost Lota b t u were exposed by dissection in the anesthetized and curarized animal, the fixation fluid [1%

Fig. 2. When the cupula is taken off, the rhomboid surface of sensory epithelium is exposed. The framed area corresponds to that magnified in Fig. 3.

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osmium tetroxide buffered with veronal acetate according to Rhodin (1954)l was applied in situ with a pipette. The organ was then dissected free with its bony capsule, rinsed in Ringer, and dehydrated with 70% alcohol. When appropriate areas had been exposed for scanning microscopy the tissue was frozen in liquid propane at -192°C and freezedried. After mounting with glue on metal holders the specimens were covered with gold by vacuum evaporation and examined in a Cambridge Stereoscan microscope. A similar procedure applies to epidermal lateral line organs which were taken from the salamander, Necturus m u l o s u s . Specimens for transmission electron microscopy were fixed in osmium tetroxide, dehydrated in alcohol, embedded in Epon ( Luft, 1961) , sectioned in an LKB Ultrotome, and examined in a Siemens Elmiskop I.

B. Canal Organs The sensory area is located at the bottom of a bony half-cylinder ( Fig. 1).At this point the lumen of the canal is narrowed by the presence of a septum; here the cupula reaches its maximum height and partially occludes the lumen. If volume displacement occurs between the two

Fig. 3. Bundles of sensory hairs project from the sensory surface.

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sides of the septum the cupula will be subjected to shearing displacement along the axis of the canal. The narrowing of the canal lumen at this point may provide amplification of displacement amplitude. The cupula is a rhomboid dome, the matrix consisting of a finely fibrillar substance as seen in the transmission electron microscope. The surface outline clearly demonstrates the striated appearance resulting from the columnar aggregation of cupular material. Concentric shelves of surface architecture may reflect periodicity in the growth of the cupula. With the cupula dissected away the surface of the sensory epithelium becomes available for inspection (Fig. 2 ) . Sensory hair bundles from several hundred hair cells extend perpendicular to the sensory epithelium (Fig. 3 ) . They are about 7 p high, counting the kinocilium, and their diameter is about 1.5p. The surface of supporting cells exhibit numerous microvilli. Each sensory hair bundle is composed by about 35 stereocilia which are arranged in a stepwise fashion, the length increasing toward an

Fig. 4. In the center are two sensory hair bundles which have their kinocilia ( k ) facing opposite directions. To the right is a bundle where the stepwise increasing length of the stereocilia ( s ) can be clearly seen. Bulbous swellings on one kinocilium are artifacts.

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asymmetrically placed kinocilium ( Fig. 4). Neighboring cells have opposite orientation, a well-known fact from transmission microscopy, with every other cell facing toward the tail or toward the head (Fig. 4). At the periphery of the sensory epithelium is a zone of mantle cells which border endothelial cells lining the canal wall (Fig. 5 ) . The endothelial cells exhibit a striking feature of lamellar microvilli arranged in a labyrinthine pattern. These cells are probably secretory and contribute to the formation of the canal lymph. At some distance farther away from the sensory area crypts appear in the epithelial lining ( Fig. 6 ) . These are likely to represent orifices of glands or goblet cells.

Fig. 5. Bordering the sensory epithelium is a zone of mantle cells ( m ) , below this the epithelial lining of the canal wall takes over. The area "se" is magnified in Fig. 6.

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C. Epidermal Organs The epidermal organs in Necturus maculosus are arranged in so-called “stitches,” comprised of groups of 2 5 neuromasts (Fig. 7). Each neuromast contains 6-10 hair cells which reach with their apical ends to the bottom of a shallow pit from which the tall and cylindrical cupula extends for about 150 p (Fig. 8 ) . Hair cells in this animal are unusually large, probably because of the large size of the nucleus; cytoplasmic organelles are of ordinary size. As in Xenopus (Murray, 1955) each stitch is inner-

Fig. 6. Adjacent to the sensory area is a secretory epithelium where openings lead to goblet cells or glands.

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vated by two myelinated nerve fibers which are afferent and a few thin myelinated nerve fibers which carry efferent impulses. Physiologically one whole stitch serves as one functional unit rather than the individual neuromast since each nerve fiber branches in a tram-line fashion to contact all neuromasts within the stitch. The sensory hair bundle springing from each hair cells contains about 15 stereocilia and a single kinocilium. The stereocilia are somewhat less well developed than in canal organs, they are not arranged in the same orderly fashion, and their height is less. Within each neuromast neighboring hair cells also face opposite directions, along the axis of the stitch. 111. SENSORY EXCITATION IN THE HAIR CELL

When the hair cell is mechanically excited by cupula displacement electric potential changes occur inside the cell. This response, the receptor potential, can be recorded with intracellular microelectrodes as

Fig. 7. Three epidermal neuromasts of Necturus maculosus occupy the bottom of a shallow ditch. From each organ a fragile cupula extends. “Mulberry” cells are supeficial cells of the epidermis.

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Fig. 8. Section through one neuromast in Necturms maculosus: hc, hair cells; c, cupula; sc, supporting cells; and e, epidermis.

was recently successfully done in the epidermal organs in Necturus maculosus (Harris et al., 1970), which is a suitable organ for this kind of work because of the large size of its hair cells. Such potentials can also be recorded from the canal organ of Lota lota although the hair cells here are quite small.

A. Methods in Physiological Study Figure 9 shows the experimental arrangement for recording from Necturus hair cells. The tram-line pattern of innervation of the neuromasts within the stitch allows nerve action potentials excited from one neuromast by vibratory stimulation (by the capillary tube A ) to be recorded in the neighboring organ (by electrode C ) . This way the sensitivity of the organ recorded from (with the intracellular electrode B) can be monitored throughout the experiment. In the canal organ the cupula can be driven directly by coupling a glass tube to the top of the cupula, in this situation the stimulus amplitude, direction, and phase relations can be well controlled. The use of a stepmotor to drive the microelectrode aids the penetration of the cell wall. That the origin of the response is a

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Fig. 9. Schematic drawing which illustrates an experiment for recording of intracellular receptor potentials in Necturus rnaculosus. While the left organ is stimulated by motion of a capillary tube A, an electrode B containing vital stain is inserted into a hair cell ( h ) in this organ. Nerve action potentials evoked from this organ spread in the myelinated nerve fibers ( n ) , they antidromically invade a neighboring neuromast where they are recorded by another microelectrode C. From Harris et al. ( 1970). e = epidermis, s = supporting cells, a = afferent ending, k = kinocilium.

hair cell is ascertained by electrophoretic injection of a blue dye, Niagare Skye Blue 6B, which fills the microelectrode; the cell can be seen by direct vision in the microscope during the experiment or it can be identified by histological and electron microscopic procedures. The interior of the hair cell bears an interior negative electric charge, 15-25 mV in Lota, which slowly deteriorates during the recording as a consequence of the puncturing of the cell wall. This is faster in the small cells of Lota (maximum 5 min) than in large Necturus cells (maximum 1 5 2 0 min) . Mechanical stimulation evokes an electric response with the same frequency as the stimulus. The receptor potential is quite small (ca. 800 pV in Necturus and maximum 1.5 mV in Lota). It is advanta-

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geous therefore to increase the signal-to-noise ratio by averaging with a computer.

B. Properties of the Receptor Potential Several criteria indicate that this is a true biological potential and no epiphenomenon to mechanical motion of the electrode or the tissue, one is the fact that methylene blue abolishes the response reversibly when applied in the vicinity of the cupula while it leaves the resting potential of the cell unchanged. The response reappears when the methylene blue is washed away. The receptor potential is highly directional. If the direction of stimulus displacement is rotated 360" around the compass rose the receptor potential in canal organ hair cells varies as a cosine function

A

Fig. 10. Traces A and B are receptor potentials recorded intracellularly from two different hair cells in the canal organ of Lota h a . They are seen to be 180° out of phase, Time scale is at the bottom, above this is the stimulus driving voltage.

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of stimulus direction: A hair cell which is depolarized by cranial displacement is hyperpolarized by caudal displacement whereas displacement perpendicular to the axis of the canal gives little or no output. Different hair cells have responses of opposite polarities as seen in Fig. 10 which shows recordings from two hair cells 180” out of phase. In the lateral line, as in the inner ear, microphonic potentials can be recorded with gross extracellular electrodes. The hair cells have been suggested as the responsible generators, a theory which now seems to be confirmed. It should then represent the summed output of all hair cells “seen” by the electrode. Such summation of extracellular current may be simulated in the computer by summating, point by point, the responses A and B in Fig. 10. The result, as seen in Fig. 11, is a signal twice the frequency of the stimulus. This is in fact characteristic of the externally recorded microphonic potential in lateral line canal organs which is thus the distortion product between hair cells of opposite polarity.

A+B

B

A

Fig. 11. Each of the curves A and B are summed averages of receptor potentials from several hair cells like those in Fig. 10. The upper curve A f B is the result from adding these two curves point by point.

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These findings intimately relate to ultrastructural findings on the basis of which the observed receptor potential characteristics have been predicted earlier (Flock and Wersall, 1962). As seen in Fig. 4 each hair cell is asymmetrically polarized by the presence of a kinocilium in one end of the bundle and by the stepwise increasing length of the stereocilia toward the kinocilium. Neighboring cells face opposite directions, toward the head or toward the tail alternately. There is strong indication that this morphological polarization underlies the directionality of the receptor potential in such a way that bending of the sensory hairs in the direction in which the kinocilium is leading causes depolarization of the hair cell whereas opposite bending causes hyperpolarization. It is a task for future researchers to investigate the mechanism of generation and the possible role of the receptor potential; at the present time some possible mechanisms might be considered.

C. Origin of the Receptor Potential The appearance of a receptor potential is an early sign of excitation of receptor elements in sense organs, this applies to the eye, the organs of olfaction, and to the inner ear and other mechanoreceptors throughout the animal kingdom. It is likely to play a causative role as an early step in the sensory process by which hair cells detect mechanical motion of molecular dimensions. It is of importance to inquire what is the underlying mechanism capable of such extraordinary performance. At least two possible mechanisms suggest themselves; one relates to the mechanical sensitivity of the cell membrane in mechanoreceptors such as the Pacinian corpuscle and one to the mechanical sensitivity of cilia. The receptor potential can be recorded extracellularly as well as intracellularly. Electric current thus flows in a path that engages the hair cell; when the hair cell is depolarized it enters the apical end and leaves through the cell wall over the rest of the cell. The current thus passes the sensory hair bundle and the apical cell membrane where the mechanical excitatory energy acts; this is the likely location of a sensitive mechanoelectric transducer. The plasma membrane that covers the stereocilia is a triple-layered membrane (Fig. 12) which comprises a sizable increase of area exposed to mechanical energy. Perhaps the membrane accommodates gates for electric current or ion flow. Gating may be governed by mechanical interaction directly in the membrane, or it may be controlled by a transducer at some other site. The hair cell bears a single kinocilium characterized by an internal

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Fig. 12. The stereocilia are surrounded by a triple-layered plasma membrane which is continuous with that of the cell's apical surface.

Fig. 13. Schematic drawing showing the ultrastructure of the basal portion of the kinocilium with its basal body and one neighboring stereocilium. The arrow indicates the direction of excitatory deformation. From Flock ( 1965).

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structure of nine peripheral double-barreled tubules surrounding a central pair of simple tubules (Fig. 13), the peripheral tubules have an intracellular extension which constitute the basal body. From the wall of the basal body a basal foot projects in a direction away from the stereocilia or rather away from the cuticular plate in which the rootlets of the stereocilia are planted. This structure is quite similar to that of other types of cilia. The sensitivity of cilia to mechanical deformation is well known and has been particularly studied by Thurm (1968). He concludes from experiments on mussel cilia that the basal portion of the cilium, particularly the shaft and perhaps the basal body, are the principal mechanically sensitive portions. The eff eclive stimulus is a shearing displacement parallel to the cell surface. Intracellular potential changes have been recorded in ciliated cells during mechanical stimulation. However, Thurm concluded on the basis of his experiments that mechanical sensitivity and beat induction in the cilium are not dependent on a change in membrane potential: This is rather a secondary effect perhaps produced by some output from the basal body. To summarize, it seems that mechanically sensitive cilia have some effector output, chemical or electrical, which is generated by a sensitive transducer, which may then be directed to induce a ciliary beat or to cause a membrane potential change depending on the requirements. One possibility is that the receptor potential is generated in this way by the action of some agent produced by perhaps the basal body on the apical cell membrane. Resulting changes in membrane permeability may allow the movement of ions along their electrochemical potential gradients and thus cause potential changes recorded as electrical events by the microelectrode.

IV. TRANSMISSION AT THE SENSORY SYNAPSE

In sense organs containing primary receptor cells the sensitive site is an integral part of the sensory neuron (Pacinian corpuscle, etc.). Here the receptor potential acts to directly trigger nerve action potentials. In secondary receptor cells such as hair cells, spike initiation is probably not caused directly by the receptor potential but via synaptic transmission between the neuroepithelial sensory cell and the innervating afferent nerve endings. In this case the role of the receptor potential has been a more controversial question. The hair cells are innervated by nerve endings from bipolar sensory neurons which contact the bottom of the cell (Fig. 14). This synapse is characterized by the presence, inside the hair cell, of a dense synaptic

.h, FLOCK

256

Stc

AfhmnI nerve

Efferent nerve

ending (sensory)

ending (inhibitory)

,

I

Fig. 14. The hair cell is innervated by afferent nerve fibers as well as by efferent inhibitory ones.

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body surrounded by vesicles with a diameter of 350 A (Fig. 14). These vesicles are similar to presynaptic vesicles in motor end plates and at other sites in the nervous system where neurochemical transmission occurs. It is thus likely that sensory transmission is mediated by a transmitter substance of yet unknown pharmacology. A second type of nerve endings also contact the hair cells, these are endings of efferent nerve fibers and are characterized by the presence of synaptic vesicles inside the nerve ending and by a subsynaptic sac inside the hair cell in apposition to the nerve ending. The transmitter has not been identified although it is likely to be acetylcholine which has been demonstrated to occur in the efferent system of the inner ear (Schuknecht et al., 1959). The liberation of transmitter substance at a neurochemical synapse results from depolarization of the presynaptic terminal caused by the nerve action potential traveling down the axon into the terminal. That the release mechanism is in fact governed by the presynaptic potentialand not a consequence of other changes occurring in the presynaptic membrane when it is depolarized by the action potential-has recently been demonstrated by Katz and Miledi (1967). They found, in muscle end plates and in squid giant synapses where electrical excitability was blocked by tetrodotoxin, that transmitter release could be evoked by electric pulses delivered to the presynaptic terminal. It remains to be seen whether the receptor potential in the hair cell may have a similar function of governing the release of the transmitter at the afferent synapse. One indication that potential change is causative in transmission is the fact that efferent nerve endings inhibit afferent nerve impulses (Russell, 1968) via their action on the hair cell. Efferents have been seen to terminate on hair cells only (Hama, 1965; Flock, 1965); no synaptic contacts have been seen between efferent nerve endings and afferent terminals, and so inhibition is by presynaptic action on the hair cell. In similar situations within the nervous system inhibition is brought about by a hyperpolarizing effect of the efferent synapse or by shunting of current which would otherwise have excited the afferent synapse. Thus, here, it is by manipulation of the presynaptic potential that inhibition is brought about. On the other hand, the receptor potential is quite small-even at high intensities of stimulation it rarely exceeds 1 mV. Is it possible for such a small potential change to have an effect on transmitter release? Certain fishes are equipped with electroreceptor organs which derive from the lateral line system. These organs are extremely sensitive to external potential fields. In some types of electroreceptor organs the afferent discharge rate is linearly related to graded stimuli (see Bennett, 1965, and this volume). Excitation of the afferent terminal is likely to be by chemical transmission, the voltage across the presynaptic membrane

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determining the rate of transmitter release. The sensitivity of these synapses to change in electric potential is such that if the afferent synapse on hair cells works by a similar mechanism then the receptor potential recorded in hair cells is large enough to be causative in the synaptic process.

V. INITIATION OF NERVE IMPULSES

In the neuromuscular junction, as well as in other neurochemical synapses, each synaptic vesicle contains a fixed amount of transmitter substance capable of producing one unit of potential change in the postsynaptic membrane-a miniature postsynaptic potential. Each miniature potential represents a depolarization of the postsynaptic terminal which travels along its nonmyelinated portion down to the point where the myelin sheath starts. This site is electrically excitable; when a threshold level of depolarization is reached the nerve action potential is triggered. Each miniature potential is generally of subthreshold size; however, the integrative capacity of the nonmyelinated terminal makes possible the summation of several miniature potentials to reach threshold. The spontaneous release of transmitter is random in time and so the firing of a nerve potential is probabilistic. What is said here may apply also to excitation of sensory nerve fibers in lateral line organs as well as in the inner ear; in fact, postsynaptic potentials have recently been recorded from the saccular nerve in fish by Furukawa and Ishii (1967). Spontaneous firing of the nerve in Xenopus is probabilistic; that is, the histogram of successive spike intervals shows a Poisson distribution (Harris and Milne, 1966). When the organ is stimulated, nerve action potentials are triggered by each cycle of a periodic stimulus (Fig. 15); in other words, the probability of the nerve firing becomes time-locked to the stimulus. The underlying mechanism may be a synchronization of transmitter release to the stimulus causing an increased probability of nerve firing. There may as well be an actual increase in release of transmitter during part of the stimulus cycle. An increase in intensity is signaled by an increasing number of spikes occurring during each cycle as well as by shortening of the latency between the stimulus and the first spike ( Fig. 16). According to their response two types of nerve fibers can be distinguished. Some fibers are excited when the cupula is displaced in one direction along the axis of the canal, other fibers are excited by displacement in the opposite direction; this is true in canal organs (Sand, 1937) as

8.

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, Counts N

20

40

60

= ((4

80

400 msec

4-

Fig. 15. Below is an oscilloscope trace recording nerve action potentials responding to 7 cycles of a 70-Hz vibration applied to the cupula. Above is the poststimulus time histogram of 114 such stimulus presentations which shows that the nerve response is phase locked to the stiniulus ( Lotu lotu).

well as in superficial neuromasts (Gorner, 1963). Thus, each nerve fiber couples only to hair cells with the same orientation.

VI. THE CENTRAL NERVOUS SYSTEM AND FEEDBACK

The central nervous system receives information from a large number of lateral line sense organs with a wide distribution over the body. Most fishes are equipped with both types of organs: On a background of spontaneous nerve firing they signal information about environmental water motion, its velocity and direction, and the presence of vibrating

260

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-140

N.500

1

-120

I

I

totol counts.

20'9

N.500 total counts.

1036

I

(6) Fig. 16. The sets of curves above (. A ,) and ( B ) show the effect of a 17-dB increase in stimulus intensity on the nerve response locked to the rising phase of a periodic stimulus. In ( A ) the lower picture shows two action potentials evoked by ,

I

the stimulus. Above is a dot display where consecutive sweeps are displayed above each other, each spike being represented by a bright dot. There is considerable variation in the time of occurrence of the first spike as is also evident in the time histogram above. In ( B ) 4-5 spikes occur with shorter delay and regular timing

( L o b bta).

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objects such as prey. They do not respond to pressure changes per se; canal organs could if pressure gradients were to develop between distant organs. However, this is not likely to occur in acoustic stimulation since the wavelength of sound in water at the frequency fishes hear is far too long even for a sizable fish (Harris, 1967). On the other hand, a pressure field pulsating at auditory frequency would cause the swim bladder to change its volume synchronously and so cause near field displacement perhaps capable of exciting lateral line organs (van Bergeijk, 1967). Acoustic stimulation cannot thus be discarded as a possible source of input to the central nervous system via lateral line organs. Little is known about the central connections of the lateral line system, and even less is known about the neurophysiology of central nuclei and pathways. The sensory nerve fibers are extensions from bipolar neurons, the cell bodies forming a diffuse ganglion inside the brain case. They terminate in the medulla in a nucleus closely associated with the statoacoustic nucleus. Much is to be learned about information processing in central nuclei: For instance, what is the reason for the bidirectionality of the sensory input? Do sensory fibers of opposite polarity impinge upon a common central neuron to provide some kind of push-pull or differential action? The efferent innervation of the peripheral organs has an inhibitory influence on the afferent nerve discharge as shown by Russell (1968). He tentatively suggests that the cell bodies of the efferent nerve cells in Xenopus are either present in the medulla or in the ganglion. They may provide a means of setting the gain of the peripheral sense organ or they may be engaged in discrimination of adequate stimuli. It is not known whether the efferent system is under control of the central nervous system or whether it functions as an autonomous reflex arch.

VII. CONCLUSION

The lateral line mechanoreceptor organs provide a sophisticated system for detection of water motion. Each individual organ exhibits the cellular components and the basic circuitry that make the organs of hearing and equilibrium in the inner ear such delicate sensors. There is reason to believe that the process of sensory transduction in the hair cell is the same as in the inner ear; this is true also for synaptic transmission between the hair cell and the sensory nerve fiber. Inhibition of afferent input by efferent activity takes place in much the same way. Consequently, lateral line organs are not only indispensable to fish,

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they are also invaluable to the researcher who wants to investigate sensory processes in the acoustico-lateralis system at a cellular level. REFERENCES Bennett, M. (1965). Electroreceptors in Mormyrids. Cold Spring Harbor S y m p . Quant. Biol. 30, 215-262. Cahn, P. H., ed. (1967). “Lateral Line Detectors.” Indiana Univ. Press, Bloomington, Indiana. Dilkgraaf, S. ( 1963). The functioning and significance of the lateral-line organs. Biol. Reu. 38, 51-105. Flock, A. ( 1965). Electron microscopic and electrophysiological studies on the lateralline canal organ. Acta Oto-Laryngol. Suppl. 199, 1-90. Flock, A., and Wersall, J. (1962). A study of the orientation of the sensory hairs of the receptor cells in the lateral-line organ of a fish with special reference to the function of the receptors. J. Cell Biol. 15, 19-27. Furukawa, T., and Ishii, Y. ( 1967). Neurophysiological studies on hearing in goldfish. J. Neurophysiol. 30, 1377-1403. Gorner, P. ( 1963). Untersuchungen zur Morphologie und Elektrophysiologie des Seitenlinieorgans vom Krallenfrosch ( Xenopns laevis Daudin ). Z . Vergbich. Physiol. 47, 316-338. Hania, K. (1965). Some observations on the fine structure of the lateral line organ of the Japanese sea eel Lyncozymba nystromi. J. Cell Biol. 24, 193-210. Harris, G. G. (1967). Comments. In “Lateral Line Detectors” (P. Cahn, ed.), pp. 231-236. Indiana Univ. Press, Bloomington, Indiana. Harris, G. G., and Milne, D. C. (1966). Input-output characteristics of the lateral line organs of Xenopus laevis. J . Acoust. SOC. Am. 40, 3 2 4 2 . Harris, G. G., and van Bergeijk, W. A. (1962). Evidence that the lateral line organ responds to the nearfield displacements of sound sources in water. J. Acoust. SOC. Am. 34, 1831-1841. Harris, G. G., Frishkopf, L., and Flock, A. (1970). Receptor potentials from hair cells of the lateral line. Science 167, 76-79. Jakubowski, M. (1967). A method for the manifestation of lateral line canals and their neuromasts in fishes. Copeia I, 234-235. Katz, B., and Miledi, R. ( 1967). Tetrodotoxin and neuromuscular transmission. Proc. Roy. SOC.B167, 8-22. Luft, J. H. (1961). Improvements in epoxy resin embedding methods. J. Biophys. Biochem. Cytol. 9, 409. Murray, R. W. ( 1955). The lateralis organs and their innervation in Xenopus laevis. Quart. J. Microscop. Sci. 96, 351. Rhodin, J. ( 1954). “Correlation of Ultrastructural Organization and Function in Normal and Experimentally Changed Proximal Convoluted Tubuli Cells of the Mouse Kidney,” pp. 1-76. Thesis. Aktiebolaget Godvil, Stockholm. Russell, I. J. (1968). Influence of efferent fibres on a receptor. Nature 219, 177-178. Sand, A. (1937). The mechanism of lateral sense organs of fishes. Proc. Roy. SOC. B123, 47-95. Schukneckt, H. F., Churchill, J. A., and Doran, R. (1959). The localization of acetylcholinesterase in the cochlea. A d . Otolaryngol. 69, 549-559.

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Schwartz, E. (1967). Analysis of surface wave perception in some teleosts. In “Lateral Line Detectors” ( P. Cahn, ed. ), pp. 123-134. Indiana Univ. Press, Bloomington, Indiana. Thurm, U. (1968). Steps in the transducer process of mechanoreceptors. Symp. Zool. Sac. Land. 23, 199-216. van Bergeijk, W. A. (1967). The evolution of vertebrate hearing. In “Contributions to Sensory Physiology” (W. D. Neff, ed.), Vol. 2, pp. 1 4 9 . Academic Press, New York.

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THE MAUTHNER CELL J . DIAMOND

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I Introduction . . . . . . . . . I1. The Basic Anatomy of the Mauthner Neuron . . I11. The Selective Activation of Mauthner Neurons . . A . Choice of Stimulus . . . . . . . B. Choice of Recording Technique . . . . C . Selective Recording from an Individual Mauthner Cell D . The Investigation of the Effects of Mauthner Cell Excitation . . . . . . IV . The “Mauthner Reflex” . . . . . . . A . The Response . . . . . . . . B . The Inhibition of the Response . . . . C. Time-Course of the Inhibition . . . . . D . The Minimum Discrimination Time . . . . V. The Spinal Circuitry . . . . . . . . A . The Segmental Nature of the Reflex . . . B . The Spinal Responses . . . . . . . C . Understanding the Minimum Discrimination Time . VI . The Anatomy of the Spinal Circuitry . . . . A . Evidence from Light Microscopy . . . . B . The Mauthner Collaterals and the Motoneurons . C . Evidence from Electron Microscopy . . . D. Conclusion on the Identity of the . . . . . . A1 “Connecting” Unit . E . An Excitatory Cascade? . . . . . . VII . The Precision and Constancy of the . . . . . Minimum Discrimination Time A . The Function of the A1 Unit . . . . . B . The Implication of a Noise-Free System . . . VIII . The Excitation of the Mauthner Cells . . . . A . The Need for Asynchronous Firing of the Two Cells B . The VIII Cranial Nerves: “Electrical” and “Chemical” Excitation of the Mauthner Cells . . . . C . The Vestibular System and the Swim Bladder . D . An Asymmetrically Effective Vibrational Stimulus? E . The Symmetrical Stimulus . . . . . . 265

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IX. The Functions of the Mauthner Cells

B. Directionality Sensing: An Error . . . . . C. The Avoidance Reaction: A Genuine Function . . D. The Functions of the Collateral Inhibition . . . E. Functions in Different Species: Unanswered Questions References . . . . . . . . . . . .

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I. INTRODUCTION

The discovery of the giant axons bearing his name dates from Mauthner’s study in 1859. However, the association of these paired axons in the spinal cord of teleost fish and certain amphibians with two remarkable cells in the brain (cells which were first described by Mayser in 1881) was only established later (Goronowitsch, 1888). These two cells are fairly clearly related to certain brain nuclei, and especially to the VIIIth cranial nerves; their possible function is usually discussed primarily in that context. The earliest and certainly one of the most detailed and interesting examplcs of such discussion can be found in the paper by Bartelmez ( 1915). Moreover, because of their distinctive shape and synaptic morphology as seen by light microscopy, the name Mauthner cell has become associated largely with the soma-dendritic complex in the medulla oblongata. The traditional approach, therefore, has tended to relcgate the massivc spinal component of the Mauthner neuron to the “nonintegrative” role of simply relaying information from the brain to spinal motoneurons, which then drive the body muscles. This simplified view is misleading. We shall see how the characteristics of the particular spinal circuitry of which the Mauthner neurons are a part might well define the limits of probable functions of these cells. The richness of the neural inputs to, and intcrconnections between, the two Mauthner neurons in the brain is remarkable and intriguing; it has, however, to be interpreted with close regard to the findings in the spinal cord. Only then can a satisfactory estimation of the total functions of what we may call the Mauthner cell system be made. The earlier descriptions of the possible functions of the Mauthner neuron were not derived from physiological experiments but were based on (1) the anatomy of the neuron, the origins of certain inputs to it and the apparent destinations of many of its outputs; and ( 2 ) the observations that most fish have well-developed startle-responses, rapid escape-swimming reactions, and good equilibration function, Some of these descriptions may turn out to be correct, but it is well to remember that elasmobranchs, for example, are not lacking in the reflexes

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mentioned in ( 2 ) , but they do not possess Mauthner neurons (see Aronson, 1963). The following account will be largely based on data from a small number of species of teleost fish. Of course, Mauthner cell functions might well have developed in a variety of ways to suit the special needs of particular animal types. In this context the present account, which is based chiefly on experimental observations, provides a model schema of Mauthner cell function that can be used as a basis for comparison and might help to explain any apparently disparate appearances, or functions, of Mauthner cells in other piscine or amphibian species. Because of its particular specialization the Mauthner cell system presents some rather striking examples of neuronal morphology and function, which may throw light on the behavior of neurons generally. The possible wider implications of selected features which seem unusually clear and comprehensible will also be discussed. 11. THE BASIC ANATOMY OF THE MAUTHNER NEURON

The Mauthner neuron varies in shape and size in different species and in the number and disposition of its main dendrites. An interesting comparative account covering a number of teleosts and amphibians can be found in Stefanelli’s paper of 1951. At this point we need consider only those features which will help in our understanding of the physiological investigations to be described. [Further anatomical descriptions are made later in this chapter (Section VI), where appropriate reference is made to earlier studies which more recent observations largely confirm.] Figure 4 shows Bodian’s now well-known illustration of the Mauthner cell of the goldfish, which is not only relatively simple in shape but probably incorporates the most important features of all Mauthner neurons. ( I t is also perhaps the one best studied.) In a goldfish of about 12 cm from mouth to the base of the caudal fin, the somas of the two Mauthner cells are situated in the brain on either side of the midline, about 7 0 0 p apart and some 1-1;; mm below the surface of the medulla (Fig. l a ) . Viewed from above (i.e., dorsally), this region is normally overlaid by the cerebellum (Figs. 1 and 2). The somewhat oval-shaped cell soma has no definable lateral margins; on one side (lateral) it merges into the base of a single massive “lateral dendrite” which extends toward the region where the VIIIth cranial nerve enters the medulla, running slightly dorsally and caudally, as well as laterally, for 500p or more (Fig. l b ) . The opposite (ventromedial) margin of

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500p .

Fig. la-c.

the cell soma similarly merges into the “ventral dendrite” which curves downward and slightly anteriorly, also for about 500 p (Fig. l c ) . Both dendrites branch once, or sometimes twice, before they end. The axon leaves the soma dorsally and medially (Fig. I d ) and continues medially (becoming myelinated en route) toward the midline of the medulla,

9.

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200 /A.

Fig. 1. Photomicrographs of 1 5 p vertical sections of goldfish brain cut a t right angles to the long axis. Dorsal is above, ventral below: silver stained. ( a ) Level of axon hillock, right Mauthner cell; ( b ) 9 O p posterior to ( a ) , lateral dendrite, right cell; ( c ) 90 p anterior to ( a ) , ventral dendrites, both cells; ( d ) high-power view of the right cell seen in ( a ) ; and ( e ) high-power view of the right cell seen in ( b ) . R Mc, right Mauthner cell; L Mc, left Mauthner cell; Ma, Mauthner axon; VIII, VIIIth cranial nerve fibers (and roots); Id, lateral dendrite; vd, ventral dendrite; cb, cerebellum; and m, medulla.

where it crosses by the corresponding axon coming from the opposite Mauthner cell (Fig. 2 ) . Both axons then turn caudally toward the spinal cord, in which they run about 700p deep to the dorsal surface, on either side of the midline, about 200p apart and about 1 5 0 p ventral to the level of the central canal (Figs. 2 and 3 ) . They are truly giant axons (50 ,LL or more in diameter) dominating all others in a cross sec-

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M

Eighth ner\

I

muscles

Fig. 2. ( a ) Schematic drawing of the location of the Mauthner cells ( M cells ) within the medulla as seen from above (cerebellum retracted anteriorly to expose

9.

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271

tion of the spinal cord; toward its caudal end they eventually taper and disappear. En route they give off numerous collaterals. [In Protopterus the Mauthner axons may be over 200 p in diameter (see Smith, 1955).] Of all the various nerve inputs converging on to the Mauthner neuron, one (the only one in this category) is outstandingly clear, constant, and easily traceable; it is that from the ipsilateral VIIIth cranial nerve (Figs. l b and e ) , This long-established fact, the large size of many of the myelinated fibers composing this input, and the directness of their route to the lateral dendrite (see Fig. 4 ) has had an important influence on both the experimental and the speculative approach to the problem of Mauthner cell function.

111. THE SELECTIVE ACTIVATION OF

MAUTHNER NEURONS

A. Choice of Stimulus Although the Mauthner cell can presumably be excited orthodromically by a variety of stimuli and experimentally by selective stimulation of the ipsilateral VIIIth nerve, there is no component of that or any other nerve which is known to supply only the Mauthner cell, and orthodromic excitation therefore is not an adequate means of selective activation of a Mauthner cell. The only certain method, stimulation via a microelectrode which has penetrated the Mauthner cell itself, is not a practicable one for many types of experiment. The method which has found most use is the relatively easy one of directly exciting the Mauthner axon in the spinal cord, i.e., antidromic excitation. Because the axon is so large its threshold to electrical shocks delivered to the whole spinal cord is low (generally much lower than that of other fibers), and experimentally no important difficulties have been caused by coincidental excitation of other axons. The great advantage of antidromic excitation over orthodromic, however, is the precision it confers when exact timing of the Mauthner impulses is important. The presence of “synaptic noise” ( sporadic fluctuasubjacent medulla). Note decussation of Mauthner axons ( M axons). From Furshpan and Furukawa (1962). ( b ) The principal circuitry involved in Mauthner Reflex. Note: Only the left Mauthner cell, and thus the right Mauthner axon, is excited (excited cells and fibers are indicated by thick continuous lines and inhibitory fibers by dashed lines). Only the VIIIth nerve connections and collaterals deriving from the excited Mauthner cell are shown.

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Fig. 3. Photomicrograph of transverse section of tench spinal cord in tail region ( 10 p, silver stained). C, Central canal; Pm, primary motoneuron; and Ma, Mauthner

axon. Note: Ventral process from primary motoneuron on left, running down toward dorsal collateral of left Mauthner axon; also large cells above right Mauthner axon: the most dorsal one is a primary motoneuron, the other three are probably group B motoneurons .

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273

tions of membrane potential) in the Mauthner cell body and dendrites means that incoming nerve volleys, even of constant size, fire off Mauthner impulses with latencies that may vary by perhaps 0.1-0.2 msec or more, which is too imprecise for the sort of experiments described below. This particular problem is not met with when the axon itself is excited.

B. Choice of Recording Technique The only certain indication that a Mauthner neuron has been excited is the direct recording of its electrical response. The tail flip, caused by a stimulus applied either to the VIIIth nerve or to the spinal cord or by a vibrational stimulus or any other kind of stimulus, is not proof of Mauthner neuron activation; indeed, it can sometimes be caused when it is certain that the spinal motoneurons are not being excited from the Mauthner neurons. One useful response described later (Section IV, A ) , involving the activity of muscles of the eyeballs, jaw, and operculi, does not tell whether only one or both Mauthner cells are being activated. This is a point of fundamental importance; as we shall see, any trunk or tail movements associated with the synchronous firing of both Mauthner cells must be attributed to the coincidental activation of other (non-Mauthner) systems. C. Selective Recording from an Individual Mauthner Cell

Tasaki et al. (1954) first showed that stimulation of the whole spinal cord (in Japanese catfish) caused the firing in the medulla of a cell which, from its location, the ease with which it could be successfully penetrated with a microelectrode, and the short latency of the response, they took to be the Mauthner cell. However, the proof of this, and the discovery of how selectively to locate the cell, followed from the elegant experiments of Furshpan and Furukawa (1962) who made some important and fundamental observations. One of the most fruitful was that concerning the giant, predominantly negative potential which they recorded near the excited Mauthner cell in the goldfish (Fig. 5a). This extracellular potential seemed to have its origin at an extremely localized spot, somewhat less than 25 ,U in diameter, where it was maximal in size. Figure 6 shows the relation between size of this potential-the “negative spike”-and distance from the spot where it was largest (the negative spike focus). This shows that in such an experiment when the electrode tip records a negative spike of more than 15-20 mV, it is very close (probably less than 25 p ) to the focus, and it is then outside an active

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Per ikaryon Axon

b

Vlll(Xed)

loop

Ventral dendrite

Fig. 4. A sen~ischeniatic representation of the synaptic apparatus found in Manthner’s cell in the goldfish. The data of Beccari (1907), Bartelmez (1915), and Rodian (1937) indicate the following general segregation of synapses on the cell surface (the total number of endings is much larger than shown; for actual counts, see Bodian, 1937). The lateral dendrite. ( 1) Distal portion-club endings of direct vestibular root fibers ( sacculus ). ( 2 ) Proximal portion-end bulbs of collaterals of crossed vestibular root fibers; secondary vestibular fibers( ? ). The ventral dendrite. ( 1 ) End bulbs from fibers of ventral acoustic nucleus. ( 2 ) End bulbs of cerebelloreticular fibers. ( 3 ) End hulhs of crossed and uncrossed tectal fibers. (4)End bulbs of fibers from nucleus princeps trigeniini. ( 5 ) End bulbs of fibers in medial longitudinal fasciculns. The perikaryon. ( 1) Small end bulbs on axon hillock derived froin spiral fibers ( sl) ) of the medial longitudinal fasciculns [from mesencephalic nucleus of the medial longitudinal fasciculus ( ?)I. ( 2 ) Knob endings of collaterals

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275

a

b

’

\

Fig. 5. Recordings from microelectrode with its tip located near the “negative spike focus” of a goldfish Mauthner cell. Multiple exposure of a number of sweeps during penetration of the antidromically excited cell. The displacement of the lower traces from the upper resulted from the appearance of the resting potential: ( a ) negative spike (externally recorded); ( b ) positive spike (internally recorded). From Furshpan and Furukawa ( 1962).

region (i.e., a region of inward current flow), presumably associated in this case with an action potential. Furshpan and Furukawa marked the position of the focus by passing current through the metal microelectrode in order to deposit a small amount of iron there, and subsequently observed in histological sections the Prussian blue reaction spot produced by appropriate chemical treatment. The negative spike focus was shown to be so closely related to the axon hillock region of the Mauthner cell that the spike could be presumed to represent the firing of that same region. The proof of this was given by penetrating the cell at the negative spike focus, internally recording the now-positive spike (Fig. 5b), and then depositing iron electrolytically inside the cell. Histologically, the blue color was then of crossed vestibular root fibers [VIII ( X e d ) ] near axon hillock [some uncrossed fibers ( ? ) I . ( 3 ) End bulbs of fibers in medial longitudinal fasciculus. ( 4 ) End bulbs of secondary acousticolateral fibers ( ?). d, small dendrites; e, small end bulbs; h, axon hillock; LE, large end bulbs; in, myelin sheath of axon of Manthner cell; sb, bundle giving origin to spiral fibers; sp, spiral fibers in region of “axon cap”; VIII ( X e d ) , crossed vestibular fibers giving rise to collaterals which terminate as small club endings; and VIII, vestibular root fibers. Figure and data reproduced from Bodian (1942).

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P

P

Fig. 6. Spatial variation in the amplitude of the negative spike. The origin of the abscissa is the point a t which the largest spike was recorded: ( A ) recording positions within the same horizontal plane passing through the negative spike focus and ( 0 ) recording sites along a single vertical line. The curves were obtained by fitting a hyperbola to the data from each of two experiments (right and left graphs). From Furshpan and Furukawa (1962).

seen to be confined to the Mauthner cell itself and no other. The reversal of the sign of the spike after penetration showed that it was generated at the membrane of the cell, more exactly, at the axon hillock membrane.

D. The Investigation of the Effects of Mauthner Cell Excitation By using the information described above, it was possible to stimulate one or both Mauthner cells and to record their activity extracellularly with a high degree of precision, without damage to them. The experiments described below were done mainly on goldfish (Carassius auratus L.) and tench (Tincu tinca L.) (Yasargil and Diamond, 1968; Diamond and Yasargil, 1969). The fish, contained in a special chamber, was kept alive by a stream of water (containing 0.25-0.30% urethane) which flowed continuously through its mouth and out of its gills (Furshpan and Furukawa, 1962; Diamond, 1968). The anesthetized fish were sometimes paralyzed with a neuromuscular blocking agent. The Mauthner axons were excited by two pairs of steel wires, insulated except for the tips, which were positioned just outside the vertebral column; by trial and error, stimulus parameters were found which permitted the selective excitation by one pair of electrodes of one

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277

Mauthner axon and by the second pair of the other. When orthodromic excitation was required, similar pairs of electrodes were inseited through holes in the skull; thus, the electrode tips were close to the respective VIIIth nerves in the region where the nerve bundles fan out near the vestibular apparatus. For recording, conventional microelectrodes were positioned deep in the exposed medulla by micromanipulators; thus, their tips were near enough to the respective Mauthner cells on either side to record the characteristic axon hillock spikes when they were excited. Similar microelectrodes were used to record the activity of cells in the spinal cord. Muscle activity was also investigated; concentric needle electrodes recorded the electromyogram ( E M G ) of muscles on each side of the trunk or tail, in both retro-orbital regions, and of opercular muscles of either side. Ventral root activity was recorded with twin platinum electrodes. The general arrangement is shown diagrammatically in Fig. 7.

Fig. 7. General arrangement of stimulating and recording conditions (see text) : OP EMG, opercular muscle recording; VR, ventral root; LM and RM, left and right Mauthner cells, respectively.

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IV. THE “MAUTHNER REFLEX”

A. The Response The excitation of a single Mauthner cell, either antidromically or orthodromically via the ipsilateral VIIIth nerve, produces a characteristic forceful flip of the trunk and tail to the side opposite that of the cell body (i.e., to the same side as the excited Mauthner axon) (Fig. 8a and b ) . In addition, and seemingly synchronously, there is a sudden movement of both eyeballs (Figs. 9a and b ) , of both operculi (Figs. 10a and b ) and of the lower jaw (not illustrated). In the experimental chamber a brief jet of water is discharged backwards from both gill clefts. (There has been no investigation of associated fin movements which may occur.) This describes the response when only one Mauthner cell is excited. It obviously resembles, superficially at least, the startle-response of the Trunk EMG

Mauthner c e l l spike

-+ right

a[

right

left

r

I

bl

C

right

h

right

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right left

1 7 r u 2.0rnsec

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10 msec

Fig, 8. Responses recorded extracellularly and simultaneously from both Mauthner cells in brain and both right and left trunk muscle. Corresponding records are alongside each other. (Mauthner axons cross before descending in spinal cord; thus, an impulse from right cell propagates along left axon, which is ipsilateral to left muscle, and vice versa.) Vertical calibrations alongside upper pair of EMG records are 2.0 mV for all right muscle and all Mauthner cell records and 1.25 mV for all left muscle records. From Yasargil and Diamond ( 1968). a and b show responses when only one Mauthner axon was excited, and c when both were excited simultaneously.

9.

279

THE MAUTHNER CELL

M c e l l Spike

a

E y e muscle EMG

-L?? V

left

right

right

left

left right

right left

-v

"7J"c

Fig. 9. Responses recorded extracellularly and simultaneously from both Mauthner cells in brain and both right and left retro-orbital muscle. Corresponding records are alongside each other. Calibrations: vertical, 2 mV; horizontal, 1 msec for M cell records, and 2 msec for muscle records. a and b show responses when only one Mauthner axon was excited and c when both were excited simultaneously.

free-swimming fish produced by a sudden blow to the fish tank or by a sudden change of illumination (caused, for example, by passing the hand between a light source and the tank). When EMGs or ventral root records are inspected it is found that there is also an output from the spinal cord on the opposite side to that of the excited Mauthner axon (Figs. 8 and 11).The contralateral excitatory response is smaller than the ipsilateral one and it comes about 1-3 msec later. However, the overall movement is to the ipsilateral side, i.e., that of the excited Mauthner axon; the report to the contrary by Retzlaff (1957) is in error. The contralateral response, therefore, may have a role concerned with stabilization and possibly the bringing about of a brisk end to the swiveling movement of the animal. Its initiation relative to that of the ipsilateral excitation is discussed later (Section VI, E ) , B. The Inhibition of the Response

Consider now what happens when both Mauthner cells are synchronously excited. This can certainly be done orthodromically by appropri-

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M cell spike

Opercular muscle EMG

left

1 - 7

dw

right

right

O

b

I

left rioht

right + d

lef i

right left

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I

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-

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Fig. 10. Responses recorded extracellularly and simultaneously from both Mauthner cells in brain and both right and left opercular muscle. Corresponding records are alongside each other. Calibrations: vertical, 2 mV; horizontal, 1 msec for M cell records, and 2 msec for muscle records. a and b show responses when only one Mauthner axon was excited, and c when both were excited simultaneously.

ately adjusting the timing and magnitude of the two stimuli to the VIIIth nerves (cf. Retzlaff, 1957), but it can be done with far greater precision and control antidromically. The result is surprising and most clear. The tail flip, and indeed all the muscle response in the trunk and

.

Stim. to L axon

t

Stim. to R axon

Fig. 11. Effect of crossed inhibition. The left Mauthner axon stimulus was applied at various times after the right (which was applied at a fixed time). Calibrations: vertical, 2 mV for left EMG and 2.5 mV for right EMG; horizontal, 10 msec.

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281

tail, is completely absent (Fig. &). All the other components of the response, however, appear unchanged (Figs. 9c and 1Oc). The proof that the stimulus, applied at the lower end of the spinal cord, did excite an impulse in each of the Mauthner axons is given by the recordings made simultaneously at the respective axon hillocks in the medulla. It should be noted that in an investigation of this sort only responses which occur in constant association with the firing of one or both Mauthner cells are being considered. It is possible to produce muscular responses by the coincidental excitation of other (non-Mauthner) fibers, and these can be distinguished by careful observation of effects caused by stimuli just a t threshold for excitation of one or both Mauthner axons. The difficulties are greater with orthodromic stimulation because it is almost impossible then to avoid activating other cells in the medulla which are also associated with reflex movements.

What is revealed here, then, is the existence in the spinal cord of a mutual inhibitory system whose activation by an impulse in one Mauthner axon prevents the impulse in the opposite Mauthner axon from causing the usual excitation of spinal motoneurons. The inhibitory system, however, does not operate on the cranial component of the total reflex (Figs. 9 and 10). C. Time-Course of the Inhibition

Since the crossing inhibition owing to activation of one Mauthner axon can so totally and dramatically suppress the most striking excitatory response to the impulse in the other, it seems likely to be of fundamental importance in Mauthner cell function. It is surprising that, coming from the other side of the spinal cord, it acts as quickly as the ipsilaterally evoked excitation. In fact, the inhibition can be delayed slightly behind the excitation and still be as effective as when the two Mauthner axon impulses are synchronous (this is considered below). Figure 11 shows the EMGs recorded simultaneously on each side of the trunk when both Mauthner axons were stimulated. The moment of excitation of one Mauthner axon was kept constant and that of the opposite one was progressively delayed. Except at very small separation times, which are not shown in this figure but are considered in detail below, there was always the same response to the impulse in the first excited Mauthner axon, being larger on the ipsilateral side and smaller and slightly delayed on the opposite side. But a response to the second excited axon did not appear until the sepxation between the Mauthner impulses (which were simultaneously recorded and were always used for measuring time relations) was about 11 msec; the response was then a very small one. As the delay was increased this response became pro-

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gressively larger, both in its ipsilateral and contralateral components, until at a separation time of 25 msec it attained its maximum size.

D. The Minimum Discrimination Time The meaning of these results becomes clearer when a whole sequence is displayed graphically as in Fig. 12. [It is important to note that results of this kind were essentially the same whichever side was designated as ipsilateral, provided the output from the spinal cord was measured on that side, i.e., there can be no important asymmetry in the system owing to different conduction velocities or distances in the Mauthner axons (see Section IX, E, 5 ) . ] Figure 12 specifies some of the most important features of the spinal system whose activation begins with the impulse in the Mauthner axon and whose output leads to the characterL e f t muscle response (% of control) *

-100

..mu

-80

-60

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Time between two mouthier-axon spikes (rnsec)

Fig. 12. Ordinate, response of trunk muscle on left side only, as a percentage of its response to excitation of ipsilateral (left) Mauthner axon alone. Abscissa, time between the spikes in the two Mauthner axons: the origin ( a t arrow) is the point where the two axons are synchronously excited; to the right of this (negative values) the left axon spike lags behind the contralateral axon spike, and to the left of the origin (positive values) the left axon spike is in advance of the contralateral axon spike. Inset shows the part of the graph near the origin, with a X10 expanded time scale.

9. THE

MAUTHNER CELL

283

istic powerful movement which may be described as the Mauthner reflex response. One of the these features [point (4)below] embodies a principle of general interest, which will be returned to later.

(1) The muscular response to a single Mauthner axon impulse is effectively all or nothing. ( 2 ) For a period, which varies from fish to fish, of 8-15 msec after an impulse in one Mauthner axon, an impulse in the opposite Mauthner axon leads to no muscle response at all in the trunk and tail. ( 3 ) Following this period of absolute suppression of the response, there is a period which may last as long as 50 msec, during which the impulse in the contralateral Mauthner axon evokes a muscle response whose size depends on the interval between the two Mauthner spikes. As this interval lengthens the response becomes larger until it reaches its full control value (i.e., the value obtained in the absence of the ipsilateral Mauthner impulse). Individual motoneurons, therefore, are freed of the crossing inhibition at times which vary from 8 to 60 msec after the impulse in the opposite Mauthner axon, which causes it. (4)When two impulses, one in each Mauthner axon, are produced within 0.15-0.20 msec of one another (the exact time varies from one animal to the other) there is no muscle response on either side insofar as trunk and tail are concerned. This time can be called the minimum “discrimination time” of the system (inset Fig. 12). In our experiments, unless obvious deterioration of the preparation occurred, the time was fairly constant in any one fish (goldfish and tench) during repeated testing. When the two impulses are separated by times less than the minimum discrimination time, there is no indication from the spinal musculature that there were indeed any Mauthner axon impulses at all. At this separation interval, or greater ones, there will always be a unilateral movement of the trunk and tail. This movement reaches a maximum (on the side of the leading Mauthner axon spike) when the interval is more than 0.5-0.8 msec, and it then equals the response in the absence of the opposite Mauthner axon impulse. However, the response to the later spike (in the opposite Mauthner axon) is always zero in the trunk and tail muscle until the period of total inhibition is over. We can suppose that were it not for the inhibition described above, synchronous impulses in both Mauthner axons would lead to a calamitous situation for the fish, namely, the simultaneous activation of the two effectively opposed muscle masses on either side of the body. This is happily disallowed by the reciprocal inhibitory systems. But what is the basis of the remarkably low value of the minimum discrimination

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time (only 0.150.20 msec)? And, equally remarkable, how is its relative constancy achieved? This time measures the ability of the system to resolve as separate events two closely occurring spikes, one in each Mauthner axon. To answer these important questions we must know something of the spinal circuitry involved in the Mauthner cell reflex and in particular the nature and the location of the excitatory and inhibitory effects of Mauthner axon activation.

V. THE SPINAL CIRCUITRY In the experiments to be described intracellular recordings were always made from the Mauthner axon in each cord segment investigated, in order

n

M axon

Fig. 13a.

9.

285

THE MAUTHNER CELL

2

J

I

A

Fig. 13. Intracellular records from two different tench spinal cords ( a and b ) . In each set unit responses were made successively from same side of same spinal segment. In each preparation, a and b, the stimulus was at threshold for excitation of the ipsilateral Mauthner axon only. Calibrations: vertical, 20 mV; horizontal, 1 msec. both to define its position in the cord as a spatial reference point and to obtain as a temporal reference point the onset of spike activity in it. The two Mauthner axon hillock spikes in the brain were also simultaneously recorded along with the activity of cells in the spinal cord, to provide the vital information as to whether one or both Mauthner axons were excited, as well as to facilitate the experimental control of the timing between the spikes in the two axons (Yasargil and Diamond, 1968; Diamond and Yasargil, 1969).

A. The Segmental Nature of the Reflex The following considerations indicate that the reflex is organized segmentally. ( a ) The anatomical system follows a recurrent pattern along the spinal cord (see below). ( b ) When the recording electrode is inserted into muscle at points progressively further along the trunk from the level of the electrodes used to stimulate the Mauthner axon, the latency of the EMG increases detectably; converted into a “conduction velocity,” this gives a value of 50-100 meterlsec, i.e., a rate corresponding to that of the velocity o€ the impulse we measure in the Mauthner axon. Stimulation of the Mauthner axons in the cord produces

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the usual trunk and tail responses, and their inhibition, even when the brain is destroyed. It may be assumed that the crossing inhibition, like the ipsilateral excitation, is also organized at the segmental level.

B. The Spinal Responses

1. EXCITATION At any level in the spinal cord the earliest response to excitation of the Mauthner neuron is of course that in the Mauthner axon itself. The cells which fire subsequently can be usefully divided into two main groups (Fig. 13) which are distinguished by the latency of the onset of activity in the individual cells. In the first group (group A ) this latency, measured from the steepest-rising portion of the Mauthner axon spike in the same spinal segment, is usually only about 0.5-0.6 msec ( a t 12°-150C). Because of this extremely brief latency and for other reasons which will become clear later, these cells may be confidently regarded as monosynaptically activated by the Mauthner axon collaterals. However, in the second group of cells (group B ) , the response latency is more variable and always longer, usually by at least 0.4 msec (Figs. 13 and 17) . Even for the earliest activated cells in the B group therefore, there is sufficient time for an interneuron to be involved in the reflex pathway. Clearly, in such a fast and apparently direct reflex as that with which we are dealing here, the group A cells are the more interesting ones and those which might be expected to provide the answers to the questions posed above. ACTIVATED GROUPA CELLS 2. THEMONOSYKAPTICALLY There are two sorts of units (this, as will be seen, is a more appropriate terminology than “cells”) in this group, which we can call A 1 and A2 at this stage, and their relation to each other is of great interest. The electrical response of an orthodromically excited (i.e., excited via the Mauthner axon) A 1 unit shows typically two phases of activity; the first, i.e., the earliest, appears to be an excitatory postsynaptic potential (e.p.s.p.), which then fires one (occasionally two) all-or-none spikes (Figs. 13-16). Group A2 records, however, show three (or even four) distinct components of activity, and the spike proper arises not from the first of these but always from the second; the first component can usually be seen unequivocally only at high amplification (Figs. 14-16). There is a possible explanation of this puzzling finding of two dis-

9.

287

THE MAUTHNER CELL

tinct sorts of unit, both apparently monosynaptically activated from the Mauthner axon, which could help to explain some of the most important characteristics of the Mauthner reflex. There may be interesting implications here for other neural systems, too; thus, the situation will be dealt with in some detail. The first point of interest becomes apparent when records of the activity of an A 1 and A2 unit from the same side of a given spinal segment are aligned one above the other (with the two records of the simultaneously recorded Mauthner axon spikes made coincident) ( Fig. 15). It can then be seen that the first and second phases of activity of the two units occur at virtually the same time. But in the A2 unit their amplitude is greatly reduced (Fig. 15b). These time coincidences and other relevant findings presented below have suggested an interesting possibility. It is as though an electrical junction exists between the two units, thus allowing the electrical activity of the A 1 unit to be detected in the A2 unit also. On this basis, the electrically transmitted A1 response would seem to be the immediate stimulus to the A2 unit, and the excitatory action of the Mauthner axon would be exerted directly on the A1 unit. This hypothesis will be considered further. 3. THE IDENTITY OF GROUPA

AND

GROUPB CELLS

The A2 and B cells are certainly motoneurons since they can be excited antidromically by stimulating appropriate ventral roots ( Figs. l6b and 17b). However, when the antidromic and orthodromic records from the same A2 cell are compared, it is seen that the first two components of the orthodromic response are absent in the antidromic record (Fig. 16). These two components then do appear to comprise the excitatory potential immediately responsible for triggering the spike in the A2 record, which is consistent with the hypothesis described above, that they in fact represent the electrically transmitted activity of the A1 unit. What is the identity of the A1 unit? It seems to be that first fired off, apparently monosynaptically, by the Mauthner axon; and there are reasons for assuming that it is excited via the release of a chemical transmitter. Moreover, it appears to cause electrical excitation of the A2 unit, an undoubted motoneuron. On the few occasions when it was possible to make the test, A 1 units could not be excited antidromically, and only a small subthreshold depolarization was recorded in them (Fig. 17d). Of course, this failure of antidromic invasion may have resulted from damage, but it is also possible that the suggested electrical coupling of A1 to A2 units involves a less effective current flow in the

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c

Fig. 14a.

antidromic than in the orthodromic direction (Furshpan and Potter, 1959; see also Katz, 1950). Is the A1 unit actually an interneuron that connects the Mauthner axon to the motoneuron (see Fig. 18): If so, what could be the purpose of having a cell so inserted into an excitatory pathway apparently highly organized for speedy and direct reflex action? To answer these questions we must first consider further the crossed inhibition, which will allow us to understand the basis of the minimum discrimination time, and then the spinal cord histology and the location of the A l and A2 units.

4. INHIBITION a. Mechanism. The crossed inhibition appears to be chemically transmitted since (1)it is frequently associated with a hyperpolarization

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289

Fig. 14. Intracellular recordings from group A1 unit ( a ) and A2 unit ( b ) . In each case the upper trace shows the ipsilateral Mauthner axon spike recorded in same spinal segment, the middle trace the group A unit record a t high amplification, and the lower trace the same group A response at low gain. Stimulus artifacts were aligned vertically. Inclined arrows show discontinuities between phases of re'iponse: note that the first phase is often only visible in high-gain record. Calibrations: vertical, 20 and 1.25 mV; horizontal, 1 msec.

of the inhibited cell (group A and group B motoneurons, Figs. 19' and 21a); ( 2 ) for at least a part of its duration it can reduce the amplitude of ( and sometimes block) an antidromically evoked spike, presumably by increasing the membrane conductance of the motoneuron (Fig. 29) ; and ( 3 ) it can be blocked by strychnine (Fig. 20). The duration of the inhibition is variable, sometimes lasting many tens of milliseconds, whichever region of the motoneuron is investigated. This is consistent with the time-course of the crossed inhibition of the reflex which was described earlier (Figs. 11 and 12). However, if the Mauthner axon impulses are synchronous, the inhibition can always be seen to begin only slightly later, or even at the same time, as the excitatory re-

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1 23

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Fig. 15. Recordings from A1 and A2 units on same side of same spinal segment, aligned vertically in relation to the ipsilateral Mauthner axon spike (not shown in figure, see text). In each pair ( A ) , ( B ) and ( C ) the high-gain record starts above, the low-gain below, ( C ) The same unit as ( B ) , but recorded later after the cell had deteriorated. The vertical dashed lines were drawn through 1, the moment when activity in both units began; 2, the point of discontinuity between the first and second phases of the response in both units; 3, the discontinuity between the second and third phases of the response in the A2 unit. Calibrations: vertical, 40 and 2.5 mV; horizontal, 2.0 msec. From Diamond and Yasargil (1969).

sponse in the absence of the crossed inhibition (e.g., Fig. 22B). As we have seen, the very brief latency of the excitatory responses indicates that it is monosynaptically evoked. The inhibitory pathway to the motoneuron begins at the contralateral Mauthner axon, i.e., some 2 0 0 p further away than the origin of the ipsilateral excitatory pathway. Does the inhibition, therefore, also involve a monosynaptic pathway, and is the Mauthner axon the parent of two types of collateral, one being excitatory and the other inhibitory to homologous cells? There are no theoretical reasons against such a possibility, which might require that the Mauthner neuron releases different transmitters at the ipsilateral and contralateral collateral endings, or that the moto-

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Fig. 16. Comparison of ( a ) orthodromic and ( b ) antidromic responses recorded from a group A2 unit. ( a ) The high-gain trace starts below the low-gain one (arrows show points of discontinuities); ( b ) the high-gain trace starts above the low-gain one ( a slight fall in resting potential had occurred). ( c ) Low gains and ( d ) high gains, the superimposed tracings made from records ( a ) and ( b ) ; the antidromic responses are in dashed lines. (The main spike peaks were exactly superimposed to obtain these composite tracings.) Calibrations: vertical, 20 and 1.25 mV; horizontal, 1.0 msec. Based on Diamond and Yasargil (1969).

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b

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Fig. 17. Orthodromic and antidromic responses recorded from a group B cell, ( a ) and ( b ) , and a group A1 unit, ( c ) and ( d ) (different experiments); in ( c ) and ( d ) the high-gain record starts above, the low-gain below. ( a ) and ( c ) are orthodromic responses; ( b ) and ( d ) are antidromic responses. The vertical dashed line indicates the moment when the ipsilateral Mauthner axon spike began in the same spinal segment. Calibrations: 2 msec and 20 mV for ( a ) and ( b ) ; 1 msec and 1.25 and 20 mV for ( c ) and ( d ) .

neurons have different synaptic regions, one type of which responds by excitation and another by inhibition, to the same transmitter. However, very occasionally a unit has been recorded from very close to one Mauthner axon, but it was fired from the opposite Mauthner axon; the latency of the response in this unit was so short that the electrode must have been in either a collateral of the opposite Mauthner axon or an interneuron electrically excited by such a collateral. If the latter were the case then these interneurons could be regarded as providing a means of switching, without appreciable synaptic delay, from one transmitter type to another. This problem is still unresolved.

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Ventra I dendrite

Intermediate

Fig. 18. Schematic representation of part of the spinal circuitry involved in the Mauthner reflex and its inhibition. The dashed lines show features recognized physiologically but not satisfactorily identified anatomically (see also Fig. 2 b ) .

Another outstanding problem with regard to the crossed inhibition is that, to date, no convincing crossing collaterals have been found ( a t least in goldfish and tench cords) which could subserve it. Nevertheless, such crossed pathways must exist, and we can suppose that the fineness of the fibers accounts for the difficulty in identifying and following them in histological preparations. b. Presynaptic as well as Postsynaptic Inhibition? The inhibition, as assessed by the hyperpolarization associated with it, must be exerted directly on the group A motoneurons as well as (though not necessarily monosynaptically) on the group B cells (Fig. 19). However, an inspection of the records made from the ipsilaterally excited cell during the progressive delay or advance of the opposite Mauthner axon impulse, i.e., of the crossed inhibition (Fig. 21a), suggests that the inhibiting effect on the e.p.s.p. may begin before the onset of hyperpolarization (the latter can be measured of course in the absence of the ipsilateral excitation). Although this must reflect to some extent the fact that the postsynaptic inhibitory conductance change will begin before the charging of the membrane has reached a detectable level, it is possible that there may be an inhibitory component acting presynaptically, affecting the release of transmitter from the Mauthner axon collateral itself. Support for this comes from the small hyperpolarization which is sometimes recorded in the Mauthner axon some 0.6 msec after the opposite Mauthner axon spike occurs in the same cord segment (Fig. 21b). We have no evidence that can explain this as a general field effect, and it seems possible that this hyperpolarization is indeed the sign of a presynaptic inhibition whose onset is slightly in advance of the conventional postsynaptic inhibition of either the A 1 or A2 units.

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I

I

r

b

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- .

Fig. 19. Intracellular recordings from a group A1 unit [ ( a ) and ( b ) , lowest pair of traces] and a group A2 [ ( c ) and ( d ) , lowest pair of traces] following excitation of the ipsilateral Mauthner axon [ ( a ) and ( c ) , top trace], and the contralateral Mauthner axon [ ( b ) and ( d ) , second trace]. In the lower pair of traces of each set [ ( a ) - ( d ) ] , the high gain record starts above the low gain one. The inhibitory hyperpolarization in ( d ) was complicated soon after it began by the presence of a transient small depolarization. Calibrations : vertical, 2.0 mV for M cell responses, 40 and 2.5 mV for spinal units; horizontal, 2 msec.

C. Understanding the Minimum Discrimination Time The discrimination time of the spinal circuitry has already been explained as the smallest interval between two impulses, one in each Mauthner axon, which will result in a finite output to the muscles on one side (see Fig. 12). At the level of the individual motoneuron, this time can now be seen to depend upon two latencies: (1) to the moment when the ipsilaterally evoked excitatory potential in the motoneuron fires the spike in the absence of the crossed inhibition, and ( 2 ) to the moment when the crossing inhibition becomes effective (this is revealed when the two Mauthner axons are fired synchronously). The interval between ( 1 ) and ( 2 ) equals the discrimination time of that motoneuron.

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-. C

I' b

d

Fig. 20. Ventral root response [lowest trace in ( a ) - ( d ) ] to ( a ) excitation of the ipsilateral Mauthner axon; ( b ) excitation of the contralateral Mauthner axon; ( c ) simultaneous excitation of both Mauthner axons; and ( d ) repeat of excitation of both Mauthner axons some minutes after intramuscular injection of strychnine (1.0 mg/kg). In (a)-( d ) , the top trace shows Mauthner cell response resulting from excitation of the ipsilateral Mauthner axon, and the middle trace the response of the opposite Mauthner cell (resulting from excitation of the contralateral Mauthner axon). Note that the latter trace was displaced to the right. Calibrations: vertical, 0.25 mV; horizontal, 1.0 msec.

If the contralateral Mauthner axon spike is delayed by just this time or longer, then the inhibition begins too late to prevent the cell from firing. Two examples are shown in Fig. 22, one being 0.25 msec, the other 0.57 msec. The motoneurons with the smallest discrimination times will determine the minimum discrimination time of the spinal circuitry involved in the Mauthner reflex. Although the minimum discrimination time as measured from EMGs (Fig. 12) is always about 0.15-0.20 msec, the corresponding time for individual motoneurons investigated with microelectrodes was always rather longer than this. The reason for the discrepancy, apart from the failure to find with the microelectrode the motoneurons with the very small discrimination times (there will, of course, be a relatively small proportion of these motoneurons in the total motoneuron population), depends principally upon deterioration of the electrode-impaled cells in the exposed spinal cord. This causes a progressive increase to the moment when the threshold is reached and the motoneuron spike fires (see Figs. 15B and C ) . However, the moment when the crossed inhibition becomes effective is, surprisingly, hardly

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I -0.43 -0.42 -0.28 -0.23 -0.1 0

+0.221 +012

11

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Fig. 21. ( A ) Group A1 unit records (superimposed). Continuous line-only ipsilateral Mauthner axon excited. 0.00, moment when e.p.s.p. started; 0.37 msec, moment when spike was initiated. Dashed lines-both Mauthner axons excited with various intervals between them. The numbers refer to the moment when the crossed inhibitory hyperpolarization would have begun i f only the contralateral Mauthner aaon had been excited. ( Minus sign indicates inhibition following excitation, plus sign inhibition preceding excitation.) Note that the e.p.s.p. was affected at the time intervals -0.45, -0.43, and -0.42 msec, i.e., at a time preceding that when the inhibitory potential produced in isolation would have just begun. The responses at these three Mauthner impulse separations are shown at low gain in the upper part of the figure. Calibrations: vertical, 20 mV (upper), 1.0 mV (lower); horizontal, 0.5 msec. ( B ) Intracellular recording from a Mauthner axon. Upper trace, high gain; middle trace, low gain; direct stimulation. Bottom trace is a high gain record when the opposite Mauthner axon only was excited. Note small hyperpolarization. Calibrations: vertical, 10 and 1.25 mV; horizontal, 1.0 msec.

9. THE

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(msec Fig. 22. ( A and B ) Intracellular records of the activity in two different A1 units. Each set of three traces shows: above, low-gain record of A1 unit spike caused by excitation of ipsilateral Mauthner axon; middle, same, a t high gain to show e.p.s.p. and inflection point where spike fired ( t h e spike rose too rapidly to be photographed in cell on left); below, high-gain record of response when both Mauthner axons were fired synchronously. Vertical calibrations: upper, 10 mV; lower, 2 mV. The vertical lines are drawn through the points where ( 1 ) the e.p.s.p. reached the firing threshold in the absence of inhibition (right-hand line), and ( 2 ) the inhibitory process (resulting from the contralateral Mauthner axon activity) cut off the e.p.s.p. shortly after it began to rise (left-hand line). The time interval between these two lines equals the “discrimination time” for the A1 unit. It is 0.25 msec for the unit on the left and 0.57 msec for that on the right, From Yasargil and Diamond ( 1968).

ever affected. As can be deduced from Fig. 22, the result of this is a lengthening of the discrimination time of the individual motoneuron. VI. THE ANATOMY OF THE SPINAL CIRCUITRY

A. Evidence from Light Microscopy

The sections in Fig. 23 (see also Fig. 3 ) show most of the important features relevant to the Mauthner cell system in the teleost spinal cord

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Fig. 23. Photomicrographs of tench spinal cord sections ( 10 p silver stained) in trunk-tail region; different fish: ( a ) cut parallel to long axis and ( b ) cut transversely. PM, Primary motoneuron; Vd, ventral dendrite; Ma, Mauthner axon; and C, Mauthner axon collateral.

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Fig. 23b.

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and that our findings are in general accord with those of Haller (1892), Kolster ( 1898), Tagliani ( 1905), Beccari ( 1907), Tiegs ( 1931), Bodian (1952), Leghissa (1956), and Barets (1961).The short dorsal collaterals of the Mauthner axons run toward, and sometimes seem to join, the long ventral processes of certain large motoneurons, which we shall call the “primary” ones, in very close relation to the myelin sheath of the Mauthner axon (although this is obscured by the shrinkage and lack of myelin staining in these particular preparations ) . The cell bodies of these motoneurons, which usually have one or two large lateral dendrites, lie approximately in a column some 1W300 p dorsolateral to the Mauthner axon, up to 300p apart. The dorsal collaterals of the Mauthner axon are associated with these primary motoneurons in a one-to-one relationship in goldfish and tench. The ventral extension of the primary motoneuron sweeps downward to come into close apposition to the medial, or occasionally the lateral, aspect of the Mauthner axon near where the dorsal collateral occurs, and this feature largely defines this particular group of motoneurons. The ventral process gives rise to the efferent motor axon, and it has itself been variously described as axon, initial segment, and, by Tagliani and by Tiegs, a dendrite. The latter interpretation is strongly supported by its electron-microscopic appearance, and by the numerous nerve endings which make synapses on it; we shall call it the ventral dendrite of the primary motoneuron. It is not uncommon for an axon to arise from a dendrite in fish (Nieuwenhuys, 1964). Occasionally a primary motoneuron, its ventral dendrite, and the Mauthner axon collateral running to it can be seen in a single l o p section, as in Figs. 3 and 23. The motoneuron axon proper can be followed from the ventral extremity of this dendrite, sweeping ventrolaterally to join the other axons of the ventral root.

B. The Mauthner Collaterals and the Motoneurons 1. THE GROUPA CELLS

Figure 24 shows the distribution in goldfish and tench spinal cords of the recording sites of a number of group A cells relative to the Mauthner axon which was also recorded from in the same region of the cord in each instance. All measurements were scaled to the size of cord most commonly encountered, in which the Mauthner axons were 7 5 0 p below the dorsal surface. The histological drawing on the left of each diagram is based on an actual 10 p section, and the shaded areas indicate the approximate variation we found in the position of the soma and ventral dendrite of the primary motoneuron. It is clear that the distribu-

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Fig. 24. Diagrammatic cross sections of teleost spinal cord, based on light microscopic data, showing on left-hand side of sections the relationship between Mauthner axon collateral and primary motoneuron and on the right-hand side the distribution of impaled units from various experiments. The dotted areas on the left side indicate the approximate variation in the position of the motoneuron soma, ventral dendrite and axon (the latter two sometimes run around the lateral side of the Mauthner axon). PM, Primary motoneuron and Ma, Mauthner axon. Calibration line, 100 p . From Diamond and Yasargil (1969).

tion of the physiological defined A2 motoneurons corresponds to the region of the primary motoneuron, and it can be concluded that the A2 unit and the primary motoneuron are in fact one and the same cell.

2. THEA1 UNITS A most perplexing situation exists concerning the anatomical identity of these physiologically defined units. The region in which they were recorded corresponds approximately to the junctional region between

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the dorsal collaterals of the Mauthner axon and the ventral dendrites of the primary motoneurons (Fig. 24). The dorsal collateral, though often short and straight, can be relatively tortuous and long, and occasionally it forms two branches, each supplying a primary motoneuron. As we have seen, the A 1 unit appears to be chemically excited from the Mauthner axon and therefore cannot be the dorsal collateral itself. But we ourselves have never observed any cells (or fibers) connecting the Mauthner axon, or its collaterals, to the primary motoneuron, nor do the descriptions of any of the other workers mentioned above include any suggestion of such intermediary cells. Nevertheless, physiologically the A1 units appear to function in some intermediary capacity. One solution to this intriguing situation would be that the chemically transmitting synapses of an ipsilateral Mauthner axon collateral are located on a branch of the primary motoneuron. This branch (the A1 unit) would need to be connected to the rest of the primary motoneuron (the A2 unit) by a transitional region of relatively high resistance. Our own light microscopy has occasionally suggested this possibility of a dendritic branch or protuberance in the junctional region (Figs. 23b and 25). Tiegs, in a most interesting paper (1931), described a “transitional tissue,” a morphologically curious intervening region between the Mauthner axon collateral and the ventral dendrite of the motoneuron. The electron microscope, as we shall see, has been extremely helpful on this question. 3. THEGROUPB MOTONEURONS In approximately the same region (though covering a larger area) as that of the primary motoneurons, there are other fairly large cells

which send their axons directly toward the ventral root without passing especially close to the Mauthner axon. Such motoneurons (though not their axons) are seen in Fig. 3. The distribution of the group B cells of the kind which gave records like those of Figs. 13 and 17a corresponded to that of these other motoneurons, at least some of which, therefore, may be presumed to give rise to the later firing components of the all-or-nothing response to Mauthner cell excitation (see Section E below).

C. Evidence from Electron Microscopy Figures 26 and 27 summarize the evidence from both electron and light microscopy (Diamond and Yasargil, 1969; Diamond et al., 1969, 1970; Gray, 1969).

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Fig. 25. Photomicrograph of transverse section of tench spinal cord (10 p, silver stained): Ma, Mauthner axon; c, Mauthner axon collateral; and VD, protuberance on ventral dendrite of primary motoneuron.

1. THE PRIMARY MOTONEURON The primary motoneuron has conventional synaptic endings on it, and not infrequently a different type of synaptic contact where regions of close apposition of pre- and postsynaptic membranes (“tight junctions”) occur in the same bouton alongside regions with conventional synaptic thickenings and aggregations of vesicles (see Charlton and Gray, 1966). If function is to be ascribed to these structures, then these boutons might well have a mixed electrical and chemical transmission and would be, presumably, excitatory to the motoneuron. Boutons occur all along the ventral dendrite, and, interestingly, many of these have

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Ma

Fig. 26. (A)-( D ) Four typical electron microscopic appearances of goldfish spinal cord in region of Mauthner axon collateral. Ma, Mauthner axon; Mc, Mauthner axon collateral; and vd, ventral dendrite of primary motoneuron. Note: Mauthner axon myelin shown in black; Boutons on ventral dendrite and its projections; absence of Mauthner axon collateral in ( C ) and ( D ) .

the “flat” or “oval” vesicles which may be characteristic of inhibitory synapses ( Uchizono, 1965; Gray, 1969). This would certainly agree with our finding of a powerful chemically transmitted inhibitory input to the ventral dendrite. Also, there are occasional tight junctions with adjacent processes (possibly dendrites of other neurons) for which it is not possible to state whether the presumed electrical transmission would be excitatory from such a process to the ventral dendrite or from the ventral dendrite to the process itself (the latter possibility will be seen later to be of relevance here). The ventral dendrite shows some remarkable features in the region where it approaches the Mauthner axon. Unfortunately, there is always a good deal of shrinkage here during fixation, in exactly the region which is of the greatest interest in relation to the identity of the A1 unit. The evidence (Figs. 25-27) nevertheless suggests that there is a small branch of the dendrite running toward the Mauthner axon collateral, and this branch contains, near its origin at the main dendrite, structural features which are analogous to those of the “dendritic spine apparatus”

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Mauthner axon

Fig. 27. Suggested morphology of goldfish spinal cord in region of the Mauthner axon collateral.

(Gray, 1959a,b) which is characteristic of many of the small lateral protuberances known as “spines” seen especially well on the dendrites of the pyramidal neurons of the mammalian cerebral cortex and corpus striatuni. On this basis, the ventral dendrite in the fish could have a single large dendritic spine in very close relation to the Mauthner axon collateral. On this spine are occasionally seen one or more boutons with characteristic synaptic vesicles, generally of the oval type.

2. THE MAUTHNER AXONCOLLATERAL The collateral usually ends toward the outer limit of a tunnel in the myelin sheath of the Mauthner axon itself, running obliquely; thus its length may often be considerably longer than the thickness of the myelin sheath. It terminates as a dome-shaped protuberance which has the characteristic presynaptic morphology and contains synaptic vesicles of the round type. The terminal portions of the postsynaptic structure

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appear, as a consequence of shrinkage, to be broken off the main part of what is described above as the dendritic spine of the primary motoneuron. The spine bears a series of fingers at its extremity which reach out (as indicated in Fig. 27) toward the region of synaptic contact. Typical membrane thickenings can sometimes be seen along the synaptic clefts where the spine fingers invaginate the dome of the Mauthner collateral.

D. Conclusion on the Identity of the A1 (“Connecting”) Unit The physiological evidence, the location of the A1 recording sites, and the evidence from both light and electron microscopy suggest that the A1 unit is indeed a dendritic branch or spine of the primary motoneuron, and that the neck of the spine near its origin from the main dendrite is presumably the transitional region of high resistance between the synaptic region of the spine and the parent (ventral) dendrite of the motoneuron. The morphological situation is diagrammed in Fig. 27. This is, of course, an hypothesis [which has in fact been extended to a general hypothesis on the function of dendritic spines in central neurons ( Diamond et nl., 1969, 1970) 1. Nonetheless, whether the A1 unit is a dendritic spine or branch, or a very small separate intermediary or connecting cell, we believe that there is an important functional significance of the arrangement, which relates to the circuit discrimination achieved in the Mauthner reflex.

E. An Excitatory Cascade? The primary motoneurons, which the physiological and anatomical evidence indicates are the only ones directly supplied by the ipsilateral (excitatory) Mauthner axon collaterals, form only a small proportion of the total niotoneuron population (see also Barets, 19Sl). The powerful tail flip caused by excitation of a Mauthner cell must involve a good proportion of the muscle of the trunk and tail, but we are not sure exactly which muscles are activated. However, it seems almost certain that some of the tail muscle must be supplied by group B motoneurons. ( W e do not know which motoneurons suppIy fin muscles.) The ventral root response (Fig. 28) shows that there is an apparently sequential activation of two or three groups of motoneurons; presumably the first of these is the primary motoneuron group (comprised of only a few cells) while the later responses result from the group B cells. What is

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M cellresponses

Left EMG

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Fig. 28. Ventral root records during ipsilateral and contralateral Mauthner axon stimulation. The upper set of traces show the Mauthner ceIl responses: the top is the excitatory one ( corresponding to the ipsilateral Mauthner axon), stimulus applied at a fixed time; the traces below this (proceeding downward) show the inhibitory one (contralateral Mauthner axon), timed to occur a t various time intervals before and after the excitatory one. The lower set of traces show the output in the ventral root; the upperniost of these is the response to the excitatory Mauthner cell alone and proceeding downward the responses for the various Mauthner cell intervals shown in the upper set of traces. Note that the bottom ventral root record is the crossed excitatory response to contralateral Mauthner axon stimulation ( cf. Fig. 8 ) . Calibrations: vertical, 200 p V ; horizontal, 2 msec.

the excitatory pathway to the latter inotoneurons, and to those on the opposite side of the cord which are responsible for the crossed excitatory pathway (Section IV, A ) ? We cannot exclude the possibility that hitherto unrecognized Mauthner axon collaterals run to “nonprimary” motoneurons or to interneurons

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supplying them. (Indeed, we know that such collaterals must subserve the crossing inhibition. ) But there is another possibility worth considering, namely, that the Mauthner reflex involves an “excitatory cascade,” in which the primary motoneurons are responsible for passing on the excitation to other motoneurons and/ or interneurons. It is interesting to note that where the comparison was possible the effects of the crossing inhibition lasted longer on later firing motoneurons than on earlier ones; this would be necessary (if the suggested excitatory cascade were involved) for the production of the variable muscle response observed to occur toward the end of the inhibitory period. [The variable muscle response and ventral root output (Fig. 28) which occurs at short separation times between Mauthner impulses would, of course, depend upon the crossing inhibition arriving in time to prevent the firing of group B motoneurons but too late to prevent that of the primary motoneurons.] Another relevant finding was that the crossed excitation is always associated with the ipsilateral excitation, which it follows by 1-3 msec; when the latter is reduced or abolished by inhibition or fatigue (Fig. 44) so is the former, and to a roughly corresponding extent. We may speculate further on the nature of an excitatory cascade with regard to the fractionation of the response in the primary motoneuron. Figure 29 shows records of the activity in the cell body of such a motoneuron, orthodromically and antidromically excited, during the crossed inhibition evoked by excitation of the contralateral Mauthner axon. The various components of the electrical response of this cell were easily distinguishable because of its deterioration as a consequence of the microelectrode impalement. However, that these components are not experimental artifacts is indicated by the ease with which they could always be revealed by the activation of the crossing inhibition. There were four components comprising the orthodromic response in this example but only two comprising antidromic. As we have seen, the two extra components in the orthodromic records seem to be contributed by the A1 unit. As the crossing inhibition, which followed the ipsilateral excitation, was initiated progressively earlier ( Figs. 29c-e and g-i) the excitatory response was broken up in both the orthodromic and the antidromic records until first one major component disappeared (records in Figs. 29d and h ) and subsequently a second (records in Figs. 29e and i). A consideration of the antidromic responses indicates that the first response to disappear must be attributed to the cell body-initial segment region of the motoneuron and the second to the ventral dendrite. (Recordings made from a ventral dendrite itself often had only a “hump” on the falling phase to indicate the “cell body”response.) In Fig. 29e is seen the A l response as recorded in the primary motoneuron; bringing the inhibition still earlier eliminated this also (not illustrated-note the

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Fig. 29. Responses recorded from the cell body of an orthodromically excited ( b ) ( e ) and antidromically excited ( f )-( i ) group A2 motoneuron; the inhibition was produced at various time intervals relative to the ipsilateral excitation. ( a ) Response in cell when crossed inhibition was produced alone. ( b ) Response to excitatory Mauthner cell alone. ( f ) Response to ventral root excitation alone. Top trace: response in excitatory Mauthner cell. Second trace: response in inhibitory Mauthner cell. Third trace: response in motoneuron at high gain. Bottom trace: response in motoneuron at low gain. Calibrations: vertical, 2.0 mV (top two traces), 1.25 mV (third trace), and 20 mV (bottom trace); horizontal, 1 msec.

complicating response of the inhibition alone seen in isolation in record Fig. 2%). Fatigue, resulting from repetitive activation of the Mauthner axon, usually acted in a similiar manner, causing the cell body response to disappear first and that of the A1 unit last.

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It is temptins to relate the excitation of an impulse in the cell body of the primary motoneuron to the excitation of group B motoneurons. The effects of the progressively delayed crossed inhibition are consistent with this (cf. Fig. 28), and so are those of fatigue (cf. Fig. 42). Limitation of the respcinse to the ventral dendrite can be more confidently correlated with the excitation of the first-activated motoneuron group, which ought of course to be the primary motoneurons. There is, as yet, no direct evidence to support this suggestion of an excitatory cascade, in which the primary motoneuron also acts in the capacity of an interneuron for excitation of other motoneurons (or interneurons ) . We may note, however, the electron microscopic evidence of the presence of tight junctions between the primary motoneuron ventral dendrite and other processes. It is possible that here is a basis for the transmission of excitation from the primary motoneuron to other cells, perhaps leading to the crossed excitation. (Since the crossed inhibition occurs when neither A1 nor A2 units is excited, as happens with synchronous Mauthner axon impulses, it must require quite distinct collateral branches of its own.) We may note here that the motoneurons activated by excitation of the opposite Mauthner axon (the crossed excitation) are quite distinct from those involved in the ipsilateral excitation. This follows from the experiment of Fig. 20, in which block of the crossed inhibition by strychnine allowed the algebraic summation of the two excitatory responses in the ventral root; there was no “occlusion” as would have occurred if the same motoneurons had been excited as a consequence of the ipsilateral and contralateral Mauthner axon impulses.

VII. THE PRECISION AND CONSTANCY OF THE MINIMUM DISCRIMINATION TIME

A. The Function of the A1 Unit We are now in a position to suggest a likely function for the A1 unit and indeed to propose a basis upon which the most important function of the Mauthner cell system probably depcnds. The proposition is that the Mauthner axon collaterals act on a special postsynaptic region (the A1 unit) uhich is reserved exclusively for them, and this is the principal reason for their having the property of remaining uniformly excitable at all times. Becausc of this property, the effects of the impulses arriving along the ipsilateral and contralateral Mauthner collaterals

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will be constant and highly predictable. The timing of the arrival of these impulses, since their route to the special synaptic region is so short and direct, depends entirely upon the timing of the excitation of the Mauthner cells, and this is discussed later. However, the ability of the impulses in the two Mauthner axons to cause an excitatory and inhibitory interaction of such repeatable precision is largely dependent on this special feature, a postsynaptic region both protected from the usual influences which cause “synaptic noise” (see below) and isolated in order to exclude the possibility of interaction there between the effects of Mauthner inputs and those of other (non-Mauthner) inputs to the spinal motoneuron. Signals set up on either side of the proposed high resistance transitional region between the A l unit (the dendritic spine or intermediate cell) and the A2 unit (actually the ventral dendrite of the primary motoneuron) attenuate as they cross it. We have seen evidence of this in the reduction in size of the A1 e.p.s.p. and action potential when they are recorded in the ventral dendrite (Fig. 15). Their attenuation, however, is not so great as to prevent the initiation of spikes in the dendrite. What is the evidence that signals actually generated in the cell body and dendrites of the motoneuron will not cause any important effects in the A1 unit? First (Fig. 17d), even an antidromic spike in the main dendrite may not cause more than a few millivolts’ change in the A1 unit. Second, and much more significant, is the fact that in our experiments synaptic noise was never recorded in an A1 unit. Synaptic noise is the occurrence of small transient fluctuations in membrane potential believed to result usually from the sporadic arrival of impulses at nerve endings synapsing on the cell (but see Katz and Miledi, 1963). Such subthreshold synaptic activity however was recorded in the motoneuron cell body and even in the main ventral dendrite, and often this was “spontaneous.” Figure 30A shows characteristic responses caused by producing sounds in the vicinity of the fish, which presumably activated vestibular neurons in the brain. Such neurons, then, can affect the primary motoneuron in the spinal cord independently of the Mauthner cells. We have also been able to cause excitatory potentials in the primary motoneurons by stimulating axons in the spinal cord other than the Mauthner axons themselves (Fig. 30B). But no such effects were ever seen in the A1 unit itself. It seems that only the Mauthner axon collaterals make contacts on these units (and naturally there are no spontaneous impulses in such collaterals) and that signals generated by synaptic activity in the parent motoneuron attenuate to negligible levels during their electrotonic conduction across the junction into the A1 unit. Furthermore,

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2.0 rnsec (A) Fig. 30A.

inhibitory synaptic activity in the parent motoneuron will also be ineffective in reducing excitatory potentials in the A1 unit, since, regarded from the A1 unit, such inhibitory activity will have the properties of “dendritic remote inhibition” ( Diamond, 1968). The net result of all this is that the A1 unit is relatively noise free. B. The Implication of a Noise-Free System Consider Fig. 31, which shows diagrammatically how the latency of spike firing will be influenced by the presence of noise. The noise we are now considering represents activity in the motoneurons during the

0

b

Fig. 30. ( A ) Multiple exposures of responses recorded in a group A2 motoneuron during quiet sounds in vicinity of preparation. ( B ) Responses recorded in cell body of group A2 motoneuron during spinal cord stimulation below the level of the recording. a, Stimulus below threshold for excitation of either Mauthner axon; b, stimulus at threshold for excitation of ipsilateral Mauthner axon; and c, as b, but preceded by a stimulus applied to contralateral Mauthner axon. Upper trace: response of excitatory Mauthner cell. Second trace: response of inhibitory Mauthner cell. Third trace: motonenron response a t high gain. Bottom trace: motoneuron response at low gain. Note that response in a is subthreshold for firing the motoneuron, and that this is obscured by the full response in b. In c the inhibition prevents the full responses but does not affect the subthreshold response. Clearly the latter was initiated near the recording electrode, while the inhibition was exerted at a point remote from this (probably mainly on the ventral dendrite near the point of initiation of the response to Mauthner axon excitation). Calibrations: vertical, top and second traces, 2 mV, third traces, 1.25 mV, bottom traces, 20 mV; horizontal, 2.0 msec.

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Rest

HyperPO’‘

3 41

/t

_________

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _Inhibitory

noise level

Start of e.p.s.p.

Fig. 31. Representation of how noise will affect the firing latency of the A1 unit. The resting potential is indicated by the 0 mV horizontal line. Although inhibition could be effective largely because of the increased membrane conductance, it is indicated as a 3.3 mV hyperpolarization for simplicity. See text for further description.

normal (unstartled) swimming behavior of the fish. The threshold is taken as 10 mV, which though low is actually higher than that found in most of our A1 units. Excitatory “noise” is put at 3.3 mV depolarization and inhibitory noise as equivalent in its effect to 3.3 mV hyperpolarization (i.e., 33%of threshold). The e.p.s.p. is given a representative rate of rise of 25 V/sec, and this is assumed for simplicity to be uniform and unaffected by the variations of membrane potential and conductance involved in the production of noise mentioned above. On this highly simplified basis, the presence of this amount of noise could increase or decrease the firing latency by almost 0.15 msec (Fig. 31). Let us accept our experimentally determined figure of 0.15 msec as the minimum discrimination time. If the two Mauthner cells fired synchronously (and we shall see later that this is a real possibility) then, provided that the noise was below the amount mentioned above, there would be no output to the opposed muscle masses on either side of the body (see Fig. 12). Howcvcr, if this cxcitntory noise were present

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in some of the motoneurons on both sides, then the crossing inhibition would arrive too late at certain of them to prevent their firing, and the result would be obviously undesirable bilateral ( i.e., opposed) muscle activity. On the other hand, if the noise were inhibitory, then the two Mauthner impulses would need to be separated by at least 0.3 msec before any reflex movements would occur. We believe that this interval may be too great to be of use to the fish, that it is unlikely to be often achieved, and that the repeated experimental finding of 0.15 msec as the minimum separation required for effects is of significance; these points are considered in the next section.

VIII. THE EXCITATION OF THE MAUTHNER CELLS

A. The Need for Asynchronous Firing of the Two Cells From the characteristics of the spinal system, we know that the largest response to a single stimulus likely to activate the Mauthner neurons is the unilateral powerful movement resulting from the firing of only one of the two cells. But if an appropriate stimulus in the environment could cause both cells to respond, it is clearly of great importance, again taking account of the spinal system, that they do not fire synchronously or even within about two-tenths of a millisecond of one another (Fig. 12). This need for asynchronous firing must be regarded as of fundamental importance in Mauthner cell function. We must now consider the sorts of physiological stimuli which could cause the Mauthner cells to fire, the properties of these stimuli and the sensory pathways, and the brain circuitry of which the Mauthner cells are a part, which could help to bring about the time separation between the Mauthner impulses required for successful reflex effects.

B. The VIII Cranial Nerves: “Electrical” and “Chemical” Excitation of the Mauthner Cells From anatomical studies, it is certain that there is an important direct input to the Mauthner cells from the ipsilateral VIIIth nerve andalthough less easy to recognize histologically-a probable one from the contralateral VIIIth nerve also (Fig. 1). Of the greatest interest here are the prominent VIIIth nerve myelinated fibers which contribute the large “club endings” on the lateral dendrite (Figs. 1, 4, and 34A). Furshpan and Furukawa (1962) showed that these are excitatory to the

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1

Fig. 32. Responses recorded in Mauthner cell following stimulation of the ipsilateral VIIIth nerve. ( A ) Electrode in cell body. Superimposed traces made at various stimulation intensities. Note spike firing from early e.p.s.p. The later e.p.s.p.’s seen as delayed hump( s ) in subthreshold traces. Calibrations: vertical, 10.0 mV; horizontal, 0.5 msec. From Furshpan (1964). ( B ) Electrode in lateral dendrite, 325 p from axon hillock. Stimulus intensity near threshold for excitation of Mauthner cell spike. The latter fires from the second e.p.s.p; since this was smaller than the first, the spike must have been generated at a distant point (i.e,, the initial segment of the Mauthner axon) where the first e.p.s.p. was smaller than the second. This means that the first e.p.s.p. mainly resulted from synaptic activity near the recording electrode in the lateral dendrite. Calibrations: vertical, 5 mV; horizontal, 1 msec. From Diamond ( 1968).

ipsilateral Mauthner cell, and the evidence now indicates that their synapses are electrically transmitting (Furshpan, 1964). Figure 32 shows responses recorded intracellularly from two different Mauthner cells when their ipsilateral VIIIth nerves were excited directly by stimulating electrodes. Quite often in such experiments there are at least two phases of e.p.s.p., and the Mauthner impulse can arise from either of these, e.g., the first in Fig. 32A but the second in Fig. 32B. The earliest e.p.s.p. can have a synaptic delay which is almost immeasurably brief. Figure 33 shows evidence that this early e.p.s.p. results from the excitation of VIIIth nerve fibers synapsing directly on the lateral dendrite (see also Fig. 32B and legend) and that the synaptic transmission is electrical in nature. The early e.p.s.p. is very similar in time course to the impulse recorded directly by an electrode inside an VIIIth nerve fiber itself. The fastest conducting fibers, presumably the large myelinated ones forming the club endings, have a relatively low resistance coupling to the lateral dendrite since an electrical signal in the dendrite is detectable in

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O.5msec

Fig. 33. Intracellular recordings from VIIIth nerve fibers. Two types of fibers were distinguished: ( 1) by the shape and the latency of the response to stimulation of the VIIIth nerve ( A 1 and B1) and ( 2 ) by the finding that antidromic excitation of the ipsilateral Mauthner cell caused a potential change in the A fiber ( A 2 ) but not in the B fiber ( B 2 ) . In C simultaneous recordings were made from an A-type fiber and from the ipsilateral Mauthner cell body when the Mauthner axons were excited by a shock to the spinal cord; both potential changes had the same threshold. From Furshpan (1964).

them (Fig. 33-A1, A2, and C ) . (This signal was actually the passively transmitted potential resulting from an impulse generated at the axon hillock of the Mauthner cell.) In electron microscopic studies (Robertson et al., 1963) the club endings are seen to make tight junctions with the lateral dendrite (Fig. 34B), consistent with an electrically transmitting synaptic mechanism. The very early e.p.s.p., therefore, must be the actual activity generated in the large club endings, recorded postsynaptically in the Mauthner cell itself.

Fig. 34. ( A ) Phase contrast light micrograph of section of a lateral dendrite showing the synaptic bed (block arrows) and several club endings (thin arrows). 2.5% OsO, fixation; KMnOI stain. Marker is 10 p. ( B ) Section of club ending ( C ) on lateral dendrite of Mauthner cell ( M ) . A synaptic disc like the one designated by the arrow but from another micrograph is enlarged in the inset. Note the extracellular inatrix material ( * ) around the ending. Inset, approximately 11X magnification of rest of figure. ( C ) Section of bouton terniinaux ( b ) on lateral dendrite of Mauthner cell ( M ) . A capillary ( C ) appears to the right bounded by an endothelial

318

cell ( E ) with unknown profiles ( x ) . The endings are separated from the capillary by relatively light glia cells ( g ) . A portion of the synaptic membrane complex (arrow 2 ) is enlarged to the upper right. Inset, approximately 14X magnification of rest of figure. [ ( A ) From Robertson et al., 1963; ( B ) and ( C ) from Robertson, 1963.1 ( D ) and ( E ) Photomicrographs of two transversely cut lateral dendrites, 10p sections, silver stained: ( D ) is the proximal bulb-ending region; ( E ) is the distal club-ending region. The small endings are indicated by arrows in ( D ) and between the arrows in ( E ). Calibration lines: 10 p in ( D ) ; 20 p in ( E ). From Diamond (1968).

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Other junctions on the lateral dendrite have the conventional electron-microscopic appearances of chemically transmitting synapses ( Fig. 34C), and we may take it that some of these correspond to the ipsilateral VIIIth nerve enclings which do not seem to be electrically coupled (Fig. 33, B l ) . They must be responsible for some of the longer latency e.p.s.ps, the earliest of which has a synaptic delay of about 0.4-0.5 msec. These chemically transmitting endings have not been identified unequivocally, but they could include the “bulb endings” of Held-Auerbach, which cover the proximal half of the lateral dendrite (Figs. 4 and 34D) and are believed to derive directly from the VIIIth nerve, although it is not clear whether this is the ipsilateral or the contralateral one (see Bodian, 1942; Ketzlaff, 1957); they could also be the small endings which are scattered all over the lateral dendrite, and indeed over the whole cell (Figs. 4 and 34D and E ) . Many of the endings on the soma (and possibly the lateral dendrite too), and probably all those on the ventral dendrite, derive from neurons situated in a variety of regions of the brain (see legend to Fig. 4), and many of these will be activated, when vestibular excitation occurs, in addition to the direct VIIIth nerve fibers to the Mauthner cells.

C. The Vestibular System and the Swim Bladder Furukawa and his colleagues have shown that the stimulus which causes activity in certain groups of the VIIIth nerve fibers is sound, which acts primarily on hair cells in the lagena and the sacculus (Furukawa and Ishii, 1967a). It seems certain that a sudden vibrational stimulus in the vicinity of the fish will cause the more-or-less synchronous activation of both large and small myelinated fibers running to the lateral dendrite and other regions of the ipsilateral Mauthner cell, and quite probably, as we shall see, to the contralateral cell also. What then are the chances of one cell being fired in advance of the other? If the vibration originated closer to one vestibular system than the other, it would seem likely to affect the nearer Mauthner cell earlier and possibly to a greater extent than the opposite one, but there is one condition which could greatly influence this result. The diagram in Fig. 35 (taken from von Frisch, 1936) shows the relation of the swim bladder in the Ostariophysi (which includes both tench and goldfish) to the vestibular apparatus. It can be seen that vibrations could be transmitted via the body wall to the swim bladder, and then via the Weberian ossicles, the perilymphatic space and the endolymphatic transverse canal, directly and probably equally to the lagena and sacculus

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Sc'h

Fin. 35. Connection between swim bladder and labyrinth by the Weberian ossicles in Osturiophysi: Black, Weberian ossicles; Sch, swim bladder; Si, sinus impar ( perilymphatic space); C tr, canalis transversus (endolymphatic space); S, sacculus; L, lagena; U, utriculus; H, brain (posterior part being removed); I, incus; St, stapes; and M, malleus. From von Frisch (1936).

of both sides. Furthermore, even without the intermediation of the swim bladder, a vibrational stimulus in the environment located symmetrically with respect to the two vestibular systems would be expected to affect them more or less equally and synchronously. Here then, are two situations which make it likely that the Mauthner cells could often be excited simultaneously, an event, as we have seen, which would be singularly unproductive of the powerful tail flip caused by the excitation of a single Mauthner cell. Before dealing with this apparently unpromising situation, let us consider the asymmetrically effective stimulus.

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D. An Asymmetrically Effective Vibrational Stimulus?

1. THEEXCITATORY INPUTS Consider the situations when ( a ) a sudden vibrational stimulus affecting the vestibular systems directly is located nearer one side of the head than the other, and when ( b ) the stimulus acts via the swim bladder but the linkage of this to the two vestibular systems is sufficiently asymmetrical for one (say the ipsilateral) to be affected more than the other. In each case the excitatory VIIIth nerve volley to the ipsilateral Mauthner cell might be expected to be very slightly larger than that to the contralateral cell; furthermore, it would also be set up slightly in advance of that in the contralateral VIIIth nerve, although for moderate-sized goldfish the time difference could hardly be more than a very few microseconds (see Section IX, B). The inference from these considerations however is that unless the asymmetry involved is quite gross, the e.p.s.p. in the ipsilateral Mauthner cell would at best begin fractionally earlier, and rise slightly more steeply than that in the contralateral. Without the involvement of other factors, it seems highly improbable that, with a vibrational stimulus, asymmetrical effects alone could cause differences in the excitatory inputs to the two Mauthner cells sufficient to result in the 0.15-0.20 msec separation in the times of their firing needed for the production of the reflex trunk and tail movement. 2. THECROSSED INHIBITION OF THE MAUTHNER CELL There is onc potentially useful system here which must now be considered, a crossed inhibition acting on the opposite Mauthner cell, which can be observed when an VIIIth nerve is excited electrically ( Furukawa arid Furshpan, 1963). Figure 36 shows how this crossed inhibition can canccl out the effects of ipsilateral VIIIth nerve excitation and how these mutually opposed VIIIth nerve influences can be balanced experimentally to control the firing of the Mauthner cell. Is it possible, again considering the asymmetrical situations describcd above, that this inhibition could be responsiblc for producing the necessary time separation between the firing of the two Mauthner cells? By the same reasoning as was used above for the excitatory VIIIth nerve activity, the inhibitory volley derived from the ipsilateral vestibular apparatus would be expected to be slightly greater, and initiated slightly earlier, than that from the contralateral. That is, when both vestibular

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b

0

C

- '/

.*

e

Fig. 36. Responses in goldfish Mauthner cell to stimulation of VIIIth cranial nerves. ( a ) Contralateral VIIIth nerve excited alone (note inhibitory depolarization owing to C1- leakage into cell from microelectrode). ( b ) Ipsilateral VIIIth nerve excited alone. ( c ) Contralateral stimulus precedes ipsilateral; spike blocked. ( d ) As ( c ) , but ipsilateral stimulus increased. ( e ) As ( d ) , but contralateral stimulus increased. ( f ) As ( e ) , but ipsilateral stimulus further increased. [Spike fires in ( f ), but is reduced in amplitude.] Calibrations: vertical, 10 mV; horizontal, 1.0 msec.

systems are activated asymmetrically, the VIIIth nerve inhibition acting on the contralatera1 Mauthner cell would be slightly more effective than the corresponding inhibition of the ipsilateral cell. This effect would be in the right direction to contribute further to the relative delay of the contralateral Mauthner cell firing mentioned above. Let us see if this is supported on quantitative grounds. The VIIIth nerve inhibitory mechanisms resemble in many respects those of a collateral inhibition produced by excitation of a Mauthner cell itself. These latter mechanisms have been more fully analyzed and are discussed below, At this stage we can state that a major component of the VIIIth nerve inhibition acts by conventional chemical transmission involving an increase in the C1- (and possibly K') conductance of

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the postsynaptic membrane of the Mauthner cell (Fig. 36). It begins some few tenths of a millisecond after a single VIIIth nerve volley is initiated on the opposite side and may last 10-30 msec or even longer. The earliest phase of this inhibition probably involves a direct VIIIth nerve pathway, the latency of its action allowing only for the synaptic delay at the Mauthner cell itself (unless an electrically excited interneuron were interposed en route) ( Furshpan and Furukawa, 1962). Obviously only this particularly early inhibitory action is relevant to the present discussion, and the probable role of the later components is considered in Section IX, D. We are especially concerned with very small fractions of 1 msec. Exactly how soon can this crossing inhibition become effective? The fibers responsible for the crossed inhibition probably include those ending as “unmyelinated club endings” at the axon hillock region (see Fig. 4 legend). Suppose these conduct even as fast as 5-10 meters/ sec (which we may also accept for the conduction velocity in those ipsilateral VIIIth nerve excitatory fibers which transmit chemically). The crossed inhibitory pathway is about 1 mm longer than the ipsilateral excitatory pathway in a moderate-sized goldfish. The inhibitory volley would therefore arrive at the contralateral cell some 0.1-0.2 msec after that cell received the excitatory volley in its own VIIIth nerve “chemical” fibers, and it can be estimated from Furshpan’s data (1964), some 0.3-0.4 msec after the excitatory volley in the VIIIth nerve electrically transmitting fibers. Since the electrically transmitted e.p.s.p. is capable of initiating a spike within about 0.2-0.3 msec of its onset, there seems no conceivable way in which its effects could be interfered with by this crossing inhibition. We must remember that we are dealing with the asymmetrical situation, and therefore the inhibitory volley will arrive fractionally earlier at the contralateral Mauthner cell than the calculation allows, and the excitatory volley fractionally later. Moreover, in a small fish the crossed inhibitory pathway is less than 1 mm longer than the ipsilateral excitatory one. Thus, particularly in small fish, we have the possible result that just before the electrically transmitted e.p.s.p. fires the spike in the contralateral Mauthner cell (the vibrational stimulus is taken to be more effective on the ipsilateral side), the crossed inhibitory volley arrives there from the ipsilateral vestibular apparatus. But there uill be a minimum synaptic delay of about 0.4 msec before this inhibitory input becomas dective. Our conclusion must be therefore that the crossed, chemically transmitted inhibition almost certainly cannot interfere with the electrical excitation of a Mauthner cell by its own VIIIth nerve input, w e n in the asymmetrical situation.

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3. ELECTRICAL INHIBITION

By a chance which surely cannot be fortuitous, an extraordinary and at present unique inhibitory mechanism exists at the Mauthner cell, and its particular relevance to the present discussion lies in the fact that it involves no “synaptic delay.” It is an electrical inhibition, and it was discovered and analyzed by Furukawa and Furshpan ( 1963). The electrical inhibition is manifested as a hyperpolarization of the Mauthner cell in the axon hillock region not far from that where the impulse is generated during orthodroniic excitation. [This was shown by Furshpan and Furukawa (1962) to be probably in the axon, somewhat distal to the axon hillock.] An extracellular electrode located in this region records a transient and often irregular positive potential when the opposite VIIIth nerve or when either Mauthner cell is excited. The spatial distribution of this positive field, which is the sign of the electrical inhibition, was shown to conform closely to that of the axon cap; this is a histologically identified region around the axon hillock of the Mauthner cell, which stains somewhat differently from surrounding tissue with many staining procedures and is bounded by a roughly spherical shell of glial cells ( Bartelmez, 1915). Running through the axon cap is the narrow neck of the Mauthner axon, and it is at the outer limit of the axon cap that this axon acquires its myelin sheath. Enclosed in the axon cap are many nerve fibers and a few fine Mauthner cell dendrites. Many of the axons coming to the Mauthner cell in this region make irregular spirals round the axon neck before apparently ending on the axon hillock and cap dendrites, while other axons run through the axon cap without spiraling and also end apparently directly on the cell (see Fig. 4 ) . The diameter of the cap may be 50-100 p. The positive electric field recorded in this region when either Mauthner cell or the opposite VIIIth nerve is excited acts effectively as an external anode outside the Mauthner cell and is the direct cause of its hyperpolarization. Figure 37A shows records made within the axon cap during contralateral VIIIth nerve excitation; when the cell was fired antidromically so that the axon hillock spike coincided with the peak of the external positivity, the impulse was blocked. Clearly a very powerful inhibition was operating at that time. The mechanism of this inhibition is best appreciated from viewing Fig. 37B. In this figure it is shown how such an external positivity ( Furukawa and Furshpan’s “extrinsic hyperpolarizing potential” or EHP) caused by the activation of the collateral inhibitory pathway, actually succeeds in hyperpolarizing the local Mauthner cell membrane. When the electrode was inserted into the cell in the same region (the direction of the spike is then reversed),

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the positivity was markedly reduced although still detectable. The true trans-membrane potential is obtained by algebraically subtracting the extracellular from the intracellular potential (Fig. 37B); the result shows conclusively that during the period of external positivity the membrane was genuinely hyperpolarized. These results also indicate that the source of such a potential (the “battery”) is not in the Mauthner cell membrane but in some other structure outside it. It seems likely that the arriving impulses in the inhibitory fibers die out toward the outer limit of the axon cap, and the fiber terminals within the cap act as a passive source of external current, thus creating the positive field. Whatever the exact mechanism involved, the system does not require a synaptic delay in the conventional sense to cause an efective inhibition of the axon hillock region of the Mauthner cell. Interestingly, both the VIIIth nerve inhibition and the collateral inhibition have two components. The first is the early transient electrical one just described (the EHP), which is almost immediately followed by a conventional chemically transmitted post-synaptic inhibition. The second component can clearly be

(A)

Fig. 37A.

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Hyper polar izot ion

L Fig. 37. ( A ) The early extrinsic hyperpolarizing potential ( E H P ) evoked by contralateral VIIIth nerve stimulation blocks the antidromic spike. Extracellular recording near the Mauthner cell axon hillock. When a contralateral VIIIth nerve shock ( a ) and a spinal cord stimulus (11) were delivered during the same sweep ( c ) , timed so that the antidroniic spike would arrive at the peak of the EHP, the spike failed to appear. Both of the first two peaks of this EHP could block the spike. Calibrations: vertical, 20 mV; horizontal, 2 msec. ( B ) Hyperpolarization of the Mauthner cell during the EHP. The recordings were made with the microelectrode tip very close to the axon hillock of a Mauthner cell (negative spike, 38 mV); ( b ) was made immediately before entering the cell; the electrode was advanced slightly and it entered the cell at which time ( a ) was recorded. The stimuli were applied to the spinal cord. To determine the actual change in membrane potential ( b ) was subtracted algebraically from ( a ) . The result is shown in ( c ) ; at the peak of the EHP, the patch of membrane was hyperpolarized by 15 mV. The record in ( a ) has been retouched to remove several superimposed testing spikes. Calibrations: vertical, 20 mV; horizontal, 1 msec. From Furukawa and Furshpan ( 1963). Note that the EHP in ( B ) was part of the collateral inhibition (see text).

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seen in Fig. 41 (the “late collateral inhibition” or LCI of Furukawa and Furshpan) as a depolarization, which resulted from a leakage of chloride ions into the cell from the recording microelectrode. Similar appearances can be recorded during the VIIIth nerve inhibition (Fig. 36a). The electrical inhibition is obviously able to be effective during the period of synaptic delay required by the conventional “chemical” inhibition. We can suppose therefore that in the context of our discussion the electrical inhibition must be considered as possibly very important indeed, at least as a means of increasing the time taken by the “electrical” e.p.s.p. to reach threshold (even though the electrical inhibition might only begin during the rising phase of that e.p.s.p.). The situation can be appreciated from viewing Fig. 38, which shows the approximate time relations of the various excitatory and inhibitory mechanisms that are activated during bilateral VIIIth nerve excitation. In summary then it may be concluded that as a consequence of electrical inhibition, it is possible for a stimulus which h a the asymmetrical character described above to cause both Mauthner cells to fire, with one slightly delayed relative to the other. The delayed cell

e P S.P Elect

1

Ar-

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inhibition

Contralatcrol Mouthmr cel I

lpsilaterol Mauthner cell

Fig. 38. Time relations of e.p.s.p.’s and crossed inhibitions initiated by bilateral VIIIth nerve excitation. Lines above the horizontal line (which represents resting membrane potential ) indicate excitatory responses; lines below it indicate inhibitions resulting from nerve volleys in the VIIIth nerve contralateral to that Mauthner cell. Zero time equals arrival of the ipsilateral electrically transmitting VIIIth nerve volley. Continuous lines indicate responses when both VIIIth nerves are synchronously excited. Dashed lines indicate responses with asynchronous excitation of VIIIth nerves. The thin arrows arising from the resting potential line show the moments when each inhibitory mechanism becomes effective, in both the symmetrical and the asymmetrical conditions. The effects on both the electrical and the chemical e.p.s.p.’s are shown as discontinuities in their rising phases. The moments when impulse firing o(:curs are indicated by the thick arrows arising from the e.p.s.p.’s. Note: When an impulse fires from an electrical e.p.s.p. there would be none from the later chemical one owing to refractoriness.

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would of course be that on the opposite side to the vestibular system most affected by the stimulus. Even so, the chances of causing a delay as great as 0.15-0.2 msec must be sometimes rather slim. It may be necessary to involve yet another factor, to be discussed now, which we propose can operate even when the stimulus is entirely symmetrical with respect to the two vestibular systems, or when the stimulus is transmitted via a swim bladder which affects equally the vestibular systems on either side.

E. The Symmetrical Stimulus 1. THRESHOLD FLUCTUATIONS IN THE Two CELLS From all that has been said above it is clear that in many situations the mechanical stimulus will be transmitted equally to each vestibular system, and then often the output will be equal in both VIIIth nerves. It would seem that under these conditions the Mauthner cells would fire simultaneously, and as we already know this will cause no reflex movement of the trunk and tail. Are there other factors which could make this undesirable result unlikely? Consider the fish in its normal environment. Here is a system which is rarely bilaterally symmetrical. The fish undulates as it swims. The water turbulence on either side of the animal is unlikely to be identical in all respects, nor is the illumination presented to the two eyes; in particular, the muscular activity will not be identical on the two sides at any given instant. From considerations such as these we may suppose that in the central nervous system the representation of the two sides of the fish at any instant of time will differ. Now it is known that the Mauthner cells receive an enormous neural input, which has origins in very many parts of the brain (Fig. 4); presumably this input presents to each Mauthner cell information about the state of the animal and its environment, very probably with reference to the same side as the cell. If the state is different on the two sides, then the pattern of impulses arriving at the cells will also be different on the two sides. In this context it is worth mentioning that the sudden shadowing caused by passing the hand between a direct light source and the fish tank can produce a startle-response extremely similar to that caused by a sudden blow to the tank. The indirect optical pathway to the Mauthner cells could be involved in this response. Doubtless there are other conditions which could produce or contribute to the production of a startle-response which are dependent on inputs other than the VIIIth nerve ones on to the Mauthner cells. During normal “unstartled” behavior the effect of this information will be to

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Fig. 39. Records from Mauthner cell body during quiet talking and humming (bottom right trace) in vicinity of preparation. Calibrations: vertical, 2 mV; horizontal, 10.0 msec.

produce only subthreshold activity in the cell, i.e., “synaptic noise,” of the sort we have referred to earlier when discussing the spinal motoneurons. Figure 39 gives examples from an anesthetized, immobilized, experimental preparation of synaptic noise recorded in the Mauthner cell simply as a consequence of quiet sounds in the vicinity of the fish. Obviously, in the normal state, when the central nervous system must be receiving an enormous input as a consequence of the very many stimuli about it, this synaptic noise must be very large indeed. Since it will be different on either side at any instant, then the sudden superposition of the excitation resulting from a sudden vibrational stimulus, even a “symmetrical” one, cannot be equally effective in both cells. This would also apply for optical stimuli. One cell must fire before the other. Taking all Factors into consideration it seems likely that it will be only on rare occasions that a vibrational stimulus actually will cause the two cells to fire simultaneously. We suppose that usually there would be an interval between the two Mauthner impulses and that this would often be at least one- to two-tenths of a millisecond, i.e., the interval necded for the spinal system to operate, resulting in the powerful tail flip described above. 2. A “LASTRESORT”?

Even if there were, despite all the factors discussed above, simultaneous firing of the two Mauthner cells, a useful end result could still be achieved. We have already seen how the cranial component of the Mauthner cell reflex response is not associated with a crossing inhibi-

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tion like that in the spinal cord relating to the trunk and tail movement (Figs. 9 and 10). If this cranial response occurred, then the sudden intake of water into the mouth and the sudden expulsion of water from the gills could still cause a movement by a “jet propulsion” effect, which might well serve to remove the animal from immediate danger even in the absence of the tail flip.

IX. THE FUNCTIONS OF THE MAUTHNER CELLS

A. Swimming and Equilibration: Two Improbable Functions 1. FATIGUE IN THE MAUTHNER CELLSYSTEM

a. Excitatory and InhibitoTy Mechanisms. Certain parts of the neuronal system involved in the Mauthner reflex show marked “fatigue” with repetitive activation [ i.e., their response ( s ) becomes progessively reduced]; they are: ( i ) the collateral inhibition of the Mauthner cells in the brain (both the electrical and the chemical components); (ii) the excitation of the spinal and cranial motoneurons; and, to a lesser extent, (iii) the crossed inhibition in the spinal cord. Figure 40 shows an experiment in which the progressive reduction of the collateral electrical inhibition (the EHP), with increasing frequency cf stimulation of the Mauthner cell causing it, paralleled that of the tail muscle response on the side of the excited Mauthner axon. Figure 41 shows fatigue of the collateral chemical inhibition ( L C I ) of the Mauthner cell, and Fig. 42 shows fatigue affecting the ventral root response in the trunk region. Sometimes many seconds ( u p to 10) may be required for complete recovery of any of these component systems after even a single activation. Some of the fatigue, certainly of the excitation of the primary motoneurons, seems to be of the presynaptic mechanism (Fig. 43), although a form of postsynaptic “desensitization” cannot be excluded. Since the recovery timc is very variable and sometimes can be as small as 1-2 sec, this fatigue might be partly dependent on a reduced local blood circulation following operation. However, even in the best preparations, e.g., those in which only the brain and not the spinal cord or ventral roots was exposed (as in the preparation which gave the results shown in Figs. 40 and 44) fatigue in all the systems mentioned above was always readily demonstrable. Interestingly, the crossed inhibition in the spinal cord was less susceptible to fatigue than was the excitation (Fig. 44). The Mauthner cell and its axon can follow rates of stimulation of 100/sec or higher however.

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Fig. 40. Fatigue in the Mauthner cell system. Three stimuli were delivered in each test. Top trace: Mauthner cell spike, antidromically excited. Second trace: EHP recorded outside opposite Mauthner cell; only the response to the third stimulus of each triad is shown. Lower traces ( a ) - ( e ) : EMGs from trunk muscle. Superimposed traces of all three responses in each test triad. Three stimuli delivered to Mauthner axon at 1/10 sec in ( a ) , 1/5 sec in ( b ) , 1/3 sec in ( c ) , 1/2 sec in ( d ) , and l/sec in ( e ) . Calibrations: vertical, 5 mV ( M cell), 2 mV (EHP), 1.25 mV (EMG); horizontal, 10 m e c for EMG, 2 msec for remainder.

b. Startle-Rc!sponse; Swimming and Equilibration. Now if fatigue is, as our results suggest, a genuine property of the system, it becomes very important in relation to the question of Mauthner cell function. Attempts to elicit the startle-response repeatedly, by tapping the fish tank or by

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Fig. 41. Fatigue of the LCI. In each record ( a ) - ( c ) two successive stimuli were delivered to the Mauthner axon and recordings made from the Mauthner cell body. ( a ) Single pair of stimuli; the reduction in amplitude of the second spike during the depolarizing inhibitory potential (resulting from C1- leakage, see text) is a measure of the conductance change during the collateral inhibition. ( b ) The pair of stimuli were repeated once a second; a few superimposed traces are shown. ( c ) Repetition rate of stimulus pairs was increased to two per second. Note the reduction in ( b ) and the abolition in ( c ) of the inhibitory potential and the conductance change owing to fatigue. The horizontal trace in each set shows the resting potential recorded without stimulation of the Mauthner axon. From Furukawa and Furshpan (1963).

sudden surface splashing, usually fail unless many seconds are allowed to elapse between successive stimuli. Indeed, it seems that this need for a recovery period between stimuli (delivered to the freely swimming animal) can be reasonably correlated with the fatigue in the neural

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0.5sec

Fig. 42. Ventral root responses caused by ipsilateral Mauthner axon excitation at various stimulation frequencies. Interval between successive stimuli shown at left of each trace. Calibrations: vertical, 200 pV; horizontal, 2 msec (compare Fig. 28).

systems described above. Neither the excitation of the Mauthner cell itself by electrical stimulation of the VIIIth nerve, nor a t least one major component of the VIIIth nerve response to vibrational stimulation (Furukawa and Ishii, 1967a) is particularly susceptible to fatigue. But if the effects caused by Mauthner cell excitation are so readily fatigued, it becomes difficult to ascribe to those cells any function which requires their activation more frequently than about l/s e c at most. One function so excluded would be their involvement in normal swimming, during which ihe frequency of side-to-side tail movements, especially in small fish, can be many times a second. Furthermore, the response to Mauthner cell excitation is, as we have seen, a powerful all-or-nothing tail flip to one side [except at the restricted periods ( i ) at the end of the crossed inhibition and ( i i ) during the very brief interval of interaction between the effects of two very closely spaced Mauthner impulses, see Fig. 121. The result of this flip

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a

Fig. 43. Responses in group A1 unit to single stimulus applied to ipsilateral Mauthner axon [( b ) at low gain and upper trace in ( a ) a t high gain] and to repeated stimulation [at 1/6 sec (full response), 1/5 sec (largest subthreshold response), and 1/4 sec, 1/3 sec, 1/2 sec, Usec (smallest response)] of Mauthner axon [subthreshold responses in ( a ) , traces superimposed]. Calibrations: vertical, 2.5 and 20 mV; horizontal, 1 msec.

is the characteristic “swiveling” motion which moves the animal a little away from its original position. A simple flick of the tail might be expected to cause only a simple rotation of the animal about a vertical axis. However, if the effect of the water expulsion from the gills is also taken into consideration (see Section IV) then the total movement would be expected to resemble that which seems to occur during the startle-response, the result of a rotation and a forward motion. Our understanding of this reflex would benefit greatly from a kinematic analysis of the startle-response. It is the author’s opinion that a simple alternation of excitation of the two Mauthner cells would not result in normal coordinated swimming movements, even those seen in “escape-swim-

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Stim. to L axon L EMG

t

Stim. to R axon

Fig. 44. Fatigue of excitatory responses and crossed inhibition owing to repetitive stimulation of the Mauthner axon (see Fig. 11). ( A ) The left Mauthner axon stimulus was applied at various times after the right (which was applied at a fixed time). Records superimposed, ( B ) As ( A ) , except that the right Mauthner axon was excited at 2/sec until its excitatory responses (both ipsilateral and contralateral) were fatigued out. Note that the crossed inhibition resulting from the repetitively excited Mauthner axon, though shortened in duration, was not eliminated as were both the excitatory responses. Calibrations: vertical, 2.0 mV for left EMG and 2.5 mV for right EMG; horizontal, 10 msec.

ming” which in appropriate conditions often follows the startle-response. The crossed inhibition in the spinal cord is very powerful and would certainly cancel out any concomitant excitatory activity of the primary motoneurons. Similarly the ipsilateral excitation is unlikely to be susceptible to inhibitory systems other than that deriving from the opposite Mauthner axon; this excitation is exerted near the origin of the efferent axon itself, on the ventral dendrite of the primary motoneuron. The explosive and prepotent character of the ipsilateral excitation and contralateral inhibition at the level of the “final common path” in the spinal cord seems quite unsuitable for controlled coordinated swimming and even less suitable for “equilibration” functions. The author has no d a c u l t y in accepting the role of the Mauthner cells as being

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entirely to do with a simple but powerful escape reflex, a mechanism for avoidance of predators (see below). If the excitatory cascade does exist (Section VI, E), it is still possible for the primary motoneurons, and the subsequently activated group B motoneurons, to be utilized for swimming; but the presumed coordination required would be best achieved by controlling the motoneurons from non-Mauthner systems. That way the individual “cascades” ( i.e., those involving only one primary motoneuron) would provide the minimum functional units. When the Mauthner axons are involved, all the primary motoneurons and all the subsequently activated group B cells together constitute the minimum unit. Of great relevance here are the findings of Sims (1962) on the larvae of Xenopus laevis. After section of their spinal cords the animals showed, not surprisingly, a loss of locomotory movement below the level of the lesion and had no startle-response. Subsequently their normal swimming ability returned. Histological examination showed that a great deal of regeneration had occurred in the spinal cords. However, below the level of spinal section the Mauthner axons appeared to have degenerated irreversibly. The startle-response, when it reappeared in the recovered animals, never involved the part of the tail innervated from cord levels below that of the original lesion but only that from above. This is telling support for the rejection of the suggestion that Mauthner cells are involved in swimming, and strengthens the conclusions reached in Section IX, C below.

c. The Fatigue. It is of interest that the fatique in the collateral inhibitory systems in the brain is also probably located in the Mauthner collaterals rather than the interneurons which are almost certainly involved in the inhibitory pathways; recordings from such interneurons (cf. Furukawa and Furshpan, 1963) show that their excitation ceases during repetitive activation of the Mauthner cell, at frequencies which cause fatigue of the collateral inhibition (cf. Fig. 40). We have observed that repetitive activation of both Mauthner axons synchronously still causes fatigue in the neuronal circuit, even though there is no output from the spinal cord (resulting from crossed inhibition). When, in the train of stimuli, one is omitted on one side only, there is no motoneuron discharge in response to the impulse in the opposite Mauthner axon, the usual recovery period is required. This shows that the phenomenon is not owing to a “Renshaw inhibition” initiated from excited motoneurons. It seems possible that the phenomenon of fatigue in the various Mauthner cell functions may reflect the relatively enormous integrative and distributive function which is required of this single central neuron.

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Our conclusion that fatigue is a genuine property of the Mauthner cell is strengthened by our observation that the VIIIth nerve electrical and chemical inhibition and excitation of the Mauthner cells was quite unaffected at frequencies of stimulation higher than stimulation frequencies which very rapidly caused all the fatiguable systems mentioned above to cease operating completely in the same experimental preparations.

B. Directionality Sensing: An Error In Section IV, C we saw how the minimum discrimination time measurcs the ability of the spinal circuitry to resolve (as separate events) two impulses, one in each Mauthner axon; this time can be as small as 150 pst'c. Consider a sound, originating to one side of the head, on a line with both vestibular systems, If we ignore the influence of the swim bladder and accept the situation discussed in Section VIII of an asymmetrically effective stimulus, we can estimate approximately whether the sound could cause the nearer Mauthner cell to fire an impulse 150 psec or more iii advance of the opposite cell. Only if this happens can there be a response from the animal giving information as to the side from which the sound originated. The sound would travel through water, or goldfish tissue, at about 1500 meters/sec; if the vestibular systems were 15 mm apart it would affect the nearer one some 10 psec before the other. Even if we allow for some loss in the amplitude of the stimulus in its transmission through (and around) the head, and thus for a very slight effect on the relative magnitude of the resultant VIIIth nerve volleys (Section VIII, D ) , it is difficult to believe that the 10 psec differential could be stretched to 150 psec. The system is clearly not designed for directioiiality sensing of sound. ( I n the following section we mention the possibility of optical stimuli reinforcing sound as an orientating mechanism.) The suggestion made by Moulton and Dixon (1967) that the Mauthner cells provide a basis for directionality sensing must therefore be rejected; moreover since Mauthner cell activity was measured 'by the occurrence of a tail flip, which our observations show can result from the activation of other cclls in the brain and spinal cord, their evidence must be regarded as inadequate.

C. The Avoidance Reaction: A Genuine Function When is the Mauthner reflex likely to be of greatest value? One likely proposition relates to birds of prey, and this was first suggested

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to the author many years ago by A. R. Ness of University College London. (The author is indebted to Mr. Ness for much information on these birds.) Many species of teleost fish live, or spend much of their time, in relatively shallow water, and as such are the natural prey of most species of fish-eating birds. Some birds pick or scoop up fish (shearwaters, frigate birds, and white pelicans), and here we may suppose that optical stimuli to the fish may be especially important, more so than vibrational ones, in contributing to a startle-response. This would apply too in the case of swimming birds which dive from the surface (cormorants, auks, and penguins), but there could also be an important effect on the fish from the shock wave. However the group of birds against which the startle-response would seem to offer the greatest protection must surely be the diving or plunging birds, both those which pick up with the beak (kingfishers, terns, gannets, Tropic Birds, and brown pelicans) and with the feet (osprey). It can be calculated that the minimum time which elapses between a diving bird hitting the water surface (and thus providing the mechanicaland probably optical-stimulus) and arriving at the level of a fish not far from the surface, would be a few tens of milliseconds. The interval between mechanical stimulation (of the vestibular system) and the early development of the Mauthner reflex movement would be about the same. The reflex response seems to move the fish just a few centimeters away from its original position (usually it finishes u p to one side rather than ahead or behind), but this could be enough to cause the bird to miss its target. Some casual observations by the author on the responses of freely swimming goldfish to surface splashing suggest that the startle-response is usually immediately followed by escape swimming; the initial characteristic swiveling movement, the presumed Mauthner reflex, was however readily distinguishable, and to the observer there seems to be a clearly detectable brief lapse of time before true swimming begins. The latter might well require some orienting cues. The startle-response serves simply to put the fish slightly away from the position it was in when the bird first sighted it and made its dive. On this basis it would be immaterial which way the fish moved, and all that we have said about the Mauthner cell reflex implies that it would not matter which cell fired first. This proposition remains to be proved. It could be that optical stimuli, which are obviously capable of influencing the Mauthner cells, might contribute to an orienting startle-response if they originated sufficiently to one side or-if derived from moving sources-they produced some appropriate retinal image ( cf. Rodgers et al., 1963). This possibility could work to advantage against the un-

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welcome attention of birds like the herons and bitterns, which catch fish from a wading position. The fact that birds which prey on fish do so successfully, and improve their performance as they grow to maturity, does not invalidate the probable importance of the startle-response as an avoidance reaction particularly developed to help shallow-living fish escape such birds. It is significant that the “Mauthnerian apparatus” is absent or poorly developed in tailIess fish, in almost all species living mostly at the bottom, and in those showing eellike (anguilliform) movements; it is interesting too that Mauthner cells are absent in anuran tadpoles with small tails and most prominent in those with large ones, while on metamorphosis of these species into tailless adult forms the Mauthner cells atrophy altogether ( Stefanelli, 1951). The association of the Mauthner cell with the tail however has tended in the past to strengthen a belief that the role of the cell is in swimming. We have rejected this view in the preceding pages. The important points are the biological environment of the tailed animals which have Mauthner cells, and

the characteristics of the circuitry of which the Mauthner neurons are a part. Aquatic predators as well as birds may to some extent be put at a disadvantage by the existence of the startle-response. However, it may be that in this instance the lateral line system may be more important, and only in some species do fibers of the (anterior) lateral line nerve extend to the Mauthner cells [Aronson (1963)-see also the other origins of inputs to the Mauthner cell (Fig. 4)].

D. The Functions of the Collateral Inhibition 1. THEE F F E ~ VDURATION E OF INHIBITION Both the chemically transmitted collateral inhibition of the Mauthner cell in the brain and the crossed inhibition in the spinal cord last a few tens of milliseconds. Although we have not investigated the duration of the mechanical muscle response in the Mauthner reflex, it is probably declining by the time these inhibitions have ended (cf. Auerbach and Bennett, 1969a,b). The electrical inhibition acts very quickly (Fig. 37B ), and the total effect of the collateral inhibitions in the brain is enormously to reduce the chances of either Mauthner cell firing a second time while the reflex response is rising or is near its peak. It seems reasonable that the powerful tail flip should neither be reactivated while at its peak nor opposed by an oppositely directed one at this same time. The former possibility is disallowed by the phenomenon of fatigue, the latter by the crossed inhibition in the cord. The cranial movements

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(eyes, jaw, and operculi) require bilateral and symmetrical muscle activity, and this results from the excitation of either Mauthner cell. Only the collateral inhibition of both the Mauthner cells in the brain can minimize the possibility of the circuits involved in these cranial movements being reactivated before the peak response is passed; fortunately, this collateral inhibition acts both on the cell from which it originates and on the opposite one. Nevertheless, in view of the phenomenon of fatigue, it is not clear exactly what the function of the collateral inhibition could be. If, however, reexcitation of the Mauthner cells is a possibility, then its prevention would clearly minimize the amount of fatigue which could be caused; we have mentioned that even when the crossed inhibition is acting in the spinal cord, the excitatory neural systems still fatigue (Section IX, A, 1, c above). There would thus be a gain in the prevention of the (useless) reactivation of the circuitry at such a time, consequently reducing the fatigue in the susceptible parts of this circuitry.

2. THESOURCES OF REEXCITATION OF THE MAUTHNER CELLS Is there a likelihood of stimuli occurring which could cause reexcitation of a Mauthner cell during the increasing, or peak, reflex response, a situation which we suggest above is undesirable? A number of possibilities can be mentioned: ( a ) recxcitation, following a single startleresponse, by the mechanical disturbance of the tailflip itself; ( b ) multiple excitations resulting from multiple reflections of a sound wave from nearby structures or other fish; and ( c ) in a shoal of fish, repeated excitation by the mechanical disturbances deriving from startle-responses or sudden movements of nearby fish. In this context one might speculate as to the possibility that the first sudden movement of a fish could excite a startle-response in nearby fish, possibly coupled with optical stimuli leading to an orientated response, and that this could conceivably be the basis of the sudden changes of direction observed in shoals of freely swimming fish. It should be noted that the collateral inhibition of the Mauthner cell will not prevent its excitation with the near certainty that the crossing inhibition in the cord prevents the excitation of the primary motoneuron. Furthermore, the collateral inhibition acting on the lateral dendrite (and possibly on the VIIIth nerve excitatory system itself) lasts longer than that acting on the Mauthner cell soma (see Furukawa, 1966). Possibly, inputs to the somatic region of the Mauthner cell can initiate excitability changes even before the lateral dendrite is responsive again to the ipsilateral VIIIth nerve input. The crossed VIIIth

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nerve inhibition, electrical and chemical, will reinforce on the contralateral cell any collateral inhibition coincidently activated. Nature seems to have accepted the numerous possibilities of closely timed reexcitation of the Mauthner neurons, particularly by vibrational stimuli, as a genuine hazard to the fish. The minimization of this hazard reinforces our view that the Mauthner reflex is prepotent and suggests that the initiation of escape swimming after a startle-response (Section IX, C ) could be more valuable to the fish than the reexcitation of the Mauthner reflex with its effective preclusion of coordinated swimming movements.

E. Functions in Different Species : Unanswered Questions (1) As indicated in the introduction to this chapter, Mauthner cell functions probably vary from one animal type to another. In this context, it is interesting to note that in the hatchetfish (Gasteropelecus) other giant nerve fibers intervene between the Mauthner axons and the motoneurons, and the reflex response to tapping the aquarium is a jump of the fish into the air, achieved by the simultaneous contraction of pectoral fin musculature on both sides and axial musculature on one side (Auerbach and Bennett, 1969a,b). Surprisingly, no spinal crossing inhibition was reported. In this fish, too, a phenomenon analogous to that of “fatigue” ( described above) was observed. The Mauthner axons excite the giant axons chemically, and they in turn excite the motoneurons electrically (here there was good evidence of the excitation of motoneurons by other motoneurons resulting from electrotonic coupling between them). In the hatchetfish therefore the giant (non-Mauthner) axons are analogous to the A 1 units we have described. Comparative studies on a variety of different animals will undoubtedly show that the Mauthner cells and those that they act upon can vary in their functions (cf. Rovainen, 1967), and such studies will clarify the whole problem of the role of Mauthner cells in general. ( 2 ) Certainly one important type of evidence lacking at present is a full kinematic analysis of startle-responses with simultaneous recordings from the Mauthner cells themselves rather than the recording of inadequately defined reflex movements or electrical potentials in the cord or muscles ( cf. Berkowitz, 1956; Wilson, 1959). ( 3 ) The linkage of the swim bladder to vestibular systems, already referred to, could provide more than simply a means of transmission of mechanical stimuli originating in the environment. The results of Furukawa and Ishii (198713) suggest that possibly hearing sensitivity could be influenced, or even controlled, by changing the abdominal pressure.

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Clearly the depth of the fish might be an important consideration and could perhaps influence the production of startle-responses to stimuli associated with aquatic predators. More experiments are needed on these important points. (4) The type of muscle activated in the Mauthner reflex is not known. It would certainly be of interest to have information on this and also on how the same muscles could be activated in other, non-Mauthner reflex activity (see Hudson, 1968). In addition the behavior and sources of activation of fin muscles is largely unknown, certainly in relation to startle-responses [except in the case of thc pectoral fins in the hatchetfish, see ( 1 ) above]. Comparative studies would be of great value here. (5) It has been suggested that in any one animal there could be differences in the size and shape of the two Mauthner neurons and their axons (Moulton and Barron, 1967). This, of course, would be very useful in helping to bring about a time difference in the initiation of the Mauthner impulses and in their speed of conduction in the cord. However, without statistical information the variability of such histological appearances in fixed tissues can hardly be adduced as good evidence for true anatomical differences in the living animal. Physiologically, as we have said, no differences are usually found between the two neurons or their axons. This point does however deserve further study. ( 6 ) The morphology of the inputs to the Mauthner cells and their distribution on the cell soma and dendrites varies in different species ( cf. Otsuka, 1962, 1964). Here there may be further clues as to functional characteristics. Again a comparative analysis of the startle-responses of different. fish and their correlation with the morphological features relating to the Mauthner cells might give more understanding of the whole of the Mauthner cell system and the reflex with which it is concerned. There are numerous other questions which we have raised in this chapter and other investigations in which the Mauthner cells may be (and have already been) of great value. We may mention here their usefulness in quantitative studies on synaptic pharmacology (Roper et d., 1969). Developmental studies, too, may be more conveniently carried out on the larger Mauthner neurons and their axons than on most other central neurons (Stefanelli, 1951; Hibbard, 1965; Moulton et d., 1968; Piatt, 1969), as may chemical investigations (Edstrom and Sjostrand, 1969). Clearly there is an important future for the Mauthner cells as the neurons of choice in vertebrate central nervous systems, especially when the investigation requires the ability to distinguish between somatic and dendritic regions (Diamond, 1968); we can expect these cells to

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Otsuka, N. ( 1962 ). Histologisch-entwicklungsgeschichtliche Untersuchungen an Mauthnerschtm Zellen von Fischen. Z . Zellforsch Mikroskop. Anat. 58, 33-50. Otsuka, N. ( 1964). Weitere vergleichend-anatomische Untersuchungen an Mauthnerschen Zellen von Fischen. Z . Zellforsch. Mikroskop. Anat. 62, 61-71. Piatt, J. (1969). The influence of VIIth and VIIIth cranial nerve roots upon differentiation of .Mauthner’s cell in Ambystoma. Dcuelop. Biol. 19, 608-616. Retzlaff, E. (1957). A mechanism for excitation and inhibition of the Mauthner’s cells in Teleosts. A histological and neurophysiological study. J. Comp. Neurol. 107, 209-225. Hobertson, J. D. 1,1963). The occurrence of a subunit pattern in the unit membranes of club endings in Mauthner cell synapses in goldfish brains. J . Cell Biol. 19, 201-221. Robertson, J. D., Bodenheimer, T. S., and Stage, D. E. (1963). The ultrastructure of Mauthner cell synapses and nodes in goldfish brains. J. Cell Biol. 19, 159-199. Rodgers, W. L., and Melzack, R. (1963). “Tail Flip Response” in Goldfish. J. C o m p . Physiol. Psychol. 56, 917-923. Hoper, S., Diamond, J., and Yasargil, G. h4. (1969). Does strychnine block inhibition post synaptic:ally? Nuttire 223, 1168-1169. Rovainen, C. M. (1967). Physiological and anatomical studies on large neurons of the central nervous system of the sea lamprey. (Petromyzon murinus) in Miiller and h4authnc:r cells. J. Neurophysiol. 30, 1001-1023. Sinis, R. T. (1962). Transection of the spinal-cord in developing xenopus laevis. J. Emhryol. Ex&. Morph. 10, 115-126. Smith, I. C. (1955). Giant nerve fibres in protopterus. J. Physiol. ( L o n d o n ) 129, 42P43P. Stefanelli, A. ( 1951). The Mnuthnerian apparatus in ichthyopsida; its nature and function and correlated problem of nenro-histogenesis. Quart. Rev. Biol. 26, 17-34. Tagliani, G. (1905). Le fibre del Malitliner nel midollo spinale dei vertebrati inferiori ( Anainnii). Arch. Zool. 2, 386-437. Tasaki, I., Hagiwara, S., and Watanabe, A. (1954). Action potentials recorded from inside a Mauthner cell of the catfish. Japan. J. Physiol. 4, 79-90. Tiegs, 0. W. (1931). A study of the neurofibril structure of the nerve cell. J. C o m p . Netirol. 52, 189-222. Uchizono, K. ( 1965). Characteristics of excitatory and inhibitory synapses in the central nervous system of the cat. Nature 207, 642-643. von Frisch, K. { 1936). Uber den Geliiirsinn der Fische. Biol. Rev. 11, 210-246. Wilson, D. M. ( 1959). Function of giant Mauthner’s neurons in the lungfish. Science 29, 841-842. Yasargil, G. hf., and Diamond, J. (1968). Startle response in teleost fish; an elementary circuit for neural discrimination. Nature 220, 241-243.

10 ELECTRIC ORGANS M . V. L. B E N N E T T

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

I. Introduction . . . . . 11. Electric Organs and Electrocytes . . . . A. Methods . . . . . . B. Membrane Properties . . . . . C. Marine Electric Fish . . . . , D. Freshwater Electric Fish . . . . . E. Some Quantitative Considerations . . . F. Adaptation and Convergent Evolution in Electric Organs G. Embryonic Origin and Developmerlt of Electric Organs H. Electrocytes as Experimental Material . . 111. Neural Control of Electric Organs . . . . . A. Pathways and Patterns of Neural Activity . . B. Synchronization of Electrocyte Activity . . C. Organization of Electromotor Systems . . IV. Conclusions and Prospects . . . . . . References . . . . . . .

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347 355 355 357 362 380 448 450 455 457 460 465 475 478 483 484

I. INTRODUCTION

Electric organs are organs specialized for the production of an electric field outside the body. They are found only in fish but apparently have evolved independently in some six different groups ( Fig. 1 and Table I ) . Electric fish are conveniently divided into two types, strongly and weakly electric. The discharge of a strongly electric fish is so large that the fish is painful to handle; the electric organ presumably or demonstrably functions as a weapon either defensively against predators or offensively in securing food. A weakly electric fish produces potentials that are too small to have value offensively or defensively; their organs function ( a t least in freshwater species) as part of an electrosensory system. The fish detects objects by means of the distortions they cause in the field set up by the electric organ. The sensing elements are the electroreceptors 347

348

M.

Electrophorus Sachs'

V.

L. BENNETT

Molopterurus

Main

9 Astroscopus

(0)

Gnothonemus

Gymnorchus

(

- ,

which are described in the following chapter. Electrosensory systems can function in communication between fish. Some strongly electric fish also have weakly electric organs. Strongly electric fish must have been known to primitive man, and Torpedos are said to have been used by the Romans in a primitive, and probably subconvulsant, form of shock therapy ( Kellaway, 1946). Recognition that the discharge was electric came soon after the development of the Leyden jar allowed widespread study of electricity by scientists

10.

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349

and experience of shocks. The Torpedo and electric eel were studied by many early physiologists, but the stargazer escaped the attention of the scientific community until just before Dahlgren and Silvester described its organ in 1906. Recognition of weakly electric fish was relatively delayed because of the imperceptibility of their discharges, and the electric activity of many was unknown until the 1950s (Lissmann, 1951, 1958; Coates et al., 1954; Grundfest, 1957). Howcver, all the groups had been recognized from morphological evidence by the early 1900s although their organs were sometimes classified as “pseudoelectric.” Historical references are given in the earlier reviews by Grundfest (1957) and Keynes (1957), and the historical essays of Kellaway (1946) and Mauro (1969) are charming. Relatively brief reviews of recent work are given in Bennett (1970) and Grundfest ( 1967). Electric fish have been studied in part because of their remarkable physiological abilities and the ease of detection of their discharges. Also, it has been hoped that they might reveal a great deal about normal function. The argument is that evolution may have exaggerated some aspect of organ or cell that makes a general phenomenon more understandable or easier to study. The giant axon of the squid is an outstanding example of a cell that has evolved in such a way-toward increased size-that makes feasible many kinds of experiments that are much more difficult in other tissues ( Hodgkin, 1964). The generating cells of electric organs are modified from muscle fibers except in the sternarchid family of the larger group, the gymnotids. In the sternarchids the myogenic part of the organ has been lost and thc organ is modified from nerve fibers; that is, it is neurogenic. The evidence of origin will be discussed in Section II,G,l,f. In a number of species the generating cells are flattened and for this reason have been termed electroplaque( s ) , electroplate( s ) , or clectroplax( es). However, in many of the more recently described organs, the generating cells have quite complex shapes. Although electroplaque still seems a natural term for flattened cells, the author has introduccd the more general term electrocyte to refer to any generating cell whatever its shape (Bennett, 1970). Electric organs generally are rather gelatinous, and a large fraction of their volunic is extracellular space. They contain a considerable amount of connective and other accessory tissues as well as blood vessels and motor nerves that control the discharge. As will be seen below the connective tissue can be important in channeling the flow of current. Electrocytes work on the same general principles as ordinary nerve and muscle cells: potentials are generated across membranes. In all known cases the potentials result from selective permeability and passive movement of ions down their concentration gradients. But it is likely

Table I Groups of Electric Fishc Common name

Family

Genera and species

Strength of organ discharge

Distribution Marine, cosmopolitan

Skates, ordinary rays

Rajidae

Raja, many species, a Weak number of other genera not known to be electric

Electric rays, torpedos

Torpedinidae

A number of genera, many species

Strong, up to 60 V or 1 kW, some perhaps weakb

Marine, cosmopolitan

Mormyrids, elephant-nosed fish (many lack enlarged chin or snout)

Mormyridae

A number of genera, several with many species

Weak

Freshwater, Africa

Gyrnnarchus

Gymnarchidae

1 species, G. niloticus

Weak

Freshwater, Africa

Gymnotid eels; electric eel and knifefish

Electrophoridae

1 species, Electrophorus Strong, more than Freshwater, South electricus 500 Vc America

Gymno tidae

1 species, Gymnotus curapo

Weak

Freshwater, South America

Sternopygidae

4 or 5 genera, a num-

Weak

Freshwater, South America

ber of species Rhamphichthyidae

2 monospecific genera

Weak

Freshwater, South America

Sternarchidae

About 9 genera, a number of species

Weak

Freshwater, South Smerica

Electric catfish

Malapteruridae

1 species, Malapterurus Strong, more than electricus 300 V d

Stargazers

Uranoscopidae

1 electric genus, Astroscopus; several species

Strong? about 5 V Marine, Western in air from small Atlantic animals8

Arranged phylogenetically; see Greenwood et al. (1966) for more detailed classifications. From Bennett (1970). From Bennett et al. (1961). c From Albe-Fessard (1950a). From Remmler (1930). * From Bennett and Grundfest (1961~). a

w

B

352

M. V. L. BENNETT

that some electrocytes will be found to have electrogenic pumping [movement of ions that involves net current flow and is linked to reactions such as adenosine triphosphate ( ATP) hydrolysis (see Hodgkin, 1964; Albers, 1967)l. When the membranes on opposite faces of the generating cells are at the same potential that at rest is the resting potential, no current flows (Fig. 2A). When the membranes are at different potentials, current flows in a circuit that involves the two membranes, the cell cytoplasm, and the external medium (Fig. 2B). Figure 2C represents current flow around an electrocyte when the two faces are at different, but uniform, potentials ( dotted lines). These currents are associated with potential changes in the external medium, and surfaces at equal potentials ( equipotential surfaces) are also diagrammed in Fig. 2C (solid lines). The current flow and field that an electric fish sets up around itself is essentially like that of Fig. 2C but larger since many cells in series and parallel are active at the same time. If an object such as a hand is present in the external medium, a potential difference is present across it and current flows through it. The current from a strongly electric fish is of sufficient magnitude to excite nerves, muscles, or receptors in the hand. An object distorts the electric field if it is of different conductivity than the medium, and the distortions, if large enough, can be detected by a fish's electrosensory system (see Chapter 11, this volume). The relatively large size of external potentials generated by electric organs (Table I ) as compared to other excitable tissues is not a result of larger membrane potentials although they may be slightly larger in a few instances. Rather the large outputs are a result of ( a ) arrangement of a cell's membranes in such a way as to maximize current outside the cell; ( b ) synchronous activity of many cells arranged in series and parallel; ( c ) to some degree, lower membrane resistances; and ( d ) accessory structures tending to channel current flow. The different kinds of adaptation will be discussed in connection with the various types of organ. The types and patterns of electric organ discharge can be divided into several categories. The strongly electric organs all produce essentially monophasic pulses. They are all active intermittently, as indeed they must be, for the power outputs are so large that the fish can maintain them for only short periods. The organ is normally silent and is generally, if not exclusively, discharged in response to appropriate external stimuli. These stimuli may be tactile, chemical, electric, or perhaps visual. Responses are single pulses or trains of pulses usually of fairly constant size ( Fig. 3A,A'). Generally, weakly electric organs of freshwater fish continually emit pulses of rather constant size. The pulses may be monophasic, diphasic,

10.

353

ELECTRIC ORGANS

A

+ + + + + + + + ( = ) - - -

+ + + + + + + +

Resting, no current flow

+ + + + + + + +

+ + I + Upper face active

D

7 External

potential

Indifferent electrode Face 2

r C

1'

r----------

I

active face

I

Monopolarly recorded potential external to active face

L-----7---i

j

'

t

Fig. 2. Equivalents of electrocytes during rest and activity. ( A ) Diagram of a cell at rest; equal potentials are opposed. ( B ) When the upper face generates an overshooting action potential, two potentials act in the same direction and current flows as indicated by arrows. ( C ) Equipotentials (solid lines) and lines of current flow (dotted lines) around a thin electrocyte indicated by the heavy horizontal line. If the polarity of the cell corresponds to that in ( B ), the potential is negative above the thin horizontal line, which is the zero isopotential, and positive below it. For reasons of mathematical simplicity the diagram is for very long ribbon shaped cell of zero thickness. The membranes are assumed to act as current sources corresponding to a shell of magnetic dipoles. The isopotentials are separated by equal increments and meet at the two edges of the cell. ( D ) Electrical equivalent of a resting cell. The resistance of the external current path can be represented by re,, rez, and res. ( E ) Equivalent of an active cell. The small arrows indicate the direction of current flow.

354

M. V. L. BENNETT

Fig. 3. Patterns of electric organ discharge. ( A ) An electric catfish 7 cm long. Potentials recorded between head and tail in a small volume of water with head negativity upward. Mechanical stimulation evokes a train of five pulses ‘ which attain a maximum frequency of 190/sec. (A‘) Single pulses can also be evoked (faster sweep speed). (B-D) Weakly electric gymnotids immersed in water, discharges recorded between head and tail, head positivity upward. (A) A variable frequency gymnotid, Gymnotus; pulses are emitted at a basal frequency of approximately 35/sec. Tapping the side of the fish at the time indicated by the downward step in the lower trace causes an acceleration up to about 65/sec. The acceleration persists beyond the end of the sweep. The small changes in amplitude result from movement of the fish with respect to the recording electrodes. ( B ) Faster sweep showing the pulse shape. ( C ) Sternopygus, a constant, low frequency gymnotid. The pulse frequency is about 55/sec. The horizontal line indicates the zero potential level. ( D ) Sternarchus, a constant, high frequency gymnotid. The frequency is about 800/sec. The horizontal line indicates the zero potential level. Calibrations in volts and milliseconds. From Bennett ( 1968a).

triphasic, or even more complex. The patterns of emission fall into two categories. In one, the responses are brief pulses separated by long intervals. These species generally accelerate their discharges when presented with almost any kind of stimulus (Fig. 3B,B’). Acceleration results in an increased rate of testing the environment but may also represent a signal to another fish (Bullock, 1970; Black-Cleworth, 1970; Moller, 1970) . In the second category the duration of the pulses is as long or longer than the intervals between them (Fig. 3C,D). Generally pulses are emitted at a very constant frequency that can be very high. Recently, it has been found that in most species weak electric stimulation at a frequency close to that of the discharge causes small shifts in frequency ( Watanabe and Takeda, 1963; Bullock, 1970). These changes apparently represent an attempt to avoid “jamming” of the electrosensory system by the applied signal. Other small changes of frequency may function in communication ( Bullock, 1970; Black-Cleworth, 1970). These two discharge patterns of weakly electric organs have been termed variable and constant frequency (although the terms now need a modifier such as relatively). They have also been called buzzers and

10.

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355

hummers from the sound one perceives when the electric pulses are fed into a loudspeaker system. Variable frequency species include the mormyrids and many gymnotids. Constant frequency species include the remainder of the gymnotids and Gymnurchus. Gymnarchus, mormyrids, and several gymnotids are able to cease discharging for brief periods. This response appears to represent hiding by keeping quiet electrically and may also function in communication. Discharge patterns of weakly electric organs in marine fish are intermittent, but are poorly known under natural conditions.

11. ELECTRIC ORGANS AND ELECTROCYTES

A. Methods An introduction to methods of establishing the properties of electrocytes may be helpful at this point. Microelectrodes are placed intracellularly and at various sites externally. Potentials at the tips can then be recorded by suitable amplifiers and associated equipment. A recording is termed monopolar if it represents the potential difference between the electrode tip and a distant grounded electrode in the vessel containing the preparation. The distant electrode is often called the indifferent electrode because it is sufficiently far from the active cell that moving it around does not detectably affect the recorded potential. One may use a differential amplifier to subtract the potential recorded through one microelectrode from that recorded through another; the result is differential recording. Monopolar recordings can also be subtracted after the experiment to obtain the same result. In discussing properties of single electrocytes it is useful to have an equivalent circuit. As a first approximation one can consider an electrocyte to be a flattened cell with uniform potentials across each face that differ from each other during activity. Because of the uniformity of potential, each face can be given a simple equivalent circuit like those used for other membranes. (Of course a “naked” cell could not have a discontinuous change in potential at the edge of the cell for this would require an infinite current density. However, an electrocyte is often situated in an insulating connective tissue sheath that adheres closely to its edges, and the potentials over the faces are indeed quite uniform. In any case the nonuniformities are not important for most considerations of membrane properties.) Separate branches of the equivalent circuits for each membrane can be assigned to different ion species, each branch with a particular internal (equilibrium or Nernst) potential and a con-

356

M. V. L. BENNETT

ductance that may or may not vary during activity. Alternatively, each membrane can be considered to consist of a single branch with a single internal potential and conductance, both of which can change. The membrane capacity, which may or may not be significant in electrocytes, is in parallel with the other branch or branches of the membrane equivalent. Generally, but not always, the resistance of the cytoplasm is negligible, and in the equivalent circuit of the entire cell the two membranes are directly connected together but oriented in the opposite direction. The circuit of the electrocyte can then be represented as a three terminal network, two terminals just external to each face and one inside the cell (Fig. 2D). The external terminals can be considered to be connected by a resistive path through the tissue and medium surrounding the cell. Another resistance leading to the indifferent electrode is required if monopolar recording is employed. This resistance is connected into the resistance of the external path thereby dividing it into two components. The placement of the connection depends on the symmetry with respect to the indifferent electrode of the material making up the external resistance. There are thus three resistances in the external path which are labeled T,,, re2,and res in Fig. 2D. Consider what is recorded when one membrane of a cell generates a spike and the other membrane does not change its properties (Fig. 2E ) . Appropriate differential recordings show the potentials across each face and across the external medium. The recording across the active face gives the internal potential of the membrane less whatever internal voltage drop there is due to current flow through the opposed, inactive face and external path. Differential recording across the inactive face gives the passive voltage drop across this face that results from current flow. The potential across the external path is the voltage drop across the external resistances and also the difference between the potentials across the opposed membranes. Monopolar recording outside the two faces gives potentials of opposite sign; for a conventional depolarizing response the recording is negative going outside the active face and positive going outside the inactive face. The potentials are smaller than when differential recording is used because they represent voltage drops across part of the resistance around the external circuit ( r e l and rr2 in Fig. 2D). A monopolar recording by an intracellular electrode cannot distinguish between the two faces. The spike is smaller than that recorded across the active face by the voltage drop across T , ~in Fig. 2D. The spike is larger than the passive drop across the inactive face by the voltage drop across rez. Obviously the same kind of inferences can be made from monopolar and differential recording since monopolar records can be subtracted to

10.

ELECTRIC ORGANS

357

give the equivalent of differential recording. However, differential recordings often are more easily interpreted and electronic subtraction is much easier than the same process done graphically. An additional advantage is that interfering potentials resulting from activity of distant cells tend to be about the same size at different monopolar electrodes and are thus subtracted out by differential recording. It is useful and often essential to pass stimulating currents through the faces. When current is passed from an intracellular electrode to an indifferent electrode, it flows outward through both faces in amounts depending on their resistances, capacities, and any responsiveness they exhibit as well as on the external resistances. Each face is depolarized and a response of one face may obscure a response of the other. For this reason it may be desirable to pass current between an external electrode outside one face and the indifferent electrode. (Use of two external electrodes, one outside each face, would be preferable in some ways but has not been carried out because of added technical complexity.) In this case part of the applied current flows inward through one face and outward through the other. Since this current polarizes the faces in opposite directions, it is generally simpler to evaluate the responsiveness of the two faces. Electrocytes have been studied both in situ and in isolated tissue bathed in suitable physiological saline. For in situ work normal organ discharge can in many species be stopped by spinal section. Curare which blocks vertebrate neuromuscular transmission also blocks nerveelectrocyte transmission. It can be used to immobilize a fish as well as to block neurally controlled activity of myogenic electrocytes, but of course it prevents study of nerve-electrocyte transmission. In a number of instances it is possible to apply currents to columns of cells or even to part of the intact animal. By means of a bridge circuit the potential resulting from voltage drops across fixed resistances in series with the active tissue can be subtracted from the records, and information similar to ( and supplemental of) that obtained from single cells can be obtained ( Albe-Fessard, 1950b; Bennett, 1961; Bennett et al., 1961). The success of experiments of this kind is dependent on the series arrangement of the active membranes.

B. Membrane Properties The surface membranes of electrocytes exhibit a number of different passive and active properties. In considering these properties, it is convenient to think of the cell surface as containing various kinds of sites

358

M. V. L. BENNETT

where different and more less-specific ions can enter or leave the cell. These sites are intermixed in varying degrees and proportions to account for the different kinds of membrane activity. The membrane separating the sites has a bimolecular lipid core that presumably is of very high resistance and inactive in ion movement. Most of the membrane capacity can be assigned to this part of the surface. For most cells the specific capacitance is about 1 pF/ cm2 while membrane resistance-reflecting the number and nature of sites for ionic movement-can vary over a range of six or seven orders of magnitude. This concept of localized sites of ion movement has received strong support from recent experiments using artificial bimolecular lipid membranes. These membranes are similar to ordinary membranes in dimensions and capacitance but have an extraordinarily high resistivity. A number of compounds lower the resistance into the physiological range by providing sites for movement of ions or by acting as carrier molecules (e.g., Cass et d.,1970). In impulse responses of electrocytes there appear to be three types of sites that change their properties as a function of membrane potential. When the membrane is moderately depolarized, the conductance at sodium sites increases (sodium activation) allowing influx of sodium and further depolarization. This process is responsible for the active rising phase of the spike. If depolarization is maintained, the sodium conductance decreases again (sodium inactivation) and this change is a factor in return of the membrane potential to its resting level. At the resting potential the inactivated sodium sites gradually recover their ability to increase in conductance when the membrane is depolarized. (Any remaining activated sites rapidly return to their low, resting conductance at the resting potential and can be rapidly activated again without further delay.) One kind of potassium site in electrocytes increases in conductance when the membrane is depolarized (potassium activation or delayed rectification), but the change is delayed compared to sodium activation. The increased potassium conductance tends to restore the cell to the resting potential where the conductance gradually returns to normal. If the membrane is kept depolarized, the increase in potassium conductance can also reverse (potassium inactivation), a process which is generally much slower than either the reversal at the resting potential or sodium inactivation. So far these changes are like those of ordinary nerve (Hodgkin, 1964; Hille, 1970). Another kind of potassium site decreases in conductance when an outward current is passed through it. This change is very rapid in onset and reverses very rapidly when the outward current is reduced. It resembles and probably has the same mechanism as anomalous or outward rectification in muscle

10.

ELECTRIC ORGANS

359

(Adrian et al., 1970; cf. Bennett, 1970). It can also be considered a kind of inactivation ( Bennett and Grundfest, 1966). The resting membrane potential is apparently largely determined by potassium selective sites and the concentration gradient of potassium, although the cells may also be significantly permeable to chloride. In some electrocytes large hyperpolarizations cause the conductance of the resting membrane to decrease which can lead to what have been termed hyperpolarizing responses ( Bennett and Grundfest, 1966). Since the conductance decreases by a large fraction of the resting value, these responses must involve a change in the potassium selective sites of the resting membrane. There is no known physiological significance of these responses. In myogenic electrocytes another kind of a response is mediated by sites sensitive to the neurotransmitter acetylcholine released by the presynaptic nerve fibers. The transmitter increases the permeability at these sites, presumably to sodium and potassium ions, and the resulting current flow generates a postsynaptic potential (PSP). The change in conductance is virtually independent of the membrane potential, a property which has been termed electrical inexcitability ( Grundfest, 1957; Bennett, 1964). An important consequence of there being an electrically inexcitable conductance increase is useful in establishing the chemical mediation of a postsynaptic response (Bennett, 1966). If the membrane is made sufficiently inside positive prior to the release of transmitter, the PSP current that would have depolarized the cell reverses and flows to make the inside of the cell less positive; the response is inverted. The potential at which the current reverses (loosely speaking the equilibrium potential by analogy with Nernst potentials) is generally slightly negative to the zero potential; this is the primary reason for believing that permeability is increased to both sodium and potassium (see N. Takeuchi, 1963). Sites of the kinds described are combined in a number of ways in membranes comprising the faces of different electrocytes. These combinations are as follows: (1) Spike generating membrane. This membrane contains sodium sites, and may or may not have potassium activation. Probably anomalous rectification is present in spike generating membranes of all myogenic organs. PSP generating sites may or may not be present. ( 2 ) Membrane exhibiting delayed rectification without spike activity. This membrane appears to be spike generating membrane that has lost the sodium mechanism. PSP generating sites may or may not be present. ( 3 ) Postsynaptic potential generating membrane. Postsynaptic poten-

360

M. V. L. BENNETT

ca

12 3

re

1

1

2

3

i ii

1

2

3 123

r3’

Fig. 4.. Activity of different kinds of electrocytes and their equivalent circuits. The upper part of each circuit, labeled only in A, represents the innervated or stalk face; the lower part represents the uninnervated or nonstalk face. The resistance of the extracellular current path is represented by re. The potentials that are recorded differentially across the two faces ( V , and V,) and across the entire cell ( V , ) are drawn to the right of each circuit (intracellular positivity and positivity outside the uninnervated face shown upward; note that Vi - V, = V e ) . Placement of electrodes for recording these potentials is indicated in C. The successive changes in the membrane properties are shown by the numbered branches of the equivalent circuits, and their times of occurrence are indicated on the potentials. A lower membrane resistance is indicated by fewer zigzags in the symbol. Return to resting condition is omitted. ( A ) Electrocytes of strongly electric marine fish and discshaped electrocytes of rajids. The innervated face generates only a PSP. ( B ) Cupshaped electrocytes of rajids. The innervated face generates a PSP and the uninnervated face exhibits delayed rectification. ( C ) Electrocytes of the electric eel and a few other gymnotids. The innervated face generates an overshooting spike. ( D ) Electrocytes of mormyrids and some gymnotids. Both faces generate a spike, and V, is diphasic. In some the spike across the uninnervated face is longer lasting and the second phase of V, predominates. These potentials are

10.

ELECTRIC ORGANS

361

tial generating sites may be the only responsive elements in a membrane as well as occurring intermixed in membrane of the preceding kinds. (4) Low resistance inexcitable membrane. This membrane has a low resistance owing to a large potassium conductance, and it generates a resting potential; its properties do not change when the potential across it is changed. In some electrocytes there may be a significant C1 conductance. ( 5 ) High capacity inexcitable membrane. This membrane has few sites for ionic movement, and its resistance is high. As a result, during activity virtually all of the current through it is capacitative. Its capacity per unit area of plasma membrane is probably similar to that of other membranes, that is, 1 pF/cm2. Where membrane of this kind has been studied by electron microscopy, the surface is highly convoluted on an ultrastructural level, and therefore the capacity per unit area referred to macroscopic area is larger than the values for ordinary cells. [A similar situation obtains in muscle because of the transverse tubular system (cf. Gage and Eisenberg, 1969).] In electrocytes of the gymnotid Sternopygus one membrane has an effective capacity so large that it probably results from shift in ionic distributions on either side of the membrane (see Section 11, C, 1, d ) . In different electrocytes the two faces are made up of different combinations of the foregoing kinds of membrane. The equivalent circuits at rest and during activity are diagrammed in Fig. 4;they will be discussed in detail in sections concerning the different kinds of fish in which they occur. There is good indirect evidence that sites mediating the different components of electrically excitable responses are independent of each other on a microscopic scale and that they are also separate from PSP generating sites (which often occur in the same general region of the cell). To be sure the spatial resolution of electrophysiological techniques may not allow measurement of the separation of different kinds of membrane responsiveness. Nonetheless, physicochemical considerations and separability of different kinds of electric activity under various electrical and pharmacological treatments strongly support the idea of separate

shown by dashes. ( E ) Electrocytes of the electric catfish. The stalk face is of higher threshold and generates a smaller spike (indicated by a smaller battery symbol) and the external potential is entirely negative on the nonstalk side distant from the stalk. ( F ) Electrocytes of Gymnarchzrs. The uninnervated face acts as a series capacity and the external response has no net current flow. The summation of a second response (2’,3’) on the first is shown by dashes. Electrocytes of sternarchids and Eigenmanniu may operate similarly. From Bennett ( 1970).

362

M. V. L. BENNE’IT

channels mediating different response components (see, especially, Hille, 1970; but also Bennett, 1961; Grundfest, 1966). Another argument for separability of different components is the separate occurrence in different cells and in macroscopically different regions of a single cell. Electrocytes provide much of the comparative evidence of this kind. A frequent characteristic of electrocytes is a kind of impedance matching between the faces (Bennett, 1961). The lower the membrane resistance the greater the electrical output of the cell, but also the greater the leakage at rest, and the greater the exchange between Na’ and K+ across spike generating membrane during activity. It would thus be inefficient to have one face of much lower resistance than the other (or of much lower resistance than the series resistances of the external medium and cytoplasm). One finds that a low resistance inexcitable face generally is of lower resistance than the opposed, excitable face at rest (Fig. 4A,C). The excitable face reduces its resistance during activity so that under these conditions the resistances of the two faces are more closely matched. In electrocytes where both faces become active and decrease their resistances, the resting resistances are more or less equal. Often this kind of impedance matching has morphological correlates, which will be discussed in respect to individual cases and in Section 11, F.

C. Marine Electric Fish There are three groups of marine fish possessing electric organs, the torpedinids (electric rays), the rajids (skates or rays), and members of the teleost genus Astroscopus (one group of stargazers). Because of functional similarities between the electric organs it is convenient to consider these three groups together, although other characteristics make them the “lowest” and the ‘%ighest” fish possessing electric organs. The electrocytes of Astroscopus, the torpedinids, and some of the rajids are the simplest known. There is only the one response component in the innervated face and no response in the uninnervated face. 1. Astroscopus

The electric stargazers are a small group of several species that occur along the western coast of the North and South Atlantic. There may also be a representative on the Pacific side of the Panamanian isthmus (Dahlgren and Silvester, 1906). These fishes are unusual in being somewhat flattened dorsoventrally. Their eyes are located on the dorsal surface of the head and look virtually straight upward, hence the name stargazer. Their habit is to burrow into sand leaving only their eyes

Fig. 5. Innervation and structure of electrocytes of the stargazer. The upper picture shows the dorsal surface of a single cell teased from formalin fixed material and stained with methylene blue. A nerve bundle enters from the lower left and forms profuse, sometimes anastomosing branches. Finer branches are not resolvable. Capillaries ( c ) are also seen. The lower micrograph is a vertical section through the organ, dorsal surface uppermost, following osmic acid fixation. The innervated, dorsal surfaces are smooth; the uninnervated, ventral surfaces have long papillae. Two nerve bundles are seen on the left. The edge of a cell between two others (lower right) illustrates the characteristic irregular layering of the cells. Just to the right of center is a vertical fissure (arrow) which may represent edge to edge apposition of two cells. From Bennett and Grundfest (196lb). 363

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protruding when they are practically invisible except for the two small black spots of their pupils. From this position they gulp down unwary minnows passing overhead. The electric organs lie just behind the eyes ( Fig. 1) , and they are in point of fact modified from extraocular muscles (Dahlgren, 1914, 1927). Other members of the same family, Uranoscopidae, are very similar in appearance but lack electric organs. The electrocytes are large flattened cells that lie in the horizontal plane and are densely innervated on their dorsal surface by branches of the very large oculomotor nerves (Fig. 5 ) . The ventral surface has many short processes or papilli that markedly increase its surface. This surface is further increased by many small invaginating tubules or canaliculi (Mathewson et al., 1961; Wachtel, 1964). About 150-200 layers of cells are arranged in series, one above the other (Dahlgren and Silvester, 1906). Each layer of cells in parallel consists of about four large cells (approximately 5 mm in diameter in a 20-cm fish) surrounded by about 10 smaller ones. A single large cell can also spiral around to overlap itself to some extent. The organ discharges are pulses making the dorsal surface negative (and presumably also making the inside of the mouth positive, although this has not been directly verified). Responses to handling the fish range from single pulses up to trains of several tens of pulses at frequencies of about 5&100/sec. Trains of pulses are also emitted when the fish is capturing small minnows (Pickens and McFarland, 1964). The pulses are about 5 msec in duration. The amplitude is somewhat variable, particularly at the beginning of a train but is up to about 5 V recorded from a fish 20-30 inches in length with its dorsal surface in air (Fig. 6 ) . The voltage across the organ is reduced somewhat if the discharge is evoked while the fish is immersed in seawater. The pulses are generated synchronously by the two organs. The effectiveness of the discharge in aiding the capture of prey has not been demonstrated, but the unstimulated fish emits pulses rarely, if ever, at other times. Large fish can reach 40 cm in length, and the discharge is sufficiently strong that it is easily detected when the fish are handled (Dahlgren and Silvester, 1906). Even a small fish can cause mild discomfort if one's hands are quite wet and have a number of minor cuts (personal observation). Only the innervated (dorsal) face of the electrocytes is active during organ discharges (Bennett and Grundfest, 1961b). The responses are PSPs and are monophasic depolarizations of about the Same shape and duration as the organ pulses. The innervated membrane does not respond at all to depolarization but behaves linearly; it is electrically inexcitable. The response that is evoked by stimuli applied directly to the organ is

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Fig. 6. Patterns of electric organ discharge of Astroscopus. ( A,B) Simultaneous records from the two organs in each of two different fish. A probe electrode was placed on the skin over each organ with the dorsal surface of the fish in air. The reference electrode was on the ventral surface and grounded; dorsal negativity up. ( C ) Same fish as in B, but recording with the animal covered by seawater. (D-F) Simultaneous recordings as in A but from a fish which had had one of its electric organs denervated. The electrode on the denervated organ (lower traces) registered only a small pickup of the activity of the other organ.

mediated by the presynaptic nerve fibers. The uninnervated face is of very low resistance and generates only a resting potential. (Presumably it is inexcitable, but this point has not been established critically because the membrane is of such low resistance that it may not have been depolarized sufficiently to excite it. However, one doubts that this membrane would have electrically excitable sites when they would be nonfunctional and when the innervated face lacks them.) The equivalent circuit during responses is shown in Fig. 4A. At rest the two faces generate equal potentials and no current flows. During activity the resting potential virtually disappears across the innervated face and the conductance of this face to (presumably) sodium and potassium greatly increases. Current flows inward across the innervated face and outward across the uninnervated face. [It is to be expected that the innervated face actually generates a potential of -10 to -20 mV (see N. Takeuchi, 1963). Note that the potential in the circuit is provided by what we have termed the inactive face.] Monopolar recordings on which this description is based are shown in Fig. 7. (The straight horizontal line is a reference trace.) A brief stimulus is applied to the dorsal surface of the organ by means of a pair of small wire electrodes 1 msec after the start of the sweep. The recording trace goes off screen for about 0.2 msec during the stimulus

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_F

--

D

-A H

A B C D E F G

H

-'+I; I msec

Fig. 7. Activity of a single electrocyte of Astroscopus. Monopolar recording zjizjo. Stimuli are applied by a pair of fine wire electrodes close to the site of microelectrode penetration. The diagram on the right indicates positions of the recording electrode in successive records inferred from appearance and disappearance of resting potential and changes in response amplitude and sign. From Bennett and Grundfest ( 1961b). ill

and then returns more slowly toward its initial potential before the response begins with a latency of about 1 msec. As the electrode penetrates the cells a regular sequence of potential changes is observed that serves to identify electrode position as well as to characterize the responses. Immediately dorsal to the most superficial cell the response is a monophasic negativity of about 15 mV amplitude ( A ) . The sign of the potential indicates that the underlying membrane is passing inward current. As the electrode is advanced the steady potential shifts about 90 mV negative which represents the resting potential across the innervated membrane ( B ) . Simultaneously the response becomes a positive-going or depolarizing response of about 60 mV indicating that the electrode has crossed an active membrane. If it were differentially recorded across the innervated face (record B minus record A ) the response would be about 75 mV in amplitude. As the electrode is further advanced, the steady potential shifts back to its initial value signaling passage of the electrode through the cell into the underlying extracellular space ( C ) . The resting potentials developed by the two faces are equal, and thus no current flows through the cell at rest. The response recorded outside the innervated face is virtually identical to that recorded in the cell. This indicates that the resistance of the uninnervated face is very low compared to the resistance in the external path. The response recorded differentially across the entire cell would of course be almost exactly like that recorded across the innervated face. When the electrode is further advanced, the steady potential again shifts negative indicating penetration of the second cell ( D ) . Simultaneously the response amplitude decreases by about half indicating that the innervated membrane of this cell cornprises a significant fraction of the resistance of the external path. Further advances show shifts in the steady potential as the electrode leaves

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and enters cells (E-H). The amplitude decreases as the electrode crosses innervated faces but not as it crosses uninnervated faces because of their relatively high and low resistances, respectively. The neural mediation of these responses is indicated by the latency which remains about 1 msec even if very suprathreshold stimuli are used. If spike generating membrane were present, a much shorter latency could be obtained (an example is given in Fig. 18). Neural mediation is confirmed by pharmacological data and the effects of denervation (see below). Responses like those of Fig. 7 result from single nerve fibers for their amplitude varies in an all-or-none manner as stimulus strength is varied. Other experiments involving passage of current during responses show that the resistance of the innervated faces is decreased during neurally mediated activity but is not affected by depolarization alone. Thus during activity the resistance of the innervated face moves toward that of the uninnervated face in the kind of impedance matching discussed in the preceding section. The resting resistances of the two faces correlate with the degree of surface elaboration seen in the fine structural studies; the lower resistance membrane is much more elaborated ( Mathewson et al., 1961). (The negligible voltage drop across the innervated faces need not obtain in responses of the entire organ when all the cells are active in series and much larger currents flow. In organ discharge there would also be a smaller potential across the innervated face because of its internal resistance. ) The PSPs of the electrocytes are cholinergic as they presumably are in the muscle fibers of origin. They are greatly reduced by the blocking agent curare and prolonged by the anticholinesterase eserine. The cells are depolarized by acetylcholine, presumably the actual transmitter, and the related compound carbamylcholine. Denervated cells retain their ability to respond to these drugs, but the response to electrical stimulation disappears confirming its neural mediation. The microelectrode experiments demonstrate that unitary responses of the electrocytes are essentially of the same duration, shape, and sign as the organ discharges. If 150 cells in series give rise to a 5 V pulse about 30 mV per cell is required, a value exceeded somewhat in the microelectrode experiments. The discrepancy is explicable as a result of greater current flow during synchronous activity and failure of some of the cells to fire. Furthermore, cell counts, organ discharges, and PSP amplitudes may have varied in the different animals used for the measurements. Although most of the presynaptic fibers are active during a response (see Section 111),some do fail to fire on occasion as indicated by the variable response amplitude, and a few inactive cells in series

368 M. V. L. BENNETT

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Fig. 9. Innervation of torpedinid electrocytes. From the main organ of Narcitie. (Upper) A single cell teased out from forinalin fixed material and stained with methylene blue. Four nerve fibers run across the surface from different points on

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would markedly increase the internal resistance of the organ. In spite of the quantitative discrepancy the principles of series summation and synchronous activation are well illustrated by this electric organ.

2. TORPEDINIDS The electric rays are a cosmopolitan group of marine fishes. The electric organs of the genus Torpedo are diagrammed in Fig. 1. They are large flattened organs on each side of the head that extend completely through the “disc” from dorsal to ventral surfaces. The Torpedos are rather slow moving, and the use of the organ discharge in capture of much faster swimming prey has been studied in the European species, Torpedo murmorata (Belbenoit, 1970). When a fish comes near a Torpedo resting on the bottom, it swims forward and upward and then emits pulses. Small fishes can be stunned. The Torpedo drops back to the bottom over any immobilized prey. If it has been successful, it consumes the prey while continuing to emit pulses at a low frequency. The delay between movement and discharge indicates that the organ does not function in prey detection. The detection of prey appears to be mechanical or perhaps electrical. The effectiveness of the organs in predation is confirmed for the American species, T . nobilianu, by the presence of large and rapidly swimming fish in the stomach contents (Bennett et al., 1961). Each electric organ in T. nobiliuna is made up of some 500-1000 closely packed and roughly circular columns of electrocytes that run from top to bottom of the organ (Fig. 8). The electrocytes are thin (10-30 p ) discs and have the same diameter as the columns; about 1000 are stacked one above another as in a role of coins. The columns can be quite large in diameter, 5-7 mm in a large Torpedo (1 meter across ) . The cells are profusely innervated on their ventral surfaces. Nerves run in the interstices between columns and 5-7 nerve fibers enter the space between cells at roughly equal intervals around the periphery; each fiber innervates a sector of the cell (Fig. 9). The nerves arise from the electromotor lobe in the medulla (perhaps including parts of the seventh, ninth and tenth cranial nerves, Fig. 8). Electron microthe periphery. The fibers branch profusely, but each innervates a separate segment of the surface. The inset shows schematically the columnar arrangement of the electrocytes. (Lower) A region from the lower right of the isolated cell is shown at higher magnification. Nuclei of the electrocytes are stained as well as many fine nerve branches. Modified from Bennett and Grundfest (1961a) and Grundfest (1957).

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scopic examination reveals that the innervation is very dense (Fig. 10). The organ discharges of a large Torpedo are monophasic pulses, positive on the dorsal surface and about 5 msec in duration and SO V in amplitude recorded in air (Bennett et al., 1961). The internal resistance of the organs is low, and the power output at the peak of the pulses can exceed 1 kW. Because the electrocytes of Torpedo are very thin, it is difficult to use two or more electrodes for differential recording across the two faces and the entire cell. However, a single electrode can be advanced into a column of cells, and successive changes in resting potential and response can be used to determine electrode position as described in

Fig. 10. Fine structure of torpedinid electrocytes ( Torpedo murmorutu). The central portion of the figure shows a perpendicular section through three electrocytes ( E P ) , dorsal surface upwards. The ventral surface is densely covered with nerve endings ( n ) . On the left is shown a region of the uninnervated surface at higher magnification. Several external openings of the many canaliculi are seen ( A ? ) . The rectangles (Bt) indicate branch points of the canaliculi. Basement membrane material extends deeply into the canalicular network. On the right is shown a higher magnification of the innervated surface. Nerve profiles contain vesicles ( v ) as well as small granules ( g ) , possibly glycogen, and lie embedded in the electrocyte. Occasion folds ( j ) extend quite deeply into the postsynaptic cell. From Sheridan et al. (1966).

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respect to Astroscopus (Fig. 7). The responses of the electrocytes of Torpedo are essentially the same as in Astroscopus (Bennett et d.,1961). The active face is the innervated face, which however is on the ventral side. Again, this face does not respond to depolarization; it is electrically inexcitable. The response that is evoked by external stimulation is a monophasic depolarization, that is, a PSP mediated by activity of the presynaptic fibers. Its origin as a neurally mediated response is attested to by the irreducible delay when stimulating with closely applied electrodes and by the block of the response when curare is applied to the innervated face. In a column of tissue the response can be inverted by polarizing currents [the first tissue in which PSPs were shown to invert ( Albe-Fessard, 1951; Bennett et al., 196l)l. The uninnervated face is of very low resistance and generates only a resting potential. The area of this face is greatly increased by numerous invaginating canaliculi ( Fig. 10). The maximum response amplitude observed in a single cell is about 90 mV, which is somewhat larger than the recorded resting potentials. However, the cells are very thin and the canaliculi extend across at least half the cell thickness; a supposedly intracellular electrode might always be partly in the extracellular space outside the uninnervated face. Probably the full resting potentials are not recorded and like other PSPs those in Torpedo do not overshoot the resting potential. The duration of the PSPs is about 5 msec. Their latency with locally applied stimuli is 2-3 msec, an appreciable fraction of which may be conduction time in fine branches of the innervating fibers. The PSPs in Torpedo electrocytes are cholinergic in that they are blocked by curare and dihydro-P-erythroidine and prolonged by the cholinesterase inhibitors eserine and physostigmine. Also, the cells are depolarized by actylcholine and carbamylcholine. The synthesis of organ discharge from the responses of the single electrocytes simply requires synchronous activation as in Astroscopus. The duration of the PSPs is about the same as that of the organ discharge, and the amplitude of the PSPs is sufficient that the number of cells in series could produce the discharge amplitude. Narcine is a smaller relative of Torpedo. I t has a bilateral electric organ, the “main” organ, that resembles that of Torpedo both morphologically and physiologically ( Bennett and Grundfest, 1961a). Nurcine has an additional (bilateral) electric organ, the accessory organ, that lies at the posterior margin of the main organ (Mathewson et al., 1958). This organ differs in one important respect from the main organ. The responses to low frequency stimuli are very small, but when the nerve is stimulated at up to 100/sec, the responses are considerably augmented

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and prolonged. However they are still somewhat smaller than in the main organ of Torpedo. Although the normal discharge of the accessory organ is unknown, these findings suggest that the discharge involves repetitive and fused responses of the single cells similar to what is observed in the rajids (see the next section), It seems likely that the accessory organ is used either in an active electrosensory system or in communication, although there is no direct evidence for this suggestion. The torpedinids like most other elasmobranchs have ampullae of Lorenzini, which are electroreceptors (see Chapter 11, this volume). A number of other small torpedinids are known, some of which live in the deep seas and are blind (Lissmann, 1958). Possibly their main electric organs are used in electrolocation. It is not known whether these species have accessory organs. 3. RAJIDS

The skates or ordinary rays are a large cosmopolitan group of marine fish comprising six or more genera and many species. They are weakly electric, but unlike freshwater species they emit pulses only infrequently. The discharges are sufficiently inconspicuous that a major taxonomic work on the group “Fishes of the Western North Atlantic” (Bigelow and Schroeder, 1953) makes no mention of the fact that these fish are electric. Other rays (suborder Myliobatoidea ) apparently lack electric organs. The electric organs of rajids are located in the tail in the center of the most lateral bundle of longitudinally running muscle fibers (Fig. 1). The organs are spindle-shaped and run most of the length of the tail. They are much greater in length than in diameter. The electrocytes are oriented anteroposteriorly and are innervated on their anterior faces. Each cell lies in a small connective tissue compartment. Two types of electrocytes have been described, the cup-shaped and the disc-shaped (Fig. 11). However, these terms are not particularly descriptive of the morphological differences. Cup-shaped cells lie at the anterior margin of their connective tissue chamber. Often they are convex posteriorly, which accounts for their name. Both faces are relatively smooth at the light microscopic level of resolution. Electron microscopy reveals a relatively small number of tubules invaginating into the innervated face and a somewhat greater number in the uninnervated face (Mathewson et al., 1961). Disc-shaped cells lie nearer the posterior of their chambers. The posterior, uninnervated faces have a large number of protuberances tens of microns in diameter and length (Fig. 11).Probably there are more invaginating tubules in these faces than in cupshaped cells. Both classes of cells contain striated filamentous material

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Fig. 11. Anatomy of rajid electrocytes. Longitudinal sections of electric organ from R. erinacea, which has cup-type cells (A,C) and from R. eglanturia, which has disc-type cells (B,D). Rostra1 surface up, hematoxylin and eosin stain, A,B, low power; C,D, high power. Cup-type cells are usually smooth on both surfaces ( A ) although there may be a few processes on the caudal face in some cells ( A , lower right). The cells lie against the caudal walls of the connective tissue chambers ( c ) that contain them. The caudal face of disc-type cells usually has many processes although in some regions it may be relatively smooth (B, lower right). The cells lie anteriorly in their connective tissue chambers ( c ) . Both types of cell contain striated material in a central area ( C , D ) . There is often a short process or stalk ( s ) that is a remnant of the muscle fiber from which the cell develops (Ewart, 1892). From Bennett ( 1961).

(Fig. 11, Wachtel, 1964) that reveals their myogenic origin. The response properties of the two kinds of cell are somewhat different. The disc-shaped cells are physiologically similar to those of other marine electric fish, but the cup-shaped cells are more complex (Bennett, 1961). Some intergrading of the two extreme physiological types apparently does occur, but too few species have been studied to be sure of the extent of the correlation between form and physiological functioning. There is evidence that the differences between cup- and disc-shaped electrocytes are associated with other morphological characteristics and that the rajids can be divided into two groups (Ishiyama, 1958). The organ discharges are monophasic and head negative. The fish

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can only sometimes be provoked into discharging by mechanical stimulation. Under these conditions the organ discharges are usually irregular and variable in size and duration (Fig. 12). The maximum pulse amplitude is of the order of a volt recorded in air, but tens of millivolts if the fish is in seawater ( D ) . Sometimes more regular pulses are emitted (A), and preliminary results with long-term recording from animals in holding tanks suggest that “spontaneous” discharges or those evoked by light touch under these conditions are more constant in amplitude (A, B. Steinbach, unpublished data). In any case the discharges that have been observed are long lasting compared to the responses of individual electrocytes, and the discharges most probably involve fused repetitive activity of many cells. Unlike the electrocytes of strongly electric marine fish, cup-shaped electrocytes respond to depolarization. However, the response involves only delayed rectification. There is no sodium activation or other electrically excitable component leading to a regenerative response; the response to graded depolarizing pulses is graded. An example is shown in Fig. 13. For small depolarizing (outward) currents the cell behaves linearly with the same resistance as for hyperpolarizing current. The resulting potentials rise slowly. and more or less exponentially to a steady state value; the slowness of rise results from charging of the membrane capacity. For larger depolarizing currents there is an early peak of depolarization which then decreases as the rectification “turns on.” The peak depolarization is always less than the steady state poten-

Fig. 12. Discharge of rajid electric organs. (A-D) Raja erinacea, which has cup-type electrocytes. ( A‘,B’ ) Raja eglantaria, which has disc-type electrocytes. Discharges are evoked by vigorous prodding and recorded differentially between tip and base of tail, caudal positivity up. Upper trace, higher gain. In both forms, discharge is asynchronous and variable in amplitude and duration. All records are in air except D, for which the animal is immersed in seawater while regularly responding as in B. The discharge is greatly reduced in amplitude. From Bennett (1961).

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Rqa

20 mV

..+J

x.'.

Fig. 13. Intracellularly applied polarization of cup-shaped electrocytes from

R. eriinacea. ( A ) Superimposed records of responses (lower traces) to depolarizing currents (upper traces). For larger currents, the voltage reaches an initial peak, then falls to a much lower steady value. ( B ) Records as in A in response to hyperpolarizing currents. The voltage change increases approximately exponentially toward a steady level. Graph: voltage-current relationship for the same cell as A and B, but using additional data. The relation is linear for hyperpolarizing current. For larger depolarizing currents, the initial peaks fall somewhat below the potentials they would have reached if the cell had the same resistance as for hyperpolarizing current. The potentials at the end of the pulses are much lower and continue to decrease as further current is applied. Modified from Bennett (1961).

tial would be that corresponded to the resistance for small depolarizing and hyperpolarizing currents. If a current pulse is terminated on the rising phase of the initial depolarization, the potential immediately begins to return toward the base line; there is no tendency of the potential to continue in the depolarizing direction (Fig. 14). These findings lead to the conclusion that there is no regenerative component in the response. The increase in conductance is indicated not only by the reduction in potential during the current but also by the more rapid drop in potential following cessation of the current after the conductance increase has been produced ( Fig. 14). The conductance increase caused by a brief stimulus lasts a few tenths of a second. It is associated with a small depolarization from the resting potential (Fig. 14D). The nature of the permeability change underlying the conductance increase is unclear. There is suggestive but incomplete evidence that it may be an increase in C1 permeability ( Bennett, 1961; Grundfest, 1967).

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Fig. 14. Responses of cup-shaped electrocytes to depolarizing pulses of different strengths and durations ( R. erinacea ). Lower trace: intracellular voltage. Upper trace: current applied through second intracellular electrode. ( A-C ) Superimposed records of constant current pulses of different durations, magnitude increasing from A to C. ( D ) Superimposed records of a large and a small pulse of the same length; the traces cross following the end of the pulses. Whatever the current strength or duration, the voltage starts t o decrease immediately on cessation of the current. Following the peaks, the voltages decrease more rapidly than if the pulses are stopped before them. The time course of the decrease deviates from exponential because the cells are not isopotential. In D a small depolarization is seen to be maintained following the larger stimulus. From Bennett (1961).

When the nerve supply to an electrocyte is stimulated, a PSP is produced that is graded in several all-or-none steps indicating that the cell is innervated by several fibers (Fig. 15C). The PSPs are cholinergic (Brock and Eccles, 1958). The larger PSPs activate the delayed rectification, and these PSPs decay more rapidly because of the shortened time constant of the cell. In most cells the delayed rectification is found primarily in the uninnervated face; it thereby acts to increase external current flow in the medium around the electrocytes. At rest the resistance of innervated and uninnervated faces is relatively high. Depolarization by PSPs generated in the innervated face increases the conductance of the uninnervated face and thus allows more current to flow out through this face and around the cell. (Delayed rectification in the innervated face is maladaptive; it increases microscopic current loops in this face and by loading the PSP generating membrane tends to reduce current flow around the cell.) The effect of the delayed rectification on external potentials can be illustrated with reference to Figs. 4B and 15. The external response to a small PSP that does not turn on the delayed rectification is a singlepeaked potential which is positive outside the uninnervated face (Fig. 15C ) . However, the peak of the external potential is earlier than that of

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Fig. 15. Effect of delayed rectification on PSPs of cup-shaped electrocytes, R. erinacea. Recording and stimulating as in the diagram except that nerve stimulating electrodes are omitted. Upper trace: intracellular stimulating current. Second trace: potential across innervated face ( V , ) . Third trace: potential across uninnervated face ( V2).Fourth trace: potential across cell, posterior positivity up (Vx). A large PSP produces a posterior positive external potential that has two peaks ( A ) . The second peak is blocked when the conductance increase is prevented by hyperpolarization ( B ) that also prolongs and increases the height of the response. Small PSPs that do not activate the delayed rectification also produce external potentials with a single peak (C, superimposed traces of large and small responses). When the delayed rectification is activated by a depolarizing pulse and a PSP is evoked, the external potential is increased and the potentials across the faces are reduced ( D, superimposed responses with and without applied depolarizing pulse). The time constant of the uninnervated membrane is reduced so that all the potentials have the same time course and the peak of the external potential occurs later than the initial peak of responses when the delayed rectification has not been activated. From Bennett ( 1961).

the potential across the uninnervated face, a result that indicates that there is a capacitative component of the current. During the rising phase of the PSP the capacity of the uninnervated face is being charged and more capacitative current is flowing. At the peak of the potential no capacitative current is flowing and the current is entirely resistive. When a large PSP activates the delayed rectification, the initial part of the external record is of the same shape as that during small PSPs. However, after this period the delayed rectification turns on, outward current through the uninnervated face increases, and the external potential rises to a second peak (Fig. 15A,C). If the cell is hyperpolarized so that even a large PSP fails to activate the delayed rectification, the external response again has only a single peak that is earlier than the peaks of the transmembrane potentials (Fig. 15B). Note that in B the

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transmembrane potentials are slowly falling as they are for small PSPs. Moreover, hyperpolarization increases the amplitudes of the potentials because the PSP conductance change is unaffected while the driving force, the difference between the membrane potential and the PSP reversal potential, is augmented (see Bennett, 1961). If a large PSP is evoked after a depolarizing pulse that activates the delayed rectification, the external potential is enlarged, the voltage drop across the external response has only a single peak that occurs at nearly the same time as the peak of the transmembrane potentials (Fig. 1 5 0 ) . In addition, the external potential is enlarged, the voltage drop across the uninnervated face is reduced, and the PSP across the innervated face is also reduced because of the greater electrical load placed on it. A PSP that turns on the conductance increase causes the same changes in subsequent responses as does directly applied depolarization. The utilization of the delayed rectification to reduce the resistance of the innervated face is an interesting adaptation. It allows the fish to maintain a high resting resistance but to achieve a large external current during activity. A possible disadvantage to this mechanism is that the increase in conductance is delayed, a property that would not appear to be very significant in an organ discharge that involves fused and presumably repetitive responses. The earliest PSPs would cause the conductance increase which would remain activated during the later PSPs. The double peak of the initial external responses of single cells is not seen in organ discharges although there may be an inflection on the rising phases (Fig. 12A). The difference is undoubtedly a question of synchronization because if electric stimuli are used to evoke synchronous PSPs in lengths of organ the external responses have the same shape as the responses of single cells.

D. Freshwater Electric Fish 1. GYMNOTIDS The gymnotids are a diverse group of fish living in fresh waters of tropical South America. They are often divided into six families (Table I ) , but the relationship between the families is obvious from the similarities in body shape and in many other characteristics as well as from the possession of electric organs (Fig. 1). This group includes the electric eel, probably the best known electric fish, and a moderate number of other species that have only weakly electric organs. Because of their characteristic elongate shape, the weakly electric gymnotids

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are commonly called knife fish. The greater part of the length of the fish contains only muscle, spinal column, and electric organ. The viscera are located in the anterior end of the body, and the anus and genital papilla are just behind the chin. The anal fin begins slightly more posteriorly and runs to near the caudal end of the fish. The electric organ runs along most or all of the length of the body and is innervated by spinal nerves. A caudal fin is absent in all but the sternarchids, in which it is greatly reduced. There is little muscle caudal to the anal fin, and this region, sometimes called the caudal filament, contains mostly electric organ. Gymnotids can regenerate new posterior regions if part is removed (Ellis, 1913). Often in fish taken from the wild the posterior is regenerated indicating that there were encounters with predators in which only the front end of the fish escaped. Ordinary swimming movements of the gymnotids are quite different from those of almost all other fish. The body is held straight, and waves along the anal fin provide the propulsive force. Most species appear to swim about equally well forward and backward and often investigate objects by swimming backward toward them (see Gymnarchus, Section 11, C, 3 ) . Most of the body musculature is not involved in movement of the anal fin and is probably used only in emergency movements when the body is rapidly flexed in the more usual fishlike manner. The species of gymnotids are poorly described. Keys are available for a few areas of South America, but it is doubtful that these are complete. Tropical fish dealers, the usual source of supply of gymnotids, often bring in fish from other areas that are clearly new species. On the Rio Negro expedition of the R. V. Alpha Helix, perhaps one-third of the gymnotid species collected were undescribed taxomically. While there are difficulties in working on undescribed species, classification into genera is generally simple, and generic characteristics are apparently the most important ones from a physiological point of view. Nevertheless, it may be desirable to save specimens of a particular species studied for subsequent identification when the systematics of gymnotids is clarified. Table I1 and Fig. 16 present a key to gymnotids that is modified from that of Ellis (1913). A second system has been added, one that is nondestructive and applicable to living specimens. Accurate classification sometimes requires dissection of the specimen, but a reasonable identification can usually be achieved without it, particularly if an oscilloscope is available to observe electric organ discharges. a. The Electric Eel. The electric eel was the first electric fish for which the cellular mechanisms of the discharge were elucidated (Keynes and

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Table I1 Key to Families and Genera of Gymnotids The gymnotids are recognizable from their elongate body and anal fin; the pectoral fills are small; the caudal is present only in the sternarchids in which it is very small; other fins are absent. There are two African species, Notopterus ofcr and Xenomystus nigri, that are also commonly known as knife fish. They are similar in appearance to Some weakly electric gymnotids, and confusion might arise if place of origin were unknown. African knife fish are easily distinguished by the short “tentacle” from the anterior naris and the anal fin that extends a short distance around the tip of the tail to form a false caudal fin (as is also true of the electric eel). Notoptcrurus has a small dorsal fin. I n the following key the species name is given, if only a single species has been described.a a. Head flattened dorsoventrally, lower jaw projects beyond upper so that the large mouth opens somewhat upwards b. Strongly electric; electric organ occupies much of the body caudal to the abdominal cavity; body not scaled; anal fin extends around the end of the tail to form a false caudal. Electrophoridae. Electrophorus elcctricus bb. Weakly electric; scaled; slender cylindrical tail extends beyond anal fin. Gymnotidae. Gymnotus carapo %a. Head round in cross section or compressed laterally, lower jaw projecting little if any, mouth small or large c. Caudal fin absent; tail beyond anal fin slender and usually cylindrical; no dorsal filament d. Snout short, Sternopygidae6 e. Teeth in both jaws, color uniform or with longitudinal stripes f. Color uniformly dark except sometimes a lighter stripe along the posterior lateral line; body compressed laterally; orbital margin free, i.e., there is a distinct and deep cleft between the eye ball and adjacent skin; posterior air bladder long and conical; organ discharge more or less sinusoidal a t a frequency of 50-150/sec. Sternopygus ff. Color light; fairly transparent to pale yellow, often with several darker longitudinal stripes; body compressed laterally; eye covered by a thin membrane; posterior air bladder small, nearly spherical; organ discharge more or less sinusoidal a t a frequency of 250-600/sec. Eigcnmannia fff. Very long caudal filament, dorsal profile of head concave. Rabdolichops longicaudatusc ee. Teeth absent, color dark but patterned; brown to black mottled or with slightly diagonal banding; organ discharge consists of brief pulses separated by much longer intervals g. Body compressed laterally, depth increases from posterior of head to shortly after beginning of anal fin, then decreases (Fig. 16); profile rounded; accessory organs in head region (Fig. 32) ; lies on side when resting on a flat surface. Steatogenys elegansd gg. Body less compressed, depth near greatest a t posterior of head (Fig. 16); profile shows a protruding mouth; no rostra1 accessory organs; rarely lies on side. Hypopomus dd. Snout long and tubular. Rhamphichthyidae g. Body entirely scaled. Rhamphichthys rostratusC gg. Sides not scaled in anterior region. Gymnorhamphichthys hypostomus cc. Caudal fin present but quite small; dorsal filament (see Fig. 42) closely adherent to back but may be separated in fixed specimens; organ discharge frequency 700/sec or more. Sternarchidaee

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h. Snout long, tubular, and down curving. Sternarchorhynchus oryrhynchusf hh. Snout long, tubular, horizontal and straight i. Mouth large, opens a t least one-third the distance back to the level of the eye; upper profile of head markedly convex. Sternarchorhamphus ii. Mouth small, opens less than one-sixth the distance back to the level of the eye, upper profile quite straight. Orthosternarchus tamandua“ hhh. Snout not long unless mouth is very large j. Teeth present in both jaws; some located externally on what appear to be swollen lips. Oedemognathus exodon jj. Teeth present in both jaws but inside mouth k. Dorsal region of body virtually entirely scaled from head posteriorly to origin of dorsal filament 1. Mouth large, its angle extending a t least as far posteriorly as the anterior margin of the eye. Sternarchus 11. Mouth small, its angle extending no farther posteriorly than theposterior naris. Sternarchella kk. Scales absent from much of the body above the lateral line anterior to the dorsal filament (shaded in Fig. 16); scales near lateral line much larger than more dorsally. Porotergus jjj. Teeth absent from upper jaw m. Lower jaw toothless and with a distinct midline groove into which the beaklike upper jaw fits; middorsal region of body scaled anteriorly. Adontosternarchus mm. Lower jaw sometimes toothed and fitting into a midline groove in the upper jaw; middorsal region of body naked anteriorly. Sternarchogiton a Modified from Ellis (1913), Eigenmann and Allen (1942), and Gery and Vu-TbnTu&(1964) who should be consulted for more extensive descriptions. Sternopygus, Eigcnmannia, Hypopornus, and Steatogenys are normally grouped together as the family Sternopygidae. Properties of the electrocytes and their innervation indicates that Hypopomus and Steatogenys are more closely affiliated to the Rhamphichthyidae. Greenwood et al. (1966) observe “Peculiar specializations in [the gymnotids] are many, and a complete study may alter the family arrangement accepted here.” The author has not seen members of these genera nor are there any physiological data from them. Only one species is described in the taxonomic literature, but there is a second species described here that has somewhat different rostra1 electric organ. There is a proposed revision of Sternarchidae and Sternarchus to Apteronotidae and Aptcronotus on grounds of priority (1800 vs. 1801). As Sternarchus has been used for a long time and provides the basis of many other generic names in the family, the author stands with Ellis (1913) who uses this name and derivatives. f Although this genus has been considered to be monospecific, two apparent species of each were found on the Rio Negro expedition of the Alpha Helix.

Martins-Ferreira, 1953; Altamirano et al., 1953). The eel emits two classes of pulses, small pulses about 10 V in amplitude and large pulses some 500 V or more in amplitude. All the pulses are monophasic, head positive, and about 2 msec in duration. The voltage increases with the length of the fish, which can exceed 1.5 meters, but even a baby 7-10 cm long can emit close to 1OOV. When the animal is resting, the small

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_ _ _ _ _ - -.- -_ _ _ _ Snout short

- _..-.... Lower low only

__---._

Sternarchorhomphus mulleri (Steind

I

Snout short Eigenmonnio virescens (Vol.1

Gyrnnotus coropo (Linn)

Hypopornus brevirostris (Steind )

Electrophorus electricus (Linn )

Fig. 16. Heads of representative gymnotids. Slightly modified from Ellis (1913).

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pulses are emitted at quite low frequencies down to a few per minute. When actively swimming, the pulse rate is usually increased to about 30 or more per second. Large pulses are emitted only in high frequency bursts at a frequency of several hundred per second. The first pulse in such a burst is small, but the amplitude increases to the maximum within 2 or 3 pulses ( Albe-Fessard, 1950a). The weak pulses are implicated in the electrosensory system (see Chapter 11, this volume); the strong pulses have offensive or defensive value. The emission of moderate to high frequency pulses by one eel attracts other eels, and they also increase their rates of discharge (Bullock, 1970). The electric tissue occupies much of the cross section of the body in the posterior three-fourths of the fish, and it is divided into three (bilateral) organs. There is axial musculature dorsal to the organs and a smaller amount of muscle ventrally that controls the anal fin (Fig. 17). In the dorsal and posterior region is the organ of Sachs, and in the ventral and anterior region is the main organ (Fig. 1). Beneath these organs is Hunter’s organ. The electrocytes are more widely separated and somewhat larger in Sachs’ organ than in the main organ, but otherwise the two organs are similar (Luft, 1957; Couceiro and Akerman, 1948). The main organ generates the greater part of the high voltage discharge used offensively or defensively. The organ of Sachs generates the greater part of the low voltage pulses involved in the electrosensory system but also contributes in a minor way to the high voltage discharge. Hunter’s organ apparently functions like main organ anteriorly and Sachs’ organ posteriorly ( Albe-Fessard and Chagas, 1954). The single electrocytes are flattened in the anterior posterior axis (Fig. 17). They are ribbon-shaped and tend to run from the medial septum to the lateral margin of the fish. In an adult there are dorsoventrally about 25 in the main organ and 10 in Hunter’s organ. There are some 6000 in the anteroposterior axis ( Albe-Fessard, 1950a). Each cell is contained in a connective tissue chamber, and successive layers of cells in the anterior posterior direction are fairly accurately aligned, one behind the other. The alignment is somewhat less good in the dorsoventral direction. The electrocytes of the main organ occupy perhaps one-fifth to onehalf the chamber volume; the fraction is smaller in Sachs’ organ (Luft, 1957; Couceiro and Akerman, 1948). The cells are innervated on their posterior faces by spinal nerves. The posterior faces have a moderate number of short papilli or stalks protruding from them that increase the surface area; there are also some tubules in this face (Fig. 17). The innervation is primarily on the stalks and is much less dense than in electrocytes of torpedinids. The anterior faces have a large number of

A Rostra1

Spinal cord I Swim. bladder Caudal

Fig. 17. Structure of eel electric organ, ( A ) Diagram of gross morphology of the organ showing series arrangement of electrocytes (“plaques”) on the left and parallel arrangement on the right. From Altamirano d al. (1953). ( B ) Light micrograph of a section through a single electrocyte ( e ) . The caudal, innervated surface ( o n the left) has some small processes from it. The innervation appears quite sparse and few nerve fibers ( n ) are visible. Relatively stout processes come off the anterior, uninnervated face and there is a pronounced layer of increased density associated with the membrane. ( C ) Low and high (inset) magnification electron micrographs of the uninnervated surface showing the extensive canalicular network responsible for the density associated with this face seen in B. The canaliculi open to the exterior (arrows) and branch profusely. Basement membrane material extends into the canaliculi, ( D ) High magnification electron micrograph of the innervated face showing a vesicle filled nerve terminal ( n ) . The surface proliferation provided by canaliculi (openings indicated by arrows) is much smaller at this face. Magnification for inset in C same as that for D. Micrographs provided through the courtesy (and skill) or Drs. F. E. Bloom and R. Barnett.

5 5 r m H r-1

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papilli and an extensive network of small canaliculi that greatly increase the surface area compared to the area that a simple planar membrane would have (Fig. 17, see also Mathewson et al., 1961). The properties of eel electrocytes are only slightly more complex than those of electrocytes of the stargazer and Torpedo. The innervated face responds to depolarization by generating a spike that overshoots the resting potential by about 50 mV. The uninnervated face is of very low resistance and does not become excited. The responses of a single cell are shown in Fig. 18. Current is applied by external electrodes in order to depolarize the innervated face, and differential recording between two microelectrodes is used. In Fig. 18A-C one electrode is advanced into the cell and then into the underlying extracellular space. When the “exploring” electrode is in the cell the internally negative resting potential of -90 mV is recorded across the innervated face (Fig. 18B). An adequate stimulus evokes a spike that overshoots the zero potential by about 50 mV. The response arises from the depolariza-

Fig. 18. Responses of electrocytes of the electric eel. Right: recording differentially as one electrode is advanced through a cell; positivity of this electrode shown upward. Current passed through the cell by external electrodes in order to depolarize the innervated face. ( A ) Both electrodes external to the innervated face; no response is seen (there is a brief diphasic stimulus artifact). ( B ) One electrode is advanced into the cell. The inside negative resting potential of about 90 mV and an overshooting action potential about 140 mV in amplitude are recorded. ( C ) When the exploring electrode is advanced to outside the uninnervated face, the resting potential disappears, but the spike is essentially unchanged. From Keynes and Martins-Ferreira ( 1953). (E-F) Differential recording across the innervated face, the upper trace shows the zero potential. Stimuli hyperpolarizing the innervated face can evoke PSPs ( E ) that arise after a latency of 1-2 msec and that if sufficiently large initiate a spike ( F ) . From Altqmirano et al. ( 1955). Calibrations are the same for A-F.

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tion produced by the stimulus; the delay can be made much shorter if a stronger stimulus is given. When the exploring electrode is advanced out through the uninnervated face of the cell, the resting potential disappears demonstrating that the resting potentials across the two faces are equal (Fig, 1SC). The response amplitude however does not change. As discussed in respect to electrocytes of Astroscopus this finding indicates that the resistance of the uninnervated face is very low. Direct measurements using current pulses confirm its low resistance, which is much lower than that of the innervated face. The degree of surface elaboration seen cytologically correlates with the different resistances. Stimulation of the nerve supply evokes PSPs that can depolarize the innervated membrane to the point where it generates a spike. Current applied as in Fig. 18A-C but in the opposite direction hyperpolarizes the innervated face and does not excite it. Such stimuli can excite nerve fibers in the tissue that then produce PSPs that arise after a delay of about 2 msec (Fig. 1 8 0 ) . If enough nerve fibers are stimulated the PSP initiates a spike (Fig. 18E). The ionic basis of the action potential has been well studied in electrocytes of the eel. The cells are depolarized by high potassium solutions approximately as predicted by the Nernst relation, which indicates that at rest the potassium permeability of the cell predominates and the potential is largely determined by the intra- and extracellular concentrations of potassium (Higman et al., 1964). The inward current responsible for the rising phase of the spike is sodium dependent ( Keynes and Martins-Ferreira, 1953). Furthermore, it is eliminated by the pharmacological agent tetrodotoxin which is a specific blocking agent for the increase in sodium permeability produced by depolarization (Nakamura et al., 1965). Unlike the squid axon and many other tissues, the eel electrocytes lack K activation or delayed rectification, but they do have anomalous rectification in the innervated face (Nakamura et al., 1965). Actually, it makes sense for the cells to have anomalous rectification and not to have K+ activation. For maximum effectiveness as an electric organ, the circuit for all the current carried inward by Na' should be completed by current in the external environment, not by local currents in the innervated face. The time constant of the cells is sufficiently short that the membranc rapidly returns to the resting potential without the restoring effect of delayed rectification (as is also very nearly true of myelinated nerve fibers, see Frankenhaeuser and Huxley, 1964). The anomalous rectification decreases the conductance enough to result in a severalfold decrease in eddy currents in the innervated face,

Fig. 19. Electric organ of Gymnotus. ( A ) Cross section a t two magnifications of region 2 cm from the tip of the tail of a fish about 10 cm long; higher power view on right of the region indicated. The connective tissue tubes enclosing the four columns of electrocytes are numbered from dorsal to ventral; the section grazes electrocytes of column 1 and passes through nearly the maximum diameter of cells of column 3. The spaces dorsal to the tubes are fixation artifacts. At this level most of the body cross section is occupied by muscle. The vertebral column and spinal cord ( c ) are seen. ( B ) Photographs of a dissected unstained preparation. The cells and their main innervation are outlined on the right-hand copy, The caudal innervation of the most dorsal cells is also visible. Electrocytes of all four tubes are numbered. Modified from Bennett and Grundfest (1959).

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The excitability properties of the innervated face lead to an unusual sequence of conductance changes during the response (Morlock et al., 1969). At the start of the spike, depolarization causes sodium activation and increased conductance and further depolarization; depolarization also decreases the conductance of anomalously rectifying channels, but the net result is increased conductance, Sodium inactivation ensues, and the total conductance decreases to below the resting value while the membrane potential falls from the spike peak; at a sufficiently low potential anomalous rectification reverses and conductance rises to the resting level. This diphasic sequence of conductance changes, increase followed by decrease, contrasts to the monophasic increase seen in nerve (Cole and Curtis, 1939; Tasaki and Freygang, 1955). One might ask why the innervated membrane should not just have a high resting resistance instead of anomalous rectification. One possible reason is to provide a conductance in inactive cells for flow of current generated by active cells in series with them. As will be discussed further in Section 111, A, the cells are not all active at the same time except in the largest discharges. The voltage of these responses is accounted for by synchronous activity of some 6000 cells in series each producing over 100 mV.

b. Gymnotus. This species is weakly electric; its organ discharge is a fraction of a volt recorded in water and not much more than a volt recorded in air. Its body shape is similar to that of the eel, but the electric organ is much smaller (Fig. 1).When undisturbed and resting it normally emits pulses at about 50/sec. The pulses are approximately triphasic, initially head negative, and about 1 msec in duration (Fig. 3B, B’) . Mechanical stimuli, light, electric fields, and resistance changes can cause moderate transient accelerations of the discharge, and when the animal is feeding, the discharge frequency can briefly exceed 200/sec ( Fig. 3, Lissmann, 1958; Bennett and Grundfest, 1959; Black-Cleworth, 1970). When swimming around its tank the fish maintains a fairly steady frequency somewhat above the resting level. Gymnotus is also capable of ceasing its discharge completely for brief periods, a response that may sometimes represent hiding or “listening.” Both accelerations and cessations can be involved in communication between other members of the same species and a relatively potent stimulus for inducing cessation of firing is weak electric pulses at a frequency close to that of the organ discharge. The electric organ runs longitudinally from just behind the chin to the tip of the caudal filament. The organ lies dorsal to the anal fin but extends beyond it rostrally to the cleithrum as well as caudally (Fig. 1). The organ on one side consists of about four longitudinal columns of

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drum-shaped cells, the flat faces of which are oriented anteroposteriorly (Fig. 19). The cells are about a millimeter in diameter and 300p thick in a fish 20 cm long. The number of columns is reduced anteriorly, and there may be several additional columns caudally. In each column there is one electrocyte per segment and there are about 90 segments in the animal. Each column of cells is enclosed in a connective tissue tube that is divided into chambers by loose septa between the cells. The cells of all but the most dorsal column are innervated by a number of fibers on their posterior faces. Aside from innervation the two faces are very similar. They are quite smooth with relatively few inpocketings and canaliculi (Schwartz et al., 1971). The cells of the most dorsal column have their main innervation on the anterior face, but they have a few fibers ending on their posterior faces as well (Szabo, 1961d). As will be shown below, these cells behave physiologically like the more ventral cells but are oriented in the opposite direction. No obvious function of the posterior innervation was observed in the early physiological study of these cells (Bennett and Grundfest, 1959), but since the presence of the posterior innervation was not rwognized at that time the question could well be reinvestigated. The single electrocytes of Gymnotus generate external potentials that are diphasic (Fig. 20) in contrast to the monophasic discharges of the eel. This form of potential is produced because both faces generate spikes. The lower threshold face is the posterior, innervated face in all but the dorsal column of cells in which it is the anterior face. When the nerve supply is activated or when current is applied by an intracellular electrode, the lower threshold, innervated face fires first. Current flows inward through this face and outward through the opposite, uninnervated face which becomes excited, but with some delay with respect to firing of the innervated face. By this time the spike of the lower threshold (innervated) face is decreasing, and current flows in the reverse direction along the axis of the cell. This pattern of activity is indicated by the external recordings shown in Fig. 20. The monopolarly recorded external potentials are of opposite sign outside the two faces. External to the innervated face, the potential is negative during PSPs, and when a spike arises, goes rapidly more negative. During the later part of the monopolarly recorded intracellular spike, the potential external to the innervated face reverses to go positive, indicating that the uninnervated face has a larger potential across it than the innervated face. The potential outside the uninnervated face has the same shape and about the same amplitude as that outside the innervated face but is opposite in sign. External to the edges of the cell the potentials are very small. This feature and the opposite polarity of potentials external to the two faces

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I msec

Fig. 20. Responses of electrocytes of dorsal and ventral columns (Gymnotus). ( A ) Records from an electrocyte of the most dorsal column. ( B ) Records from a cell of the third column ventrally. Monopolar recordings outside rostral face (upper traces ) , caudal face ( middle traces ) , and intracellularly ( lower traces ) . ( AI,Bl ) Weak stimulation of the nerves evokes a depolarizing PSP which is associated with negativity external to the rostral face in A,, and external to the caudal face in B,, reflecting the different innervation of the two classes of cell. (A,,B2) Stronger stimuli evoke similar intracellular spikes in the two cells, but the diphasic external potential is initially positive outside the rostral face in A, and outside the caudal face in Bz. (AI,BS) Current passed through the intracellular electrode evokes an external response that is initially negative outside the innervated face in each case indicating that this is the lower threshold membrane. From Bennett and Grundfest ( 1959).

demonstrate the longitudinal orientation of the cell. ( I t should be noted that there will be some local circuit or eddy currents within each face that will not contribute significantly to the externally recorded potentials; for this reason the synapse in the innervated face could cause it to fire first even if it were not of lower threshold.) That the innervated face is indeed of lower threshold is indicated by the effect of intracellularly applied current, which depolarizes both faces equally if the external resistances are equal (see Fig. 2). This procedure always excites the innervated face first, even when the external resistance is somewhat greater on the innervated side. As will be shown later with respect to Gymnarchus (Section 11, C, 3 ) , a diphasic external potential can arise if one face is inactive and behaves as a series capacitance. The excitability of both faces of Gymnotus elec-

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trocytes can be clearly demonstrated by stimulating with externally applied currents that run along the axis of the cell; these currents hyperpolarize one face and depolarize the other. An experiment of this kind is illustrated in Fig. 21. When a stimulating cathode is placed external to the uninnervated face, the applied current tends to depolarize this face and hyperpolarize the innervated face ( Fig. 21B,). The uninnervated face fires first as indicated by initial positivity of the response recorded external to the innervated face. The spike recorded across the innervated face arises from a hyperpolarized level of membrane potential confirming that this face is not initiating the spike. The innervated face is excited by the activity of the uninnervated face, and negativity external to this face follows the positive phase. When the stimulating electrode external to the uninnervated face is an anode, the innervated face is depolarized and fires first; there is initial negativity outside this face (Fig. 21A1). Since the uninnervated face is hyperpolarized by the applied current, its firing is delayed compared to that evoked by neural or intracellular stimulation and the spike recorded across the innervated face has two quite distinct components. (Thc two spikc components are only barely recognizable in the monopolar recordings of Fig. 20. ) Usually if stimulation only moderately above threshold is applied as in Fig. 21A,,B,,the initially active face can excite the opposite face that is being hyperpolarized by the stimulus. However, strong stimuli can cause sufficient hyperpolarization of this face that the spike of the initially firing face cxcitvs it only partially if at all. Correspondingly, there is failure of the second spikc component recorded intracellularly and disappearance or reduction of the second phase of the extcrnally recorded responses (Fig. 21A2,B2). Although in Gymnotus the single cells generate diphasic extcrnal potentials, the overall organ discharge is triphasic. This configuration results from the opposite orientation and earlier firing of the most dorsal electrocytes (Fig. 22). These cells fire about ?d msec earlier than the more ventral cells, all of which fire synchronously. The activity of thc anterior faces of the dorsal cells results in the initial head negativity. The activity of their uninnervated faces is simultaneous with the activity of thc innervated faccs of the ventral cclls and summates with it to cause the second, head negative phase. Activity of the uninnervated faces of the ventral cclls causes the final, head negative phase. Corresponding to the number of cells active, the initial phasc is the smallest, the second phase is the largest, and the final phase is somewhat smaller than the second phase. In Gymnotus the electric organ has at its rostra1 region a small number of modified cells that fire earlier than the main organ and

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JL;

-+;

A,-

Fig. 21. Effects of axial stimulation on electrocytes of Gymnotus. Stimulation by rectangular current pulses and recording as indicated in inset except that the indifferent electrode is much farther away. Upper trace: differential recording across innervated face. Lower trace : monopolar recording external to innervated face. (A1,A2) Anodal stimulation external to the uninnervated face. ( A 1 ) The stimulus is moderately above threshold and initiates a two component spike. The external record is initially negative indicating that the innervated face generates a spike first. ( A z ) Stronger stimulation largely blocks the second spike component and reduces the positive phase in the external record; evidently the stimulus hyperpolarizes the uninnervated face sufficiently that it is only partially excited by the spike of the innervated face. (The residual positivity may represent local response in or capacitative currents through the uninnervated face, see Section 11, D, 3 . ) (B1,BZ) Cathodal stimulation external to the uninnervated face. ( B , ) A two component spike is initiated but the external record is initially positive indicating that the uninnervated face fires first. ( B z ) Stronger stimulation causes failure of the second spike component and a large reduction in the negative phase of the external record indicating failure of excitation of the innervated face. The small intracellular positivity and aysociated external negativity that develops after a latency of about 1 msec ia a PSP resulting from stimulation of the nerve supply. Modified from Bennett and Grundfest ( 1959).

apparently generate monophasic external potentials as do eel electrocytes. The detailed operation of this part of the organ has not been investigated. It resembles in its operation the rostra1 accessory organs of several other gymnotids ( see below). c. Hypopomus. At least three species of Hypopomus have been studied electrophysiologically, but the correspondence to the taxonomically named species is somewhat uncertain. Pulses are emitted at a basal frequency of 5-10/ sec, and again there are large accelerations during swimming or when the fish is stimulated. One species can maintain its discharge rate quite constant at two or more levels, the higher ones generally associated with greater activity (Bullock, 1970). Cessation of discharge has also been observed (Bullock, 1970; Black-Cleworth, 1970). The pulses are about 2 msec in duration and of the order of 1V in amplitude; they are monophasic head positive in one of the species studied and diphasic initially head positive in another.

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Fig. 22. Constitution of the organ discharge in terms of the responses of the different columns of electrocytes ( Gymnotus). Left, intracellularly recorded responses of individual cells in different columns at the three levels of the fish indicated in the center diagram. The potentials are somewhat distorted by pickup from other active cells. In each case the lowest trace is the monopolarly recorded organ discharge at the tip of the tail which is used as a time reference; the vertical line indicates onset of organ discharge. ( A ) The electrocytes of the most dorsal column (upper traces) fire about 0.2 msec earlier than the cells of the three ventral columns (middle three traces). ( B ) At this level the electrocytes of the most dorsal column also fire earlier than those in the next two columns ventrally, but the discharge slightly precedes the firing of the dorsal column in A. ( C ) Only one column continues to this level. The electrocytes are caudally innervated and probably represent those in the third column. The response arises approximately at the same time as does activity in the caudally innervated electrocytes in A. Right, organ activity recorded at the tail as a summation of the potentials produced by the different columns. Broken vertical line indicates the start of discharge of the three columns of caudally innervated cells. The rostrally innervated cells contribute a smaller, earlier diphasic potential which is of opposite sign. From Bennett and Grundfest (1959).

As in Gymnotus the electric organ runs from just behind the chin region to the tip of the caudal filament. The organ consists of 3-5 longitudinally running columns on each side in which there is one electrocyte per segment ( Bennett, 1961). The cells are cylindrical, about 0.3 mm in diameter, and 0.2 mm thick in a 15-cm fish. All the electrocytes are innervated by a small number of nerve fibers on a short process or stalk from their posterior faces. In respect to innervation the electrocytes closely resemble those of the main organ of Steatogenys (Fig. 29). In the species of Hypopomus (probably H . urtedi) with the diphasic organ discharge, the potential is initially head positive when it is

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recorded at the head of the fish or some distance rostrally to it. At the tail and caudal to it, the potential is inverted (Fig. 2 3 ) . Along the side of the fish the discharge is triphasic, probably because of asynchronous discharge of anterior and posterior cells. The single cells resemble those of Gymnotus in that both faces generate spikes and the resulting external potentials are diphasic; however, all the cells are oriented in the same direction. The response of an electrocyte to intracellularly applied depolarizing current is shown in Fig. 24 recorded both monopolarly and differentially. The innervated (posterior) face is lower threshold because it fires first under these con-

+-

I rnsec

Fig. 23. Organ discharge of Hypopornus. A 16.5 cm fish is placed in a shallow plastic tray, 45 cm long by 24 cm wide by 5 cm deep, and held by a gauze tube lengthwise against the long side, midway between the surface and bottom. Three electrodes are used. One is fixed a t the head, and one is moved at 1.5 cm intervals along the axis of the fish; these two electrodes record differentially against an electrode at the midpoint of the opposite side of the tray. The records are therefore somewhat larger but similar in form to what would be obtained by monopolar recording in a very large volume of water. The brief discharges occur at about 5/sec when the fish is unstimulated. The discharge at the rostra1 fixed electrode triggers the oscilloscope and is used to align the records in the figure obtained from the exploring electrode (positivity of this electrode u p ) . At the tip of the tail ( C ) the potential is diphasic and initially tail negative. Moving the electrode farther caudally, it becomes smaller, but is similar in form (records above C ) . Moving rostrally along the body, the potential becomes triphasic and then diphasic, initially head positive, at the tip of the snout ( R ) . The potential remains diphasic but decreases as the electrode is farther advanced (records below R ) . From Bennett (1961).

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Fig. 24. Response of an electrocyte of Hypopomus to intracellularly applied depolarizing current. Superimposed threshold pulses that do and do not excite the cell. ( A ) Monopolar recording; ( B ) differential recording as in diagrams. Upper trace: stimulating current. The transmembrane potentials in B (V, and V,) start from the same level. The external record in B (V,) is shown with positivity external to the uninnervated face upward. Both faces are excited, the spike of the innervated face is lower threshold or faster rising and initially there is negativity external to this face. The spike of the caudal face is longer lasting and larger, and there is a longer lasting negativity external to this face. The characteristics of the spikes are more clearly seen from the differential recordings ( B ) . The external records in A are mirror images, showing the longitudinal orientation of the cell. From Bennett (1961).

ditions as indicated by initial negativity outside this face and initial positivity outside the uninnervated face. Differential recording across the two faces shows clearly that, although the spikes in each face are of about the same peak amplitude, the spike generated by the innervated face is considerably briefer than that generated by the uninnervated face. Thus, the head-negative phase of the external potential is larger than the head-positive phase. External stimulation of the electrocytes also demonstrates that both faces generate spikes (Fig. 25). When current is passed in order to depolarize the uninnervated face and hyperpolarize the innervated face, the external record is initially head negative (Fig. 25A). The spike in the uninnervated face soon excites the briefer spike of the innervated face and the external record goes transiently more head positive, The longer lasting spike of the uninnervated face causes the external record to go head negative again. When current is passed that depolarizes the innervated face and hyperpolarizes the uninnervated face, the brief spike of the innervated face is evoked (Fig. 25B). However, this activity does not excite the long-lasting spike of the uninnervated face, which is apparently too hyperpolarized by the applied current. [A small head negativity occurs in the external record. This phase may represent a sub-

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-

$3

-

7 I rnsec

fl Fig. 25. Stimulation of an electrocyte by externally applied currents. Hypopomus, same cell as Fig. 24. Stimulation and recording as in diagram. Upper trace: stimulating current. Middle traces (V, and V2): potentials across the two faces starting from the same level. Lower trace: potential between the external electrodes, rostral positivity up. When anodal current is passed ( A ) , the caudal (stalk) face is hyperpolarized and the rostral ( uninnervated) face is depolarized. The rostral face is excited first as indicated by initial rostral negativity externally. This activity excites the brief caudal spike which produces a rostral-positive phase in the external record which then becomes rostral-negative again. Oppositely directed currents ( B ) excite the caudal face first. The caudal spike fails to fire the rostral face, which is hyperpolarized by the stimulus. Only a small rostral negativity occurs in the external record. From Bennett (1961),

threshold response of the uninnervated face or discharge of the capacity of this face (see Gymnurchus, Section 11, D, 3 ).] When the nerve supply is stimulated, excitation occurs first in the stalk and then propagates into the main part of the cell. The potentials in stalk and body can be quite different (Bennett, 1961) as is illustrated with respect to the species of Hypopomus with a monophasic discharge (Fig. 28). The external potential generated by the electric organ of the monophasically discharging Hypopomus remains relatively constant in shape as an exploring electrode is moved along the fish and becomes very small just caudal to the center of the fish (Fig. 26). Evidently the fish behaves more like a dipole than the diphasically discharging Hypopomus, perhaps because of better synchronization between cells but also because the discharge of the individual cells is monophasic and the total output is therefore less sensitive to slight failures of synchronization. The single cells have properties similar to those of the electric eel ( Fig. 27). However, the uninnervated ( anterior) faces have a resistance about half that of the innervated faces (Fig. 28D) and a spike in the innervated face causes an appreciable voltage drop across the uninnervated face. Still the external potentials differentially recorded across the

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Fig. 26. Potentials generated by the monophasically discharging Hypopomus. Procedure as for Fig. 23, but recorded with the fish in the center of a somewhat smaller container. The distance calibration gives recording sites with respect to the fish referred to the level at the start of the records.

a

I msec

Fig. 27. Response of an electrocyte of monophasically discharging Hypopomus to intracellularly applied depolarization. Upper trace: stimulating current. ( A,C ) Monopolar recording. Second trace: intracellular; third trace: external to uninnervated face; fourth trace: external to innervated face. (B,D) Differential recording. Second trace: across innervated face; third trace: across uninnervated face; fourth trace: across entire cell. Superimposed traces of threshold stimulation with and without a response. The innervated face generates a spike, but there is only a small potential across the uninnervated face.

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A=-.

-

B-n

2 msec

Fig. 28. Neural excitation and stimulation by axial currents of an electrocyte of monophasically discharging Hypopomus. First trace: stimulating current applied by an electrode external to the uninnervated face; next two traces starting from the same level: differential recording across the cell's faces; fourth trace: differential recording across the membrane of the stalk (one electrode external to the innervated face was used). ( A ) Three superimposed traces showing two subthreshold PSPs and one that initiated a spike. The spike in the stalk is larger than that across the main part of the innervated face of the cell. ( B ) A stronger and longer lasting stimulus excited the stalk and innervated face directly as indicated by the short latency. A PSP is still evoked and appears inverted on the peak of the spike in the stalk. ( C ) A brief cathodal stimulus causes a neurally mediated response to be initiated in the stalk (as indicated by latency and abrupt rise from the base line). ( D ) A strong cathodal stimulus prevents the stalk impulse from invading the body of the cell. A large amplitude but abbreviated spike remains in the stalk. The innervated face is depolarized by about 40 mV but fails to be excited.

cells are about 50 mV in amplitude. Corresponding to the different resistances the uninnervated face is more proliferated by tubules and canaliculi than is the innervated face (Schwartz et al., 1971). The form of the recorded spike potentials depends on the form of the stimulus. A brief pulse near threshold produces a potential that rises more slowly than it falls, whereas the opposite is true of long-lasting stimuli. The response to brief pulses more closely resembles the externally recorded organ discharge and presumably corresponds more closely to activation by way of the synapses on the stalks. During neural activation the impulse arises in the stalk and propagates into the cell body. An external stimulating electrode can be used both to evoke the neurally mediated response and to excite the cell directly. A response evoked by a brief anodal pulse applied by an electrode external to the uninnervated face is shown in Fig. 28A. Two small PSPs subthreshold for initiating a spike are seen in the stalk, but there is

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little spread to the main part of the cell. Stronger stimulation causes a larger PSP, and a spike is initiated in the stalk that is earlier, larger, and longer lasting than the spikes recorded across the faces of the cell. With a stronger and longer lasting stimulus the spike is initiated directly in the stalk (or posterior face) as indicated by the short latency of the response (Fig. 28C). At the time that the PSP occurs in Fig. 28A there is a depression in the peak of the spike in the stalk. This response is the PSP that is inverted because the potential during the spike exceeds the reversal potential of the PSP (see del Castillo and Katz, 1956). If a cathodal stimulus is applied external to the uninnervated face a large depolarization of this face can be produced without exciting it, demonstrating its inexcitability (Fig. 28D). Stimulation of this polarity hyperpolarizes the innervated face, and if a prior brief stimulus initiates a neurally evoked response in the stalk (Fig. ZSC), this activity can fail to invade the main part of the innervated face of the cell. In this event only a small potential is observed across the innervated face even though the spike recorded in the stalk remains large. The role of stalks in electrocyte function remains obscure. It will be considered again in Section 11, F. One unidentified species of Hypopomus has been studied in which external recording suggests that electrocytes in about the anterior twothirds of the body generate monophasic potentials and the posterior cells generate diphasic potentials. This possibility requires exploration using microelectrode techniques. No species of Hypopomus has been observed to have a rostra1 accessory organ like those of Gymnotus, Steatogenys, and Gymnorhamphicthys. d. Steatogenys. Steatogenys is quite similar in appearance to Hypopomus and is often confused with it by fish suppliers. The main organ of Steatogenys appears morphologically identical to that of the diphasically discharging Hypopomus (Fig. 29), and the form of discharge recorded near the tail or distant from the fish is similar (Fig. 30). The frequency of resting discharge is more like that of Gymnotus, and various stimuli cause moderate accelerations [although there is some dispute as to this point (see Bullock, 1970)l. As in Hypopomus innervation occurs on a short stalk from the posterior face (Fig. 29). Both faces of the cells generate spikes and morphologically the two faces are similar although there is a somewhat greater proliferation of the uninnervated face ( Schwartz et al., 1971). Recordings across the faces and stalk of an electrocyte of the main organ are shown in Fig. 31. A brief stimulus is applied to the nerve supply and evokes a threshold synaptic potential. This PSP is much larger in the stalk than across either face of the cell. The impulse arises in the stalk

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r,; 0.5 msec

Fig. 30. Electric organ discharge of Steutogenys ekguns. Recorded monopolarly along the side of the fish in a large dish of water. Time relations are taken from a simultaneous recording from a stationary electrode in the submental region. Records on the left are from the intact fish. The potentials are essentially diphasic in the tail region, but there is an early negative phase near the head (see Fig. 35). The early negative phase is lacking from the records on the right which were taken after removal of the rostral accessory organs. A minimum-sized potential is recorded 4 cm rostral to the tip of the tail and the diphasic potentials are of opposite sign on either side of this point.

vI msec

Fig. 31. Response of an electrocyte of the main organ of Steutogays eleguns. Recording and stimulating as indicated in the diagram. A brief stimulus is given to the nerve supply just after the beginning of the sweep and evokes a PSP just threshold for initiating a spike in the stalk (Vs, two superimposed sweeps). The spike of the innervated (stalk) face (Vt ) rises more rapidly and is briefer than that of the uninnervated face ( V , ) and the external potential ( V , ) is diphasic, initially positive outside the uninnervated face. The PSP is considerably larger in the stalk than across either face of the cell and the spike in the stalk is longer lasting.

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and rapidly propagates into the main part of the cell. The external record is initially positive on the side of the uninnervated face, which indicates that the spike in the innervated (stalk) face rises more rapidly, although this is not clear in the figure. The impulse in the uninnervated face then becomes larger and a phase of head negativity appears in the external record. This latter phase is considerably larger than the initial, head-positive phase. Steatogenys has an accessory electric organ in the chin region (Lowrey, 1913) which was called the submental filament by taxonomists ( Fig. 32). In Steatogenys elegans ( a relatively large species reaching 15-20 cm long) the submental organ consists of a single column of cells innervated on their anterior faces. The posterior part of the organ can be seen through the overlying epidermis; the anterior part lies more deeply in a fold of dermis and is not visible in the intact animal. There is an

,PO0

Fig. 32. Rostra1 accessory organs of Steatogenys elegans. ( a ) Appearance in intact fish; both postopercular organ (POO) and submental organ (SMO) are visible. ( b ) Overlying tissue is removed to show the full extent of the organs and the nerve ( N ) coming from spinal nerves to innervate them. The most rostra1 electrocytes of the main organ are also shown. Spinal cord, C ; main organ, MO; longitudinal nerve plexus, P; and rib, R.

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additional electric organ behind each operculum. The postopercular organ is for part of its length a double column of cells; all the cells are innervated on their posterior faces. These accessory organs are innervated by spinal nerves that run rostrally from the most anterior level of the main organ (Fig. 32). Electrocytes of the accessory organs have several stalks instead of the single one found in cells of the main organ ( Fig. 33). They are difficult to penetrate with microelectrodes because of the connective tissue surrounding the cells. However the isolated organs can be studied, and it can be concluded that they generate monophasic action potentials as do the cells of the eel and the monophasically discharging Hypopornus, and they have a similar marked proliferation of the uninnervated face ( Schwartz et al., 1971). When a column is stimulated by an axial current depolarizing the innervated faces, a monophasic spikelike potential is produced with a very short latency (about 0.1 msec in Fig. 34A). This response is positive with respect to the uninnervated face, and because of its short latency it can be identified as a directly excited spike. This potential is followed at a latency of about 1 msec by a smaller potential of similar sign that evidently represents PSPs and perhaps spike activity in the stalks. If a brief axial stimulus is given that hyperpolarizes the innervated face and depolarizes the uninnervated face, there is no short latency response indicating that the uninnervated face is inexcitable (Fig. 34C). This mode of stimulation does excite the nerve fibers; the delayed response is still present, but it becomes a full-sized spike since the innervated faces are not refractory as in Fig. 34A. The neural origin of the delayed response is confirmed by the effects of curare, a drug which blocks transmission at cholinergic synapses like those of nerve-electrocyte junctions. Following curare treatment the delayed response is absent for both polarities of stimulation, although the directly excited response persists unchanged (Fig. 34B,D). The accessory organs fire about 1 msec before the main organ (Fig. 35). The submental electrocytes are innervated on their anterior faces, and the potential external to the anterior end of the organ is largely negative. The postopercular electrocytes are innervated on their posterior faces, and negativity is recorded external to the posterior end of this organ. At the same time the potential in the gill opening and in the mouth is positive. These electrocytes act to make the interior of the head positive. If the accessory organs are removed, the discharge in the head region becomes the simple diphasic potential of main organ (Fig. 30). There is another much smaller species of Steatogenys (5-7 cm) which has submental electrocytes that are all innervated on a single stalk from their posterior faces. This species lacks a separate group of postopercular electrocytes. These cells also have a monophasic discharge and

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407

Fig. 33. Anatomy of submental electric organ of Steatogenys elegans. Romanes silver stain of paraffin embedded material. A longitudinal section, rostral to the right. Two electrocytes ( e ) are shown, each with several stalks ( s ) on the rostral face. Nerve fibers ( n ) come from the longitudinally running nerve trunk in the upper part of the figure to end on the stalks. The sheath ( s h ) around the organ is seen at the bottom.

function to make the interior of the head positive. The largest external potential is recorded at the posterior of the organ; there is very little potential at the anterior end of the organ under the chin. The direction of current flow with respect to the anterior posterior axis through this organ is opposite to that in the submental organs of Steatogenys elegans,

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Fig. 34. Response of the submental organ of Steatogenys elegans. Recording in a bridge circuit (Bennett, 1961). Rostra1 positivity up. ( A ) A brief stimulus depolarizing the innervated (caudal) faces evokes a brief spikelike potential at short latency followed by a smaller potential. ( B ) Following curarization ( about 0.1 mM) the brief spikelike potential is unaffected but the longer latency component is abolished. ( C ) Before curarization a brief stimulus hyperpolarizing the innervated faces evokes a response with a latency of about 1 msec. ( D ) Following curarization the same stimulus evokes no response.

but the transepidermal potentials are affected similarly because opposite ends of the organ are connected to the exterior and interior of the animal. The function of these accessory organs is presumably to increase sensitivity of the electrosensory system in the head region, but there are no experimental data.

e. Gymnorhamphichthys. Gymnorhamphichthys usually rests buried in sand during the day and emerges to swim around and feed at night. When in the sand it emits pulses at a quite constant rate of about 10-15/ sec. When actively swimming it increases its discharge rate to a new and also quite constant frequency which it maintains for prolonged periods. Disturbances also cause increase in discharge rate. It has been shown to exhibit a circadian rhythm of motor activity and (perhaps secondarily) of discharge frequency under conditions of constant darkness ( Lissmann and Schwassmann, 1965). The main organ of Gymnorhamphicthys is cytologically and, as inferred by recording from the intact fish, electrophysiologically like that of Steatogenys. Gymnorhamphichthys also has submental electrocytes as does Steatogenys. They are located beneath the dermis and thus are not obvious, which accounts for their not being seen previously. Each cell has multiple stalks from its posterior faces. External recording indicates that the submental cells fire before the main organ and generate mono-

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Fig. 35. Responses recorded in head region of Steatogenys elegans. Methods as for Fig. 30. The dotted lines indicate the sites of recording of the various traces. The three traces with arrows are recorded 2.5 cm rostral to the snout, 4.5 cm caudal to the snout and near the tip of the tail. There are large early negativities external to the rostral end of the submental organ and external to the caudal end of the postopercular organ. There are corresponding positivities just inside the mouth and gill opening.

phasic action potentials. The related genus Rhumphichthys has not been studied with respect to its electric organs. f. Sternopygus and Eigenmunnia. Sternopygus and Eigenmannia are closely related and the organ discharges are so similar that they may be considered together. The discharges consist of head-positive pulses superimposed on a head-negative base line such that there is little dc component in the organ discharge; that is, averaged over one discharge cycle, there is little or no net current flow (Fig. 3C). The frequency in Sternopygus is about 5&100/sec, and in Eigenmannia it is about 2506OOIsec. The frequency is quite constant, and ordinary forms of stimulation do not affect it. Weak electric stimuli of nearly the same frequency as the animal‘s own discharge evoke the “jamming avoidance” response, that is, a small shift in frequency away from that of the interfering stimulus (Watanabe and Takeda, 1963; Bullock, 1970; see Chapter 11, this volume). Sternopygus can also stop discharging completely under these conditions. The electric organ is located in the same position as in Hypopomus

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Fig. 36. Anatomy of the electric organ of Eigenmannia. Organ isolated from osniic acid fixed material. Dorsal up, rostra1 to the right viewed from the lateral surface. ( A ) Low power view. The five columns of electrocytes are numbered dorsoventrally. Two segmental nerves ( n ) run ventroposteriorly and give off branches to the electrocytes. A complete cell of column 4 is seen most clearly. The small spots on the cells are nuclei. The posterior, innervated ends of the cells are more darkly stained. There is little extracellular space between cells, and the cells usually overlap somewhat in the longitudinal axis. ( B ) The anterior end of a cell of column 3 lies lateral to the next cell anteriorly, and is similarly overlapped by the cell caudal to it. ( C ) Details of innervation of a cell from column 4. ( D ) Innervation of the anterior cell from column 5 in A. Magnifications the same in A and B and in C and D.

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and there is no specialized region rostrally. The electrocytes are tubular to spindle-shaped with the long axis parallel to the anteroposterior axis of the fish. The cells are 1-2 mm long and 0.2 mm in diameter in a Sternopygus 15 cm long and somewhat larger in an Eigenmunnia of similar size (Fig. 36). They are innervated on their posterior faces. There are about 6 columns on each side in Eigenmunnia and about 10 in Sternopygus. The cells are accurately aligned in columns in Eigenmannia, but they are separated by only small spaces; the amount of extracellular space between cells is much smaller than the cells themselves. The alignment is less regular in Sternopygus, but the amount of extracellular space is similarly small. It may not be obvious from records such as those of Fig. 3C that the discharge consists of head-positive pulses superimposed on a headnegative base line, and the distinction between pulse and interpulse interval may be even less obvious in the faster firing Eigenmunniu. The distinction is readily established experimentally (Fig. 37). An appropriately timed stimulus to the spinal cord can cause the complete disappearance of a pulse ( Fig. 37H) or cause a pulse to be generated earlier (Fig. 37C). The mechanism of block appears to be that the evoked volley in descending fibers finds the spinal neurons refractory from their immediately preceding response (initiated by the center in the medulla controlling the discharge, see Section 111, A), and the evoked volley also collides with the next command volley from the medulla preventing

.fuU-i i

Fig, 37. Effects of spinal stimulation on organ discharge of Sternopygus. Potential recorded differentially along caudal portion of tail, rostra1 positivity up. ( A ) Normal discharge. ( E I ) A brief stimulus t o the spinal cord is given at successively later times in the discharge cycle.

jlJUl -0 o

10 msec

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it from reaching the spinal neurons. The stimulus however has no noticeable effect on the following pulse indicating that the organ is controlled rostral to the spinal cord and that the antidromic volley does not invade the controlling nucleus. [There is actually a very slight advance of subsequent pulses ( see Bennett et d.,1967c).] In Sterrwpygus the head-negative potential between responses can also be demonstrated by section of the spinal cord which blocks further pulse activity ( Fig. 38). The head-negative potential remains immediately after spinal section but decays away over the next minute or so. Repetitive stimulation of the organ for some seconds causes at least a partial restoration of the head-negative potential which decays away again on cessation of stimulation [although somewhat more rapidly than following spinal section (Bennett, 196l)I. The origin of these potentials has been elucidated by microelectrode studies of the electrocytes. The pulse component of the organ discharge is generated by the posterior end of the cell, and the head-negative component is generated by the anterior end. The cells are long compared to

Fig. 38. Organ discharge of Sternopygus, effect of spinal section and repetitive stimulation. Potential recorded differentially along caudal portion of tail, rostral positivity up, zero level indicated by horizontal line and base line traces. Two preparations. ( A ) Two complete normal discharge cycles are shown on the left. The potential is head positive and head negative for approximately equal times. Spinal section abolishes the pulses, but a head-negative potential about equal to that during regular organ discharge remains (lower line at time 0 ) . This potential decays over approximately 2 min (right). ( B ) Stimulation of organ inactivated by spinal section. Separate stimulating and recording electrodes, head positivity up. Brief pulses are passed through a condenser to give diphasic stimuli with no dc component. The pulses are oriented so that the initial brief phase excites the innervated faces. Their response appears superimposed on the slow opposite phase ( left ). Repetitive stimulation at 50/sec for 15 sec develops a head-negative potential that on cessation of stimulation decays over 15-20 sec (right). From Bennett (1961).

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Fig. 39. Spikes and PSPs in an electrocyte of Sternopygus during organ discharge. Two intracellular recording electrodes are used, one 700 p rostral to the other. Both record differentially against a closely applied external electrode to minimize pickup from other cells. The second trace gives the potential at the caudal electrode with the upper trace as its zero level. The fourth trace gives the potential at the rostral electrode with the third trace showing its zero level. When the cell is firing regularly, as in A, the spikes and the potential between spikes are greater at the caudal end. Repeated hyperpolarizing pulses are passed through a third intracellular electrode in order to block the spikes and, after some seconds, the records become as in B. The resting potential is equal at the two ends of the cell and only PSPs are produced, which are larger at the caudal end. From Bennett (1961).

cells of weakly electric gymnotids discussed up to this point, and the potential can be quite different at the two ends of the cell. The spike and PSPs are larger at the posterior end of the cell (Fig. 39), and the threshold current is lower when applied through an electrode at this end of the cell. Longitudinal stimulation as in Figs. 21 and 25 has not been carried out. However, in the caudal filament there is little tissue other than the organ, and it can be stimulated by external electrodes. This procedure shows the posterior faces to be capable of generating a spike and the anterior faces to be inexcitable just as demonstrated for the submental organs of Steatogenys (Fig, 34). In a cell that has not been stimulated for some time, the resting potential is the same at the two ends of the cell. Repetitive stimulation causes a depolarization of the cell to develop, but the depolarization is greater at the anterior end of the cell (Fig. 40 ). On cessation of stimulation the potential of the two ends of the cells slowly equalizes again. Similar changes are seen in neural activation (Fig. 39). From these records it is clear that, during spikes, current flows from caudal to rostral in the interior of the cell, thus generating the head-positive phase of the organ discharge. When the cell has been active at the normal frequency for a sufficient period, current flows caudally in the cell in the interval between spikes thus generating the head-negative component of the overall discharge. Insufficient experimental data are available to warrant a deailed discussion, but a possible explanation of the depolarization of the anterior face can be given. During the spike sodium current flows inward through the innervated face while outward current at the anterior end is carried

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

....... ........................ ..... Peak of spike

-5

I

Fig. 40. Development and decay of slow depolarization produced by repetitive direct stimulation of a single cell of Sternopygus. Organ inactivated by spinal section. Three electrodes are used as in diagram. Brief pulses adequate to excite the cell are applied at l/sec and 50/sec. Sample records are shown above graph with the connecting lines indicating when they are taken. Upper traces: potential at the caudal end of the cell and base line. Lower traces: potential a t the rostral end of the cell and stimulating current serving also as a base line. Graph: data of complete experiment, resting potentials at the beginning taken as 0. Open circles: caudal. Crosses: rostral. Resting potentials determined at the end of the experiment are 64 mV at the rostral electrode and 67 mV at the caudal electrode. Repetitive stimulation at 50/sec causes a steady level of depolarization to develop, which is greater at the rostral end of the cell. On return to stimulation at l/sec the depolarization subsides in about 20 sec. From Bennett ( 1961).

by potassium ions. Potassium accumulates in the restricted extracellular space external to the anterior face and tends to depolarize it. This mechanisni of ion accumulation provides an explanation of why there is so little extracellular space in this organ in contrast to those of other electric fish previously discussed. The question remains as to what ions carry the outward current through the innervated face during the headnegative phase of the organ discharge. It cannot be potassium if the concentration of this ion is the same outside the two faces as it would be except for cells at either end of the organ. Spike generation in the electrocytes has an unusual feature: The resistance during the peak of the spike exceeds that at rest by a factor of 1.5 to 2. This property may be seen from the application of brief pulses between the spikes and at their peaks (Bennett, 1961) and is confirmed by ac impedance measurements from the cells of the caudal filament (Fig. 41). The sequence of conductance and permeability changes is probably as follows. During the rising phase of the spikes the conduct-

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20 rnsec

Fig. 41. Impedance changes during organ discharge of Sternopygus. The tip of the tail was placed in a bridge circuit. Upper trace: bridge signal after moderate amplification. Lower trace: bridge signal after filtering out low frequencies and much greater amplification. Amplitude and frequency of ac input 0.5V and 5 kc. The bridge could be approximately balanced for both spike peaks and between spikes ( A ) , but large imbalances occurred on the rising and falling phases. When the bridge was balanced for the falling phase ( B ), the imbalance at the spike peaks was somewhat greater than between spikes, and balance was approached during the rising phase. From Bennett ( 1961).

ance rises as a result of sodium activation. The conductance then decreases again at the spike peak because the conductance of anomalously rectifying membrane is a large fraction of the resting conductance and decreases more than enough to compensate for the increase in sodium conductance. Sodium inactivation ensues and the potential begins to fall. However, on the falling phase the conductance rises to above the maximum observed on the rising phase; this change is ascribable to potassium activation or delayed rectification. Except for the conductance increase on the falling phase this pattern of changes is like that in the electric eel for which the data quite adequately demonstrate the proposed mechanism (see Nakamura et d., 1965; Morlock et al., 1969). The similar conductances during spikes and at rest may be functional in maintaining an ac organ discharge. Exposure to media of different conductivities will load both phases of organ discharge similarly. Therefore less dc component will be associated with the new amplitude of organ discharge than there would be if the conductance during spikes were much lower than that between spikes. Conductance changes associated with organ discharge are the same in Eigenmunnia and in Sternopygus. Insufficient physiological data are available to distinguish whether the absence of a dc component in the discharge results from a mechanism like that in Sternopygus or whether the uninnervated face acts as a series capacity as in Gymnarchus and probably also the sternarchids (see Sections 11, D, 3 and 11, D, 1, g ) . The two different mechanisms might be characterized as a polarization

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capacity in contrast to a true membrane dielectric capacity. Ultrastructural data suggest that Eigenmannia may employ a dielectric capacity. The uninnervated face of its electrocytes has many fine branching tubules or canaliculi that greatly increase the surface; the situation thus resembles that in Gymnurchus (Schwartz et al., 1971). In Sternopygus, the innervated and uninnervated faces are similar and rather smooth, their areas being little increased compared to surface seen at the light microscope level of resolution. ( A question here is why there is little extracellular space in the electric organ of Eigenmanniu.) The significance of an ac discharge will be discussed in Section 11, F.

g. Sternarchids. This is the largest of the gymnotid families in terms of numbers of genera and species (Tables I and 11).The group is characterized by the presence of a small caudal fin. There is also the so-called dorsal filament that arises about the middle of the back and runs posteriorly for perhaps a third of the body length (Fig. 42). The dorsal filament is in vivo closely adherent to the back of the fish, and back and filament are contoured so that the filament is practically invisible; it may separate and become obvious in preserved specimens. The sternarchids discharge their electric organs at the highest fre-

--

=n=mmrnmm---m1--Enlarged nodes

Fig. 42. Anatomy of the electric organ of Sternarchus. The upper diagram shows the location of the organ in the fish. The middle diagram shows the organ and nerves running to it and the course of a single fiber from its origin in the spinal cord. The lower diagram represents a single fiber and its nodal structure.

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quency of any known electric fish. Depending on the species, individual, and temperature the frequency ranges from about 700 to 1700 impulses per second ( Steinbach, 1970). The discharge shape varies somewhat with the species, and will be treated separately below. The frequency is generally highly stable and is not affected by ordinary stimuli. Weak electric stimulation at closely neighboring frequencies does evoke small shifts in frequency, the “jamming avoidance” response ( Larimer and MacDonald, 1968; Bullock, 1970; see Chapter 11, this volume). Several species have been observed to accelerate their discharge briefly, emitting a “chirp” (Bullock, 1970). These changes may function in intraspecific communication and in species recognition. The electrocytes of the sternarchids turn out to be spinal neurons rather than cells of myogenic origin (de Oliveira Castro, 1955; Bennett, 1966, 1970). The best-studied species is Sternarchus albifrons because this is the one most commonly obtained by tropical fish dealers who call it the black ghost knife fish. The fish, electric organ, and electrocytes are shown diagrammatically in Fig. 42. The organ runs longitudinally just ventral to the spinal column over most of the length of the body. The axons of the spinal neurons descend from the cord and enter the electric organ (Fig. 43). They then run anteriorly for several segments, turn around and run posteriorly to end blindly at about the same level that they entered the organ. This course is established by dissection of single fibers from their point of entry into the organ until their termination. Evidently the organ has lost its myogenic electrocytes and enlarged the axons that formerly innervated them. This origin is confirmed by the existence of a nerve plexus in Gymnotus, Hypopomus, Steatogenys, and Gymnorhumphichthys in the same location as the sternarchid electric organ. In Gymnotus axons in this plexus have been traced to the (myogenic) electrocytes. The plexus is absent in Sternopygus, Eigenmannia, and Electrophorus, a fact which has implications for the phylogenetic relations within the gymnotid group. There are characteristic changes in the nerve fibers as they run along in the electric organ. In both its anteriorly and posteriorly running parts it becomes greatly dilated and can exceed 100p in diameter. Before it enters the organ and where it turns around to run posteriorly, it is of the usual diameter for a large myelinated fiber, 10-2Op. It tapers gradually before it terminates. The nodal structure also changes characteristically along the fiber (Fig. 44). The nodes become somewhat enlarged just after the fiber enters the organ. As the fiber dilates, the nodes become very narrow (although area is difficult to estimate because of the increased fiber diameter). As the fiber tapers near its most anterior part, there are several very long nodes. The nodes become of ordinary size again as the fiber turns around. This sequence repeats itself in the

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caudally running section of the fiber. There are first moderately enlarged nodes, then narrowed ones, then very enlarged ones. The organ discharge of Sternarchus consists of diphasic pulses that are initially head positive (Figs. 3D and 45). As would be expected from the neural origin of the electric organ, the discharge is unaffected by a dose of curare that is sufficient to paralyze the animal completely. The contribution of the single cells to the organ discharge is disclosed by microelectrode experiments. Just anterior to where the fiber enters the organ, a large impulse is recorded in the fiber during the headpositive phase of organ discharge (Fig. 46A). This impulse is smaller more anteriorly and the peak is delayed; thus, like the cells of Sternopygus, these cells are not isopotential. The response in this part of the fiber accounts for the head-positive phase of the discharge because current runs anteriorly along the interior of the cell at this time. In the posteriorly running section of the fiber the impulse is larger anteriorly (Fig. 46B). This activity occurs during the head-negative phase of the organ discharge and current flows in the cell in such a way as to give rise to the head-negative phase. The anatomy and recordings indicate that impulses coming from the spinal cord propagate along the electrocyte in the organ and that the activity of the anteriorly running part excites the posteriorly running part. Because the impulse is smaller at the distal end of each section of the fiber, it is likely that these regions are inexcitable like the uninnervated faces of eel electrocytes. The current paths and intracellular potentials during one discharge cycle are diagrammed in Fig. 47. There is evidence that the inactive regions of the cells act as a series capacity rather than as a series resistance. The effects when one face of an electrocyte acts as a capacity are discussed in detail in respect to Gymnarchus, for which the experimental evidence is more complete (Section 11, D, 3 ) . The most important effect is that the organ discharge Fig. 43. Electric organ of Sternarchus. Formalin fixed material, dissected and stained with methylene blue. Dorsal to the top, anterior to the right. ( A ) The electric organ ( 0 )following removal of its connective tissue sheath. Single longitudinally running electrocytes are visible. Nerves to the organ ( n ) and to more ventral tissues ( s ) are also seen. ( B ) Following dissection away of all electrocytes from nerves caudal to those shown and of most fibers entering the organ from the more caudal nerve in the figure. Three descending fibers of this nerve run approximately parallel in their origin position (arrows); two others are deflected anteriorly; the central part of a sixth is broken off shortly after entry into the organ (arrow). The more anterior nerve is undissected; its fibers diverge in the organ. Most of them lie medial to the fibers from the more caudal nerve. ( C ) Higher magnification of the left part of B. The five numbered fibers can be traced from the top left to the lower right. The smaller fibers of the ventral segmental nerve ( s ) are visible. Fiber 4 passes medial to this nerve. Calibrations on the lower right.

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Fig. 44. Structure of Stemarchus electrocytes. (A-D) Photographs of a cell isolated by dissection following osmic acid fixation. ( A ) The fiber near its entry into the organ; 4 nodes (arrows) are visible; the 3 to the left appear somewhat enlarged. ( B ) A dilated part of the fiber in its longitudinally running course; 2 nodes of peculiar structure are seen (arrows). ( C ) The thin region of the fiber where it turns

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0.5, 2.5 msec

Fig. 45. Electric organ discharge of Stemarchus. Recorded differentially between head and tail at two sweep speeds. Upper records; prior to curarization. Lower records: after a dose of curare (10 mg/kg) that completely paralyzed the fish, organ discharge was unaffected.

has no dc component. At a constant frequency of activity current flows as follows. During a spike at one end of the cell the charge on the capacity of the other end is made more positive from the negative resting level; after the spike the capacity recharges to its original level, and no net current flows over the discharge cycle. The evidence for this property in sternarchids is largely comparative (M. V. L. Bennett and A. B. Steinbach, unpublished data). The organ discharge of all sternarchids has little or no dc component. In Sternarchus this could result, as in Gymnotus, from successive firing of opposite faces or ends of the cells. However, in Sternurchorhamphus the organ discharge consists of monophasic head-negative pulses superimposed on fairly level head-positive base line with no dc component (Fig. 48A,B). The electrocytes lack a rostrally running portion; the fibers turn and run caudally on entering the organ. The monophasic appearance of the discharge is consistent with activity of only the rostra1 regions of the fibers. The head positivity between impulses could be generated by a polarization capacity as in Stemopygus. It seems more likely that the mechanism is like that of around to run posteriorly again; 5 nodes are seen; the 2 to the right appear somewhat enlarged. ( D ) The caudal termination of the fiber, at least three very large nodes (double arrows) and 3 small internodes ( i ) are seen. The nature of the adhering structures near the fiber termination is unclear. (E-F) Toluidine blue stained sections of osmic acid fixed Epon embedded material. ( E ) Arrows indicate a large node in the dilated fiber on the right and a small node in the dilated fiber on the left. ( F ) Arrows indicate a node in a narrow region of a fiber that is turning around a t the anterior limit of its course. Note that the other fibers in this section are cut transversely. Magnifications the same in A-l3 and in E-F.

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I msec

Fig. 46. Intracellular recordings from fibers in the electric organ of Sternarchus. Differential recording with respect to a closely applied external electrode to minimize contributions from other cells. Upper trace: monopolar recording of organ discharge. Middle trace: intracellular recording from anterior electrode. Lower trace: intracellular recording from an electrode in the same fiber several millimeters posteriorly. ( A ) Recording from an anteriorly running fiber segment as indicated by the larger size and earlier peak of the potential at the posterior electrode. The impulse occurs during the initial phase of organ discharge (negative going when monopolarly recorded at this level of the organ). ( B ) Recording from a caudally running fiber segment as indicated by the larger size and earlier peak of the potential at the anterior electrode. The impulse occurs during the second phase of organ discharge.

Gymnurchus, and the head-negative phase results from charging of the dielectric capacity of the caudal portion of the fiber. That the potential between pulses is quite level only requires that the time constant of the system be long compared to the interval between pulses.

Fig. 47. Diagram of potentials and current flow at successive regions of the electrocytes during activity. The upper potential tracing represents an organ discharge. The directions of current flow during the spikes in the different regions are indicated by the arrows on the right.

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In some Adontosternarchus the main organ discharge has a small head-negative phase followed by a large head-positive phase separated by a more or less level base line. However, the base line is head positive so that again there is no net current flow during a discharge cycle (Fig. 48C,D). The electrocytes have both rostrally and caudally running portions, but the rostrally running section is shorter than the caudally running section. Thus, the electrocytes present an intermediate condition between those of Sternarchus and Sternarchorhamphus, The small headpositive phase of the pulse is ascribable to the small anteriorly running portion of the fiber; the large head-negative phase is ascribable to the large posterioriy running portion. The most likely way for the headnegative level between pulses to arise is for each portion of the fiber to have a series capacity. These data do not require Sternarchus electrocytes to have a series capacity. However, if the fish is made anoxic the second phase of the organ discharge appears to fail. In spite of this change, the organ discharge still has no dc component. It seems probable then that both anteriorly and posteriorly running regions have a series capacity. In some individual Sternarchus the initial, head-positive phase is larger than the second, head-negative phase, although there is still no dc component. Correspondingly in some individuals, presumably the same ones, the rostrally running portion of the electrocytes is larger than the caudally running

6 . 2 . 5 mSec

Fig. 48. Organ discharge of Sternurchorhamphus and Adontosternarchus. (A, B ) Recorded monopolarly at the tail of Sternurchorhamphus, positivity down (equivalent to head positivity u p ) . ( C, D) Recorded differentially between head and tail of Adontosternarchus, head positivity up. Horizontal lines indicate zero potential levels. Faster sweep speed in A and C.

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portion. Unfortunately, it is not possible to test these mechanisms by activating the organ over a wide range of frequencies. The cells continue to fire spontaneously after spinal section, and synchronous low frequency activity cannot be obtained. One sternarchid, Adontosternarchus, has an accessory organ in the chin region (M. V. L. Bennett and A. B. Steinbach, unpublished data). This organ generates a potential of 20-25 mV amplitude outside the chin (Fig. 49). It is absent from the five or more other genera of sternarchids examined on the Rio Negro expedition of the R. V. Alpha Helix. It is made up of fibers that are probably modified from electrosensory nerve fibers for the fibers end in the skin in what appear to be modified electroreceptors. Consistent with its sensory origin the impulse frequency is set in the chin organ itself, and impulses proceed centripetally in the afferent fibers which can be sectioned without affecting organ discharge. Because of its peripheral origin the chin organ can fire at a somewhat different frequency from the main organ (Fig. S o ) . Since electroreceptors themselves can, under certain conditions, generate maintained oscillations (see Chapter 11, this volume), it is necessary to distinguish the neurogenic potentials of the chin organ from potentials generated by electroreceptors that of course the fish also possesses. The most important difference is morphological. The fibers of the chin organs have very dilated myelin sheaths and peculiar nodal structures that closely resemble the characteristics of electrocytes in the main organ. Electroreceptor afferent fibers are ordinary myelinated fibers. Futhermore, if an electrode is advanced into the organ from the surface, the polarity of discharge does not invert until the electrode is deep into the tissue of the chin. In contrast, the oscillations at electroreceptors invert when the epidermis is crossed. Finally, the chin organ oscillations are little affected by external loading; the frequency is nearly constant whether the chin is in air or immersed in physiological saline. In contrast, electroreceptors do not oscillate when they are electrically loaded to only a small degree; generally, they will oscillate only when the skin is allowed to dry in air.

Fig. 49. Main and accessory organ discharges of Adontostemarchus. Recorded monopolarly with respect to a distant electrode in a large volume of water. There is a relatively large potential in the chin region ( a ) and at the tip of the tail ( h ) . The potentials are smaller elsewhere over the head (b-e) and near the middle of the body (f, g ) .

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A

B

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0.5 msec

Fig. 50. Lack of synchronization of main and accessory organs of Adontosternarchw. Recording monopolarly from chin organ (upper trace) and tip of tail (lower trace). ( A ) When the sweep is triggered by the accessory organ discharge, repetitive superimposed sweeps show that the phase of main organ discharge changes. ( B ) When the sweep is triggered by the main organ discharge, the phase of the accessory organ discharge changes in different sweeps.

While measurements to date are inadequate, it is probable that discharge of the chin organ has little dc component. The function of the chin organ is obscure, but the potentials produced are large enough to activate receptors in the head region. Presumably the chin organ plays the same role as the rostra1 accessory organs of other gymnotids. 2. THEELECTRIC CATFISH The electric catfish, Malupterurus electricus, is the only silurid known to be electric. It is also distinguishable from other catfish by the absence of rays in its dorsal fin. The electric organ lies in the skin surrounding the body over most of the length of the fish (Fig. 1). It is innervated by two giant neurons, one on either side of the spinal cord in the first spinal segment (Fig. 72). Each neuron sends out a single axon that innervates the several million electrocytes on that side of the body. The electrocytes are shaped rather like lily pads. The main part of the cell is disc-shaped, about 1 mm in diameter and 2040 p thick in a fish 15 cm long (Fig. 51). From a convoluted region in the center of the caudal face (called the rosette), a stalk protrudes that is about as long as the radius of the disc-shaped part. The cell is innervated on the tip of this stalk by a branch of the axon from the giant cell. The electric organ of Malapterurus was long thought to be of glandular origin, in part because of its location and because the side opposite to the innervation of the electrocytes became negative. It thus violated the rule formulated by Pacini concerning innervation and polarity (who

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Fig. 51. Anatomy of the electric organ of the electric catfish. ( A ) The dissected ventral surface of the fish showing the organ ( 0 )and the nerve ( n e ) , artery ( a e ) , and vein ( v e ) running to it (from Rosenberg, 1928). ( B ) The body of a single electrocyte dissected out following forrnalin fixation, and stained with methylene blue. ( C ) Higher magnification of the rosette region of another cell, Romanes’ silver stain.

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is better known for the corpuscles that bear his name) and generated potentials similar in sign to those known from some glandular tissue. It turns out that the discharge polarity is explicable in terms of ordinary mechanisms of excitability (Keynes et al., 1961), and recent physiological and embryological data are consistent with a muscle origin for the organ (Johnels, 1956). The organ discharges are primarily head-negative pulses 1-2 msec in duration (Fig. 3A,A'). The head-negative pulse is preceded by a very much smaller head-positive phase that was not observed in earlier studies (Fig. 52). This early phase results from activity of the stalks as will be discussed below. Thus, the organ really does not violate Pacini's rule. The pulses are emitted infrequently by an undisturbed fish. However, when the animal is feeding or mechanically stimulated a few pulses or long trains of pulses can be emitted. Prey detection appears to precede pulse emission and the organ presumably functions in prey capture (Bauer, 1968). Inputs from taste receptors, which are

i 1.5 rnsec

Fig. 52. Electric organ discharge of the electric catfish. Recorded between head and tail of a fish about 15 cm in length in a small container of aquarium water. High gain (upper traces) and low gain (lower traces), head negativity upwards, faster sweep in A. Two superimposed sweeps, one with and one without discharges to show base line. The primary head-negative discharge is preceded by a small head positivity. Single pulses ( A) or trains ( B ) are emitted in response to touch.

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found over much of the body surface, are powerful excitants. However, the effectiveness of the discharge in stunning small fish is minimal. The amplitude recorded in air is about 150 V from an animal 30 cm in length and even a small fish 5 cm long can generate 30 V (Keynes et al., 1961). The amplitudes are considerably reduced in water but are still uncomfortably strong to someone trying to pick up the fish. The fish reaches a length of at least 50 cm, and 350 V discharges have been reported ( Keynes, 1957). It is interesting that the cells produce an almost entirely head-negative potential, although they are innervated on their posterior faces; in fact, the mechanism is not so different from that in the diphasicly discharging Hypopornus (Fig. 24). For the experiments of Figs. 53 and 54 a propagated impulse is set up by stimulation through a large monopolar electrode pressed against the main part of the cell distant from the recording site. A monopolar recording electrode detects a negative spike external to the nonstalk face (Fig. 53A) that is propagated in from the stimulation site. If this electrode is advanced into the cell, it records an inside negative resting potential and an overshooting spike (Fig. 53B). Further advance of the electrode through the cell causes a loss of resting potential and recording of a positive external potential associated with the spike. Data of these kinds show the electrocyte activity to be of conventional polarity, but the shape of the external potentials is unlike that in other fish discussed up to this point.

1 msec

Fig. 53. Responses of a single electrocyte of the electric catfish. Monopolar recordings during advance of a microelectrode through the single cell. The horizontal trace shows the d c level. Stimulation by an external electrode distant from the recording site and recording as indicated in the inset diagram. A long-lasting pulse is used to minimize interference by stimulus artifacts. ( A ) Recorded outside the rostral face, the response is largely negative but is preceded by a small positive phase. ( B ) When the electrode is advanced into the cell, it records an internally negative resting potential and an overshooting spike with a shoulder on the falling phase. ( C ) When the electrode is further advanced to lie outside the rostral face, the resting potential disappears and a positive going response is recorded. Both external recordings have inflections on their rising phases at the time of the brief peak of the intracellular recording. From Keynes et al. ( 1961).

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The origin of the peculiar shape of the external potential becomes clearer in differential recordings (Fig. 54) from which it appears that the stalk face generates a small brief spike (followed by a much smaller hump) and the nonstalk face generates a larger and longer lasting spike. The sequence of potential changes during propagation of a spike thus is as follows (see Fig. 4 E ) : outward current depolarizes each face, and the potential is initially positive outside each face as seen in the monopolar recordings of Fig. 53A,C. But negligible potential is produced across the cell because the positivities are about equal. The nonstalk face, being of lower threshold, begins to pass inward current that flows out through the stalk face depolarizing it further; the external potential becomes positive outside the stalk face and negative outside the nonstalk face. When the stalk face is excited, its activity opposes the spike of the noiistalk face, and, as a result, there is a reduction in the external potential or at least in its rate of rise. The spike in the stalk face then terminates while the spike in the nonstalk face continues. The external potential becomes much larger and then declines as the spike terminates. This sequence of firing also explains the shape of the spikes in the two faces. The spike in the nonstalk face often has two peaks; the larger potential generated initially is ascribable to reduced loading when the two faces fire together. Evidently this first maximum need not be accompanied by maximal sodium activation because the potential can rise to a second maximum after the brief spike of the stalk face. The A

B

Fig. 54. Responses of a single electrocyte of the electric catfish. Monopolar and differential recordings from three separate electrodes as diagrammed, stimulation by a distant external electrode. ( A ) The monopolar recordings are similar to those in Fig. 53. ( B ) The differential recordings show the external response to be monophasic head negative with an inflection on the rising phase ( V i ) , the response of the rostra1 face to be a relatively long-lasting spike with two peaks ( V 2 ) , the response of the caudal face to be a brief spike with a small longer lasting and probably passive component ( V3). From Keynes et aZ. ( 1961).

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residual potential across the stalk face following its brief spike is ascribable to voltage drop across this face produced by activity of the nonstalk face. What is the significance of this sequence of activity? The resistance of the two faces at rest is about equal. The brief spike in the stalk face is apparently a rapid way to turn on delayed rectification in this membrane, and by increasing its conductance to increase external current flow. Electron microscopic observations show that both faces have a moderate number of branching tubules that increase their surface areas (Mathewson et al., 1961). The proliferation is slightly greater in the nonstalk face. The similarity in areas is consistent with the suggestion that both surfaces increase their conductances during a response. Actually, the stalk face may not be capable of generating an all-or-none response. The response of this face to depolarization (applied by an external electrode) apparently is graded, and a propagated spike cannot be set up by external stimuli that depolarize this face and hyperpolarize the nonstalk face. It is even possible that this membrane only exhibits delayed rectification and totally lacks an inward current mechanism. The initial maximum seen in some external records would then be ascribable to capacitative current as in cup-type electrocytes of rajids (Fig. 15). The steep rise and fall of the potential across the nonstalk face makes this explanation unlikely, but it does not contradict available data. From the location of the synapses and the observation that spikes can propagate along the body of the cell, it appears that a PSP sets up a spike in the stalk that propagates to invade the rest of the cell. As would be expected, neurally evoked responses in the body of the cells are similar in shape to directly evoked ones. Transmission at the synapses is cholinergic in that curare blocks neural excitation of the organ and a high concentration of cholinesterase is found histochemically at the synapses ( Couteaux and Szabo, 1959). Activity of the stalk can be recorded by electrodes placed (under visual control) close to the rosette, i.e., the site where the stalk joins the disc-shaped part of the cell. External stimulation in this region can excite the cell even if the polarity is such as to depolarize the stalk face (Fig. 5 5 ) . External to the rosette opposite the stalk (upper trace) an initially positive potential is observed apparently resulting from activity of the stalk, because it is much smaller a short distance away (lower trace). If the stimulus strength is increased, repetitive firing is produced (Fig. 53B), which is never observed with stimulation of either polarity distant from the rosette. If the stimulus strength is increased further, the impulses fail to invade the body of the cell, but small biphasic

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external potentials remain that are initially positive opposite to the stalk (Fig. 55C,D). Evidently the hyperpolarization of the nonstalk face is so large that the stalk impulse cannot excite it. The predominance of the negative phases in the external records indicates that some excitation of membrane on the nonstalk side still occurs, although the activity does not propagate to the remainder of the cell. However, if the stalk impulse occurs at the end of the stimulus when hyperpolarization of the nonstalk face is terminated, the impulse in the stalk becomes able to invade the body of the cell as indicated by a large external response (Fig. 55D). A stimulus over the rosette that depolarizes the nonstalk face can also excite the cell, but the threshold is higher than for the opposite polarity of stimulation (Fig. 55E). When excited in this way the component of the external potential associated with firing of the stalk face is larger than when recorded distant from the rosette (Figs. 53 and 54). One may question why the inflection that is seen on the rising phase of responses externally recorded close to single cells is generally not seen in the organ discharge. Since propagation time over the cell is an

Fig. 55. Responses recorded near the stalk of an electrocyte of the electric catfish. External recording and stimulation as in the diagram, upper trace nearer the center of the rosette. Anodal stimuli outside the rostra1 face except in E. ( A ) A single largely negative spike is evoked that is preceded by a small but distinct positivity at the more central electrode. ( B ) A stronger stimulus evokes two responses. ( C ) A still stronger stimulus evokes two responses of the stalk but invasion of the body of the cell is delayed and blocked for the first and second responses, respectively. ( D ) A stronger stimulus evokes three responses of the stalk, but the first two fail to invade the body of the cell. The third occurs a t the end of the stimulus and does invade the body of the cell. ( E ) Cathodal stimulation can excite the cell at this site, but the threshold is higher than for anodal stimulation.

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appreciable fraction of the duration of the spike of the stalk face, the activity of the stalk face is not synchronous in the different regions of the cell. It thus fails to produce a distinct component in the overall organ discharge which at any instant is an average of the contributions of all regions of all the cells.

3. Gymnurchm The monospecific genus Gymnarchus is found in tropical Africa. It is closely related to the Mormyridae, but the mode of swimming is remarkably similar to that of gymnotids. The animal moves with a straight body by undulations of the dorsal fin. As far as locomotion is concerned, it is like an upside-down gymnotid (Fig. 1 ) . Movement appears equally easy forward and backward, and often the animal seems to investigate strange objects with the tip of its tail. The electric organ pulses are emitted at a frequency of about 25O/sec (Fig. 56). The frequency is not altered by ordinary kinds of stimuli, and “jamming avoidance” has not been observed ( Bullock, 1970). Novel stimuli that perhaps startle the fish may evoke a sudden cessation and weak electric pulses may also be effective (Lissmann, 1958; Szabo and Suckling, 1964; Harder and Uhlemann, 1967). The electric organ of Gymnurchus consists of four columns of electrocytes on each side of the body, one above the other. Each column runs to the tip of the caudal filament but their anterior extent varies (Fig. 1). The electrocytes are flattened cylinders innervated on their posterior faces by spinal nerves. Both faces are moderately convoluted. However, on a microscopic scale the uninnervated face has a large number of small canaliculi and processes that greatly increase its surface, while

-

1.2 rnsec

Fig. 56. Electric organ discharge of Gymnarchus. Recorded from an animal about 30 cm in length in a small container of aquarium water. Zero potential level indicated by the horizontal line. (A, B ) Normal organ discharge at fast and slow sweep speeds.

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the innervated face is relatively smooth and has only a few canaliculi (Schwartz et al., 1971). The electric organ discharge consists of what appear to be headpositive pulses superimposed on an almost level base line (Fig. 56). However, the potential goes head-negative between pulses and as in Sternopygw, Eigenmannia, and sternarchids there is virtually no net current flow during one complete organ cycle. (There is a small headpositive bump between the large pulses; this component is apparently a result of nerve activity.) The absence of net current flow is a result of the properties of the uninnervated face of the electrocytes. The innervated face generates an ordinary spike when depolarized either synaptically or by applied currents. The uninnervated face has a large capacity and is of high resistance and inexcitable. Current flow through it is virtually entirely capacitative. During a spike in the innervated face the charge on the capacity of the uninnervated face is made more positive; between spikes the charge on the capacity tends to return to its initial value. The resulting external potential is diphasic and initially positive outside the uninnervated face. The sequence of potential changes and the cell equivalent circuit are shown in Fig. 3E. The external medium behaves like an ohmic resistance (at the frequencies of the electric organ discharge). The external voltage is then proportional to the amplitude of transmembrane current, and the integral over time of the external voltage gives a measure of total current flow. Since the charge on the capacity of the uninnervated face before and after a spike becomes the same during a steady frequency of firing, no net current can flow through the capacity and the time integrals of the positive and negative phases of the external potentials must be equal. This they are observed to be to within the 1 or 2%accuracy of the measurement. The response of the single cells to depolarization applied by an intracellular electrode is shown in Fig. 57 recorded both monopolarly (Fig. 57A,B) and differentially (Fig. 57C,D). The monopolarly recorded potentials external to the two faces are diphasic and opposite in sign (Fig. 57A,B) establishing the longitudinal orientation of the cells. The potential outside the innervated face is initially negative indicating that this face becomes active. The differentially recorded potentials show a simple spike potential across the innervated face. The potential across the uninnervated face is also a monophasic depolarization, but its peak is much lower and somewhat later. During the falling phase, however, the potential across this face exceeds that across the innervated face, which accounts for the head-negative phase in the

434

M. V. L. BENNETT f i n t e r i 7 6

B-L-

,4---

2 ( ! !

r Posterior

i

L

u8-67--

-r-Q

Anterior

r

V"

-J-

Poster lor

2 rnsec

4 rnsec

Fig. 57. Responses of a single electrolyte of Gymnarchus. Recording and stimulation as indicated in the diagrams. Faster sweep in A and C. ( A , B ) Outside the two faces monopolar recordings are diphasic and of opposite polarity. (C, D ) Differentially recorded the response across the innervated face is a large monophasic spike. The potential across the uninnervated face V, has a much smaller peak amplitude but decays away more slowly. The resulting external potential is diphasic initially head positive.

external records. It is not obvious from these data that the uninnervated face is purely passive and acts as a series capacity. These properties are established by passing longitudinal currents with an external stimulating electrode (Fig. 58). The results may be understood with respect to the equivalent circuit shown in Fig. 59. If a long-lasting pulse of either sign is applied, there are transient potential changes across the innervated face at the onset and termination of the pulse, but the potential across this face is back very close to the resting value after 20 msec. After the capacity of the uninnervated face is charged to its new value, no current flows through it and there is no voltage drop across the resistance of the innervated face (Fig. 58A). For pulses of this magnitude records for either direction of current are symmetrical. When depolarized by over 50 mV the uninnervated face is affected by a small pulse of current exactly as when it is at the resting potential (Fig. 58B). Both these results indicate the passivity of the uninnervated membrane. Although no steady state voltage drop is produced across the innervated face by maintained current pulses applied externally, there are transients associated with charging and discharging the uninnervated face at onset and termination of the currents. If an anodal stimulus is applied outside the uninnervated face, the innervated face is depolarized

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Anterior

-3

4% F"

Posterior

Fig. 58. Responses of an electrocyte of Gymnarchtm to externally applied currents. Stimulation external to the uninnervated face and differential recording as indicated. ( A ) Oppositely directed current pulses of approximately equal amplitude produce approximately symmetrical potentials across the two faces. ( B ) When a brief pulse is superimposed on a long-lasting pulse that depolarizes the uninnervated face by about 50 mV, the brief pulse produces about the same change in potential as when it is given alone. Four superimposed sweeps with one, both, and no pulses. ( C ) The onset of an anodal stimulus depolarizes the innervated face; superimposed sweeps of threshold stimulation. ( D ) The termination of a cathodal stimulus depolarizes the innervated face; the threshold amplitude for stimulation by a longlasting stimulus is the same as in C .

by the initial surge and hyperpolarized following termination of the stimulus (Fig. 5 8 C ) . If the stimulus is cathodal, the innervated face is hyperpolarized initially and depolarized after the termination of the stimulus (Fig. 58B). Provided the pulses are long lasting compared to the time constant of the system, the voltage across the innervated face must be identical at onset and termination of stimuli of equal amplitude but opposite polarity. This is experimentally true for Gymna~chuselectrocytes; in Fig. 58 the threshold for firing at the onset of a cathodal 1-9 Anterior, uninnervoted face.

External medium

R, Posterior,

Re2

=

R, = 40 kR (400.Qcm2)

C, =

005pF(5pF/cm21

Re, f Re, = 70 kR

Fig. 59. Equivalent circuit of an electrocyte of Gymnarchzls. Intracellularly applied current ( i ) and extracellularly applied current ( I ) are indicated. Rel, Re?,and R,, represent the resistance of the external medium. Negligible current flows through the resistance of the uninnervated face and the capacity of the innervated face and these elements are shown by dashed lines. The tentative values given for membrane parameters are referred to membrane area not corrected for surface convolutions.

436

M. V. L. BENNETT

stimulus ( C ) is the same as the threshold for firing at the termination of an anodal stimulus ( D ) . Furthermore, if brief pulses are used to evoke responses of the innervated face during maintained current pulses, the amplitudes of the responses are the same as without maintained current, although they occur when the uninnervated membrane is either markedly depolarized or hyperpolarized. One may ask if the membranes of the electrocytes have unique properties that give rise to the unusual function of the uninnervated face, From the equivalent circuit (Fig. 59) it is clear that intracellularly applied current and differential recording across the innervated face allow evaluation of the resistance of this face ri. The resistances rel or rez can be evaluated by differentially recording across the cell while passing current through an electrode external to one or the other face. Where monopolar recording shows the external potentials outside the two faces to be equal, then rel = rez. The time constant of decay of potentials following either intra- or extracellular stimulation is given by ( rel ren r i ) C. Thus from the measured resistances and time constant one can obtain the capacity. Although the data available are restricted, preliminary values are given in Fig. 59. These assume a uniform planar membrane on each surface. The resistance of the innervated face is quite reasonable. The capacity of the uninnervated face is if anything too small on the assumption that its capacity is the usual 1 pF/cm2 and takes into account the great increase in membrane area resulting from the numerous tubules and processes of this face. The measurements indicate that the resistance of the uninnervated face is at least 50 times that of the innervated face. The great increase in area observed on a fine structural level means that the uninnervated membrane has a resistance of perhaps 250-500 times that of the innervated face. This is a high value, perhaps 200,000 a ern2,but it is not unprecedentedly great (Bennett and Trinkaus, 1970). If the capacity of the innervated face were the usual 1 pF/cm2 its time constant would be 0.4 msec. This value is much shorter than the time constant of the uninnervated face and is in reasonable agreement with the experimental observations. The significance of an organ discharge without net current flow is discussed in Section 11, F.

+ +

4. MORMYRIDS The mormyrids are found in freshwaters of tropical Africa. They are a large group of some 11 genera and large numbers of species. All that have been studied are weakly electric. A key to the genera is presented in Table I11 and Fig. 60. Identification as to species is generally un-

10.

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ELECTRIC ORGANS

Table I11 Key to the Genera of Mormyrids The mormyrids are identifiable as small scaled, ray finned fish without adipose fin (rayless fin caudal to the dorsal), without spines in front of the fins, and without barbels. The caudal fin is small and generally on a peduncle (Fig. 60). The mouth is small and there are teeth in both jaws. The eye is covered by a thin epithe1ium.a a. Anal very short compared to dorsal (ca. one-third the length, Fig. 60A). Mormyrus (many species) aa. Anal very long (5 times the length of dorsal, Fig. 60B). Hyperopisus aaa. Anal about same length as dorsal (no more than 30% difference) b. Ventral closer to anal than pectoral; body elongate (Fig. 60C). Zsichthys (one species, Z . henryi) bb. Ventral closer to pectoral than anal or equidistant between them c. Teeth in several rows, villiform; terminal mouth with a chin appendage (Fig. 6OD,E). Genyomyrus (one species, G. donnyi) cc. Teeth in a single row in both jaws, 10-36 in each row; no chin appendage (Fig. 60G,H) d. Nares farther from the eye than each other; mouth terminal (at the front of the head, Fig. 60F) or inferior. Mormyrops (many species) dd. Nares close together and close to the eye; mouth always inferior (Fig. 601). Petrocephalus (many species) ccc. Teeth in a single row in both jaws, 3-10 in each e. One naris close to the corner of the mouth (Fig. 60J). Stomatorhinus (many species) ee. Both nares distant from the corner of the mouth f . Dentition of upper and lower jaw similar g. Mouth inferior or subinferior without a chin appendage or elongated mouth (Fig. 60K). Marcusenius (many species) gg. Mouth terminal (Fig. 60L) or on elongated jaws, sometimes greatly so (Fig. 60N); sometimes with short or long chin appendage (Fig. 60M,N). Gnathonemus (many species) ff. A large pair of incisors in the lower jaw, without a chin appendage h. Teeth of upper jaw fine conical, anal rather shorter than dorsal (Fig. 60P). Myomyrus (two species) hh. Teeth of upper jaw obtuse or bicuspid, anal and dorsal of same length. Paramyomyrus (one species, P . aequipinnis) Modified from Poll (1957).

reliable with the possible exception of those areas for which keys are available ( e.g., Greenwood, 1956; Pelligrin, 1923). Identification of species in our earlier work (Bennett and Grundfest, 1961c) was kindly provided by Dr. M. Poll of the Muske Royal du Congo Belge, Brussels, Belgium. The organ discharges are brief pulses that are emitted somewhat irregularly at a few per second when the animal is resting, but the discharge can accelerate to 40 or more per second during swimming (Lissmann, 1958). Accelerations are evoked by most modalities of stimulation

438

M. V. L. BENNETT

Fig. 6OA-I.

10.

439

ELECTRIC ORGANS

N

Fig. 60. Characteristics of representative mormyrid genera. ( A ) Mormyrus caballus. ( B ) Hyperopisus bebe. ( C ) Isichthys henryi. (D,E) Genyomyrus donnyi; side view and chin process. (F,G) Momnyrops deliciosus, side view and dentition. ( H,I) Petrocephalus sauvagei, side view and dentition. ( J ) Stomatorhinus corneti. ( K ) Marcusenius plagiostoma. ( L ) Gnathonernus leopoldianw. ( M ) Gnathonernus petersii. ( N ) Gnathonemus numenius. ( 0 , P ) Myomyrus mucrodon, side view and dentition. From Poll (1957).

440

M. V. L. BENNETT

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441

including resistance changes and weak electric pulses (Szabo and Fessard, 1965; Moller, 1971) . Accelerations can be classically conditioned as well as operantly conditioned in an avoidance paradigm (Mandriota et al., 1965, 1968). Brief interruptions of discharge can also occur in response to novel stimuli and in interaction with other electric fish. The electric organs lie just anterior to the caudal fin (Fig. 1).They are made up of four columns of cells and each column contains 100-200 cells in series. The cells are accurately aligned one behind the other. In many species the body narrows close to the tail to form a “caudal peduncle” and almost the entire cross section of the fish in this region is made up of electric organ. Each column of electrocytes occupies one quadrant and the only other structures are spinal column, skin, and tendons to the caudal fin. The cells are innervated by spinal neurons that lie in three segments in the central region of the electric organ that extends over 8 or 10 segments. The nerve fibers end on stalks as in Mahpterurus and some gymnotids, but the stalks are much more complex (Szabo, 1958, 1961a; Bennett and Grundfest, 1961~). A large number of stalks arise from the posterior faces (Fig. 61). These stalks fuse repeatedly (binarily ) to form a greatly reduced number of stalks before they are innervated. In electrocytes of Movmyrus there may be more than 10 separate stalk systems, each with its own site of innervation. In Mormyrops there are one or two. In Gnuthonemus, Petrocephalus, and Marcusenius there is only one. There is a further complication in some species of Gnathonemus. After varying numbers of fusions the stalks turn anteriorly and pass Fig. 61. Anatomy of mormyrid electrocytes. ( A-C) Single isolated cells stained with methylene blue, ( A ) Caudal surface of a cell from Mormyrus ruma The darkly stained, thick, branching nerve trunk innervates ( i ) at least eleven separate systems of stalks ( s ) . ( B ) Rostral surface of an electrocyte of Gnathonemus compressirostris with penetrating stalks. The regions surrounding the penetrations ( p ) are darkly stained, elsewhere the stalks ( s ) and body of the cell are only lightly stained. A few stalks are seen running to the site where they are all fused and innervated ( i ) . Under high magnification details of the stalk system could be seen, and the stalks on half the rostral surface of this cell are drawn in Fig. 67C. ( C ) Nonpenetrating stalk system torn off an electrocyte of G. compressirostris and heavily stained. The innervation ( i ) may be seen surrounding the stalk ( s ) at the central region. ( D ) Parasagittal section through the electric organ of G. tamndua stained with hematoxylin and eosin. Rostral surface up. Bodies of two electrocytes, about 30 p thick and heavily stained, run horizontally across the figure. Fine stalks ( s ) , about 10 p in diameter, arise from the caudal surface, fuse, and pass through penetrations ( p ) in the bodies of the cells to the rostral surfaces where they leave the plane of section. The regions of innervation ( i ) were included in the section and lie rostral to the cell bodies. Darkly stained nuclei are seen in the stalk and in the bodies of the cells. From Bennett and Grundfest ( 1 9 6 1 ~ ) .

442

M. V. L. BENNETT

through holes in the body of the cells (Fig, 61). They undergo their final stages of fusion on the anterior side of the cells. The degree of fusion before penetration depends on the species and correlates with the size of the initial head-negative phase of organ discharge as will be discussed below (Fig. 67). A similar penetrating stalk system occurs in Mormyrops (Grosse and Szabo, 1960) and in one of two specimens of a species of Hyperopisus that I have examined. As far as is known Marcusenius and Petrocephalus have only nonpenetrating stalk systems. The form of organ discharge varies with the species. In Gnuthonemus, Marcusenius, Petrocephalus, and Hyperopisus it is essentially diphasic, initially head-positive although there is a small initial head-negative phase if there is a penetrating stalk system (Figs. 62, 67). The discharges can be quite brief, 0.5 msec or less in duration. The discharge of Mormyrops is also essentially diphasic, but the initial large phase may be head-positive or head-negative. The head-negative discharges occur in specimens in which the innervation is on the posterior side, but the stalks are penetrating (Grosse and Szabo, 1960; Fig. 62B). In Mormyrus

-.. ..

:

..v:.

I

,

I

.

"

-

0 5 rnsec

Fig. 62. Electric organ discharges of representative mormyrids. Recorded in a small volume of aquarium water between head and tail, head positivity up. ( A )

From Mormyrus. ( B ) From Mormyrops. The electrocytes of this species have penetrating stalks and are innervated on the caudal side. There are few penetrations and an initial head positive phase can be seen only with higher gain recording. ( C ) From Petrocephalus. ( D ) From Hyperopisus. The electrocytes from the specimen of this species with penetrating stalks were innervated anteriorly and had large numbers of penetrations; correspondingly the initial head-negative phase is quite large. Superimposed sweeps with ( 2 in D ) and without organ discharges to show baseline.

10.

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443

the discharge is also diphasic, but the second, head-negative phase is much larger than the initial phase (Fig. 24) and the entire response can be longer lasting (Bennett and Grundfest, 1961~;Szabo, 1961). The single cells operate in a manner similar to those of electric fish already described (Bennett and Grundfest, 1961~). Both faces of the main part of the cells generate spikes. The external potentials are diphasic to nearly monophasic depending on the relations between the two faces. The contributions of the two faces of an electrocyte from a fish producing a diphasic output are shown in Fig. 63. A microelectrode recording monopolarly is advanced through the cell from the rostral to caudal side (Fig. 63A-C). These records are then subtracted to give the equivalent of differential recording ( Fig. 63B',C'). Both rostral and caudal faces generate spikes. (In this particular experiment the separation between the two spikes is more marked than it often is, and external potentials that more closely resemble the organ discharges are usually obtained, Figs. 65 and 66.) Corresponding to the similar respon-

Fig. 63. Response of an electrocyte of Gnuthonemus. Three electrodes are used, one for intracellular stimulation (1.6 msec pulse), another for intracellular recording near the stimulation site (A-C, upper trace), the third an exploring electrode about 2 mm distant (lower trace). ( A ) The exploring electrode is external to the rostral face and records a diphasic initially positive potential. ( B ) It is in the cell and shows the resting potential and a double-peaked spike. ( C ) It is advanced to lie outside the caudal face and records a diphasic response opposite to that in A. Time calibrations begin from the peak of the intracellular spike in the upper traces. (A') Superimposed tracings of records made with the exploring electrode aligned with respect to the peak of the intracellularly recorded spike. ( B ' , C ) Potentials across the caudal and rostral faces and diphasic potential across the entire cell (rostral positivity u p ) obtained by graphical subtraction of the monopolar records. From Bennett and Grundfest ( 1 9 6 1 ~ ) .

444

M. V. L. BENNETT

siveness of the two faces, the surfaces appear similar electron microscopically; both surfaces are moderately increased by tubules, that of the anterior face slightly more so (Schwartz et al., 1971). In Mormyrus r u m the organ discharge is predominantly head negative. Both faces of the cells appear to generate spikes, although it is difficult to be sure what fraction of responsiveness on the caudal side is to be assigned to the stalks (Fig. 64).The spike of the anterior face is longer lasting and the external potential is predominantly head negative. The initial slow head-positive phase of the organ discharge appears to be a result of activity in the stalks and can be observed with external stimulation of the single cells (Fig. 64A',B'). The location of the innervation on the stalks indicates that impulses arise in this region and propagate to involve the body of the cells. Action potentials can be recorded in the stalks, and the cell to which they go can be identified by intracellular stimulation in stalks or body of the cell. Impulses in stalks are longer lasting than those in the main part of the cells (Fig. 65). The PSPs that initiate the spike activity can be

Fig. 64. Responses of electrocytes of Mormyrus. A,B and A',B are experiments with different cells. ( A ) Three recording electrodes are close to the site of intracellular stimulation. Simultaneous traces show ( from above down ) stimulating current and differential recordings across the cell (rostral negativity up) and across the rostral and caudal membranes. The spike of the caudal face is briefer than that of the rostral face. The external response is largely rostral negative and lacks the initial rostral positivity of the organ discharge. ( B ) The same recording electrodes, but the response is evoked by intracellular stimulation about 1.5 mm from the recording site. The responses are somewhat shorter but are otherwise similar to those in A. The small potentials following the spikes result from excitation of neighboring electrocytes by activity of the penetrated cell. ( A',B') Responses evoked by brief externally applied stimuli. Lower traces: recording across the cell, rostral positivity up. Upper traces: recording across the rostral face in A' and the caudal face in B . The transmembrane potentials are like those in A and B, but the external response much more closely resembles the organ discharge. Modified from Bennett and Grundfest ( 1 9 6 1 ~ ) .

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445

Fig. 65. Recording from an electrocyte stalk of Gnathonernus tamandua. The stalks are penetrating and the organ discharge is triphasic in this species. Three electrodes are used as in diagrams; the closely neighboring pair of electrodes are about 1 mm from the other one. V,, upper trace; V,, lower trace. ( A , B ) Before and after penetration of a stalk on the rostral side of the cell, stimulation in a stalk on the caudal side. Note the polarity of the triphasic external response in A. About the same potential appears to be superimposed on the broader intracellular spike in B. ( C ) Stimulation in the rostral stalk, recording in the body of the cell and in the stalk on the caudal side. The broad spike in the stalk has superimposed on it the response generated by the body of the cell. The early part of the brief response is obscure, but it clearly has a late positive phase. This polarity contrasts to the terminal negativity of the superimposed brief response on the opposite side of the cell in B. From Bennett and Grundfest ( 1 9 6 1 ~ ) .

recorded with an appropriately placed electrode and are diagrammed in Fig. 73. The difference in spike shape, the recording of PSPs, and the fact that an electrode can enter and leave a stalk before penetrating the body of its cell allow the identification of the records as from stalks. The discharges of Gnathonernus are very brief, and all regions of each face of the cells must fire quite synchronously. Direct measurement shows that a neurally evoked impulse reaches all regions of the posterior face of the cell highly synchronously. In contrast, stimulation in the body of the cell does not excite the cell synchronously. Because of the stalks an impulse initiated at one part of the body of the cell appears to be conducted along it with a very nonuniform velocity. Close to the site of stimulation (within 1 mm in the experiment of Fig. 66, right) the impulse propagates slowly, at a velocity of about 2 meters/sec. However, the impulse arrives at distant parts of the body of the cell nearly simultaneously. Evidently, the evoked impulse propagates antidromically (backward) up the stalks near the site of stimulation and then proceeds orthodromically to excite much of the cell in nearly the normal time relations. In cells of Morrnyrus the conduction velocity

446

M. V. L. BENNETT

mm

A

0

B

0.5 m m ventral

C

1.0 m m ventral

D

2 5 m m ventral

0.5 10

1%g

20 5 msec

'.O dorsal mm

0 5 msec

Fig. 66. Conduction along electrocytes of mormyrids. Three electrodes are used: one for intracellular stimulation, one for intracellular recording close to the stimulation site, and one exploring electrode that records at various distances along the edge of the cell. The records on the right are from Mormyrus rume. The response close to the site of stimulation is shown on the zero trace. Intracellular responses recorded by the other microelectrode as it is moved along the cell are below, The traces are displaced downward an amount proportional to the distances between the two recording electrodes. The spike recorded at the same time with the fixed electrode is used to position the traces in the horizontal (time) axis. Since the latency of the responses a t the stimulation site vanes, the stimulus artifacts do not align. The conduction velocity given by the slope of the broken line is 0.45 meter/sec. On the left are records from Gnathonemus tamandua. Procedure as for Mormyrus except that the exploring electrode records external to the rostra1 face. The external responses in this species are triphasic because of the large number of stalk penetrations (Fig. 6 7 ). The conduction velocity appears to be about 2 meters/sec up to a distance of 1 mm but is much faster between 1 mm and 2.5 mm. Modified from Bennett and Grundfest (1961~).

along the cells is more or less uniform (Fig. 66, left). Not only is the stalk system divided into separate parts but also the stalks are much finer and the conduction velocity in them is likely to be lower. There appears to be an evolutionary progression in going from the multiple innervation sites to the single site. Mormyrus is apparently the least complex stage after diffuse innervation (which is found in the related form Gymnarchus). In Mormyrops the number of sites is greatly reduced, and in Gnathonemus and most other genera it reaches its final limit of one. The organ discharge shows a parallel progression toward brevity, a fact that suggests more precise synchronization can be obtained when there are fewer sites of innervation. We will return to the question of synchronization in Section 111, B. The stalks play an additional roll in the form of the organ discharge. It is observed that fish possessing electrocytes with penetrating stalks all produce triphasic organ discharges; there is an initial phase of head

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ELECTRIC ORGANS

negativity that precedes the predominantly diphasic response and that is absent in fish without penetrating stalks (Fig. 67). It is explicable as a result of longitudinal currents flowing along the organ due to impulses in the stalks passing through the bodies of the cells. The sequence of changes in direction of current flow is diagrammed in Fig. 68. In agreement with this explanation the size of the initial phase is greater the greater the number of penetrations (Fig. 67). No data are available on the functional significance of the initial head negativity. It would appear to be detectable in a fish where it is quite large (such as Gnathonernus tamandua), but in species where it is very small, it would seem to be of no significance at all. Behavioral studies may help to resolve these questions. The embryological formation of the stalk system is an intriguing problem, particularly where the stalks penetrate the body of the cell. The cells are multinucleate and presumably arise by fusion of a number of cells (Szabo, 1 9 6 1 ~ )In . any case the morphogenetic movements required and the control of cell fusion would appear to be highly involved. In spite of this apparent complexity, closely related species and perhaps even different individuals of the same species can have penetrating or

0.2 msec

Fig. 67. Correlation of amplitude of initial head negativity with number of penetrations by stalks. Camera lucida drawings of one-half of a representative electrocyte from each of four species of Gnathonmus are shown with tracings of the discharge of their organs. Black area indicates zone of innervation. The potentials drawn at the same time scale but with amplitudes normalized to equal height of the head-positive phase. ( A ) Gnathonemus compressirostris, individuals which produce no initial head negativity do not have penetrating stalks. ( B ) The largest initial head-negative phase is in the discharge of G. tamundua, the electrocytes of which have many penetrations (175 in the drawing). ( C ) A specimen of G . compressirostris which has a small initial head-negative phase has electrocytes with a medium number of penetrations ( 2 5 in the drawing). ( D ) The organ discharge of a specimen of G. moorii shows initial head negativity only in high gain recordings. Its electrocytes have very few penetrations ( 5 in the drawing). From Bennett and Grundfest ( 1 9 6 1 ~ ) .

448

M. V. L. BENNETT

-

B

.TJ +

............. .;\: +

*;

;-. ............ ;

............... ".......

+ +

Fig. 68. Current flows generating triphasic pulses in electrocytes with penetrating stalks. Diagrams show a region near a single penetration during different stages of activity. Active membrane is indicated by dotted outlines. Arrows show direction of current flow. Resulting potential is shown in lower right. ( A ) Head negativity is produced when the stalk activity, initiated near the site of innervation, is passing through the penetration. ( B ) Head positivity results when the impulse in the stalk excites the caudal face. ( C ) Head negativity is again produced when the rostra1 face becomes active. From Bennett and Grundfest ( 1 9 6 1 ~ ) .

nonpenetrating stalk systems (Grosse and Szabo, 19600; Bennett and Grundfest, 1 9 6 1 ~ ) .

E. Some Quantitative Considerations The amplitudes of responses of individual electrocytes in several of the strongly electric fish tend to be somewhat larger than responses in other excitable cells. The PSPs in electrocytes of marine strongly electric fish are up to 90 mV in amplitude, and the peak is near zero resting potential in Astroscopus. This amplitude is larger than in any other known cell, but one reason is that most other cells are electrically excitable and their responses obscure the PSP. The PSP at the neuromuscular junction of frog twitch muscle fiber would also be very large; the reversal potential of the PSP is similar to that in the electric fish, and the conductance change is a substantial fraction of the resting conductance (Takeuchi and Takeuchi, 1959). In the electric eel the spike amplitude is about 150 mV, and the sodium equilibrium potential appears to be about +70 mV (Altamirano, 1955; Nakamura et uZ., 1965). This compares with values of up to about 130 mV for other spike generating cells. Voltage amplitudes of membrane responses in electrocytes are otherwise unexceptional. Of course, the externally recorded responses of individual cells of even weakly electric organs

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are generally much larger than those of ordinary nerve and muscle cells. The values of membrane resistance in electrocytes are far below the values for muscle [about 3000 fi cm2 for frog twitch muscle (Eisenberg and Gage, 1969)l. Correspondingly, the current outputs are much greater. In eel electrocytes the resting resistance of the innervated face is about 25 0 cm2 according to Nakamura et al. (1965); a value of 19 fi cm2 is reported by Cohen et al. (1969) using a different and perhaps more accurate technique. The resistance of the uninnervated face is about 0.2 fi cm2 (Keynes and Martins-Ferreira, 1953). During activity the resistance of the innervated face decreases to about 1 0 cm2 (calculated from Nakamura et al., 1965), and the peak inward current is about 60 mA/cm2. These values approach the resistance of the extracellular fluid and cytoplasm in series with the innervated face. Because of leakage and eddy currents, decrease of the membrane resistances to much below the series resistance would make the cells less efficient. In Torpedo the resting resistance across an entire cell is 5-30 fi cm2 ( Albe-Fessard, 1950b; Bennett et al., 1961). The peak current during activity is about 75 mA/cm2 and if the driving force for each cell is about 90 mV, the series resistance during activity is about 1.2 fi cm2 per cell. Although the resting and active resistances of the electrocytes are far below those of muscle membranes, the values are comparable to those of the node of Ranvier [resting resistance, 30 0 cm2; active resistance, 2 0 cm2; peak inward current 10 mA/cm2 (Dodge and Frankenhaeuser, 1959; Frankenhaeuser and Huxley, 1964)1. Furthermore, the figures for electrocytes are calculated from macroscopic areas disregarding the considerable surface convolutions, and a more realistic comparison would be referred to actual areas of plasma membrane. On the reasonable assumption that the plasma membranes of the electrocytes have a capacity of 1 pF/cm2, one may estimate the true membrane area from the capacities measured per macroscopic area, which in the eel is about 15 pF/cm2 (Cohen et al., 1969) and in Torpedo is about 5 pF/cm2 ( Albe-Fessard, 1950b). Thus, the actual membrane parameters probably differ by factors of 5-15 from the reported ones (factors which appear consistent with the increase in surface seen morphologically, references below), Weakly electric organs tend to have somewhat higher membrane resistances, several tens to several hundred Q cm2 referred to macroscopic surface. Insufficient data are available for an accurate comparison among weakly electric organs in terms of area of plasma membrane, but the membranes-with the exception of membranes acting as a series capacity-all have a shorter time constant than muscle and presumably are of lower resistivity. The response characteristic in which electrocytes are most out-

450

M. V. L. BENNETT

standing is frequency of firing. The electric organs of sternarchids discharge constantly at frequencies from 700 to 1700/ sec, Eigenmannia operates at 2sO-600/ sec, and Gymnarchus at about 250/sec. Sternop ygus discharges at 50-100/sec, but it is like Eigenmannia on a 50% duty cycle in which the interval between pulses is about equal to the pulse duration. The higher values are unequaled for maintained frequency by muscle or other nerve. In fish other than the sternarchids the nerveelectrocyte synapse is chemically transmitting ( and cholinergic ) so that chemically mediated transmission can also operate at high frequencies. However, in gymnotids and Gymnarchus the synapses between the controlling neurons, which fire at the full organ frequency, may well all be electrically transmitting ( see Section I11). The high frequency of maintained activity suggests that there could be a considerable power output per unit weight of organ. As yet no satisfactory data are available, but many of the cells contain large numbers of mitochondria suggesting a high metabolic rate (Schwartz et al., 1971). The peak pulse power of the strongly electric organs is very large, but the pulses can be emitted at a high rate only for short times. The energy output presumably represents ions running down preexisting concentration gradients that are restored relatively slowly. Electrocytes of strongly electric organs do not have high densities of mitochondria ( Mathewson et al., 1961; Wachtel, 1964; Sheridan, 1965; Bloom and Barnett, 1966), and it would be predicted that the power output per gram of cell that could be maintained would be lower than for high frequency cells. In marine electrocytes the site of production of electric energy is somewhat paradoxically the innervated face which is inexcitable in the strongly electric fish. It is across this face that the potential is found during organ discharge. In the eel the contribution of the two faces is about equal, as it is in many cells of which both faces generate spikes. For electrocytes in which one face acts as a capacitance the energy is produced entirely across the innervated face.

F. Adaptation and Convergent EvoIution in EIectric Organs The evolutionary origin of electric organs will be discussed in the following chapter, because it appears intimately linked with the development of electrosensory systems. Ignoring the intermediate stages of evolution, one notes a number of convergences in electric organ systems that extend beyond simply the production of electricity (Table I V ) . Some of these convergences have obvious adaptive value. For example, it was recognized early that electric organs are relatively

451

10. ELECTRIC ORGANS Table IV Convergences in Evolution of Electric Organsa Teleos ts

Gymnotids

No spikes, marine Organ flattened Spikes, fresh water Organ elongate Strongly electric organ Weakly electric organ Accessory weak organ Intermittently active Continuously active “Constant” frequency Variable frequency Can cease firing

-1 X x-xx-x

Monophasic, both faces respond Monophasic, one face series R Diphasic, one face series C Diphasic, both faces spike Triphasic Innervation on stalks a Characteristics are shown along the left side; different groups are shown along the top. A particular group is indicated as having a particular characteristic if a t least one member of the group has it; a question mark indicates that only suggestive evidence is available. Thicker vertical lines separate groups in which electric organs are virtually certain to be separately evolved. A convergence is indicated when a particular characteristic appears in more than one of the separately evolved groups. Other convergences, such as development of chin accessory organs, may occur within groups. I n accessory organs.

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flattened in the two kinds of strongly electric marine fish and elongated in the two kinds of strongly electric freshwater fish. This difference is ascribable to the different resistances of the environment. The low resistance of seawater requires a high current, low voltage output; the high resistance of freshwater requires a low current, high voltage output. Another striking difference between marine and freshwater electric fish is the absence of spike generating membrane in electrocytes of the three marine groups and its presence in those of the three freshwater groups. While it is natural to ascribe this difference to differences in loading by the environments, it is difficult to do so convincingly (Bennett, 1961, 1970) since each organ consists of many elements in series-parallel array and the most energetically efficient kind of membrane would appear to be the best in both environments. Yet there is almost sure to be some advantage to PSP membrane for the marine environment because in each case the muscle giving rise to the electric organ does possess spike generating membrane (Bennett et al., 1961; Grundfest and Bennett, 1901, and unpublished data). An interesting convergence is in the development of stalks. These structures are most remarkable in mormyrids but are also prominent in catfish electrocytes. Stalks are present but seem of no great importance in gymnotids. Stalks do allow a smaller PSP current to excite the electrocytes since the input resistance of the stalks is higher, but the advantages of this are far from obvious if one considers marine electrocytes where the entire output consists of PSPs. The stalks act in synchronization of Ging of mormyrid electrocytes, but the same output could be produced by diffusely innervated cells, at least in the species with longer lasting discharges and the more primitive stalk systems. Another generalization concerns the presence of a dc component in the organ discharge. All strongly electric fish emit dc pulses; perhaps this leads to more effective shocking of prey or predator. A few weakly electric fish that fire at low frequencies also emit pulses that are dc or have a large dc component, but in most weakly electric fish including all high frequency species, the organ discharge has little or no dc component. The absence of a dc component in the organ discharge allows the fish to have a dual electrosensory system in which one set of receptors is sensitive to low frequency signals arising in the environment and another set detects distortions in the high frequency field produced by the electric organ (see Chapter 11,this volume). In Gymnurchus, the sternarchids, and perhaps Eigenmannia the absence of a dc component is achieved by modification of one electrocyte face to pass only capacitative current. In Sternopygus the uninnervated face apparently acts as a polarization capacity. In others

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such as Gymnotus and most mormyrids, the opposed faces of the electrocytes are active sequentially to achieve the same effect. Other convergences in terms of pulse shape and patterns of organ discharge can be found. Mormyrids are similar to the variable frequency gymnotids, and Gymnarchus is similar to the constant frequency gymnotids. The more striking differences are sure to be associated with differences in the operation of the sensory part of the system, and some of the subtler ones may be associated with species recognition. The fine structure of electrocytes shows a few correlations with their functional properties. Where one face is low resistance and inexcitable and the other face generates PSPs or spikes, the surface of the low resistance face is greatly increased by projections or invaginations [torpedinids, electric eel, monophasically discharging Hypopomus, accessory organs of Steatogenys (Mathewson et al., 1961; Sheridan, 1965; Bloom and Barnett, 1966; Schwartz et al., 1971)l. The area of the active face may also be increased but to a smaller extent. The relative areas correlate with the changes in resistances during activity and are such that these resistances tend to become more nearly equal. The probable significance of this feature in terms of increased efficiency was noted in Section 11, B. In Gymnarchus one face acts as a series capacity and this face is markedly increased in area; the morphological relations are similar in Eigenmannia (Schwartz et al., 1971). In Sternopygus both uninnervated and innervated faces are quite smooth, which supports the hypothesis that the very large apparent capacity is a result of shifts of ionic concentrations rather than a dielectric capacity. Furthermore, the resistances during and between spikes are about equal. A number of electrocytes in which both faces generate spikes have been studied at the fine structural level [Gymnotus, Steatogenys main organ, Gnathonemus, and Malapterurus (Mathewson et al., 1961; Schwartz et al., 1971)l. In each case there is some proliferation of both faces, but the degree is always less than in the inexcitable faces of monophasically responding cells. The proliferation in diphasically responding cells is somewhat greater in the uninnervated or nonstalk face; that in all but Malapterurus is the higher threshold, later firing face. The morphological difference may be an adaptation to the fact that the earlier firing face must operate through the series resistance of the later firing face at rest and that the latter should therefore have a low resting impedance. In Gymnotus at least the time constant of the later firing face is greater than that of the earlier firing face, and a significant fraction of the early outward current through the later firing face may be capacitative. The activity of the later firing face

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occurs during the phase of increased conductance owing to delayed rectification (potassium activation) in the earlier firing face, and the area of this face need not be so great. One would not be surprised to find that delayed rectification was more pronounced in the earlier firing face, but this possibility has not been investigated. Delayed rectification is more marked in Gymnotus cells than in those of Steatogenys, which may account for the smaller degree of proliferation of both surfaces in Gymnotus as compared to Steatogenys. Although some nerve-electrocyte junctions are continuously active at very high rates, no morphologically distinctive features have been found (Schwartz et al., 1971). All investigated do not appear significantly different from neuromuscular junctions. It is a bit disappointing that current electron microscopic techniques have not revealed any further correlations with function. One cannot yet distinguish between a high resistance inexcitable membrane and a low resistance excitable membrane. This failure probably reflects the small fraction of membrane surface actually involved in ionic movements ( cf. Hille, 1970). Perhaps freeze-cleaving techniques, which allow one to examine relatively large areas of membrane e n face, will provide sufficient resolution. After all, it is to be expected that ionic channels, although themselves small, have associated with them protein molecules well within the range of resolution of the electron microscope. An important kind of adaptation in electric fish is the arrangement of connective tissue. In many electric organs, connective tissue appears to provide an insulating sheath that channels current flow along the axis of the organ. An example from Gymnotus is given in Fig. 69. In Torpedo the resistance of the skin on the dorsal and ventral surfaces of the organ is lower than that over the rest of the body (Bennett et al., 1961). This difference tends to maximize current in the external medium and minimize current flowing through the animal’s body. An old but frequently recurring question is why strongly electric fish do not shock themselves. Their resistance to their own discharges as well as those of other fish is no doubt contributed to by the heavy fat and connective tissue layers that surround much of the nervous system. For example, the electromotor axons of Malapterurus are rather small, but they are surrounded by a sheath so that their apparent diameter is about 1 mm (in a fish about 15 cm long). This sheath becomes attenuated as the fiber branches, but it nevertheless extends beyond the synaptic terminals and part way down the electrocyte stalks (Mathewson et al., 1961). One would not be surprised if the hearts of strongly electric fish were less easily driven into fibrillation by shock or if the electroconvulsive threshold were lower for their neural tissue, but no data have been obtained. Another answer to the question of why electric fish do not shock

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Fig. 69. Influence of connective tissue on external fields of electrocytes of Gymnotus. The diagram shows portions of the connective tissue tubes of the three ventral columns of electrocytes, two of which are indicated by heavy lines in each tube (see Fig. 19). Septa between chambers are shown as dotted lines. Rostra1 is toward the top. The lower cell in tube I11 was stimulated intracellularly and a third electrode explored the external field. Upper and lower records of each pair represent the response before and after penetrating the connective tissue tubes. The diphasic external response is initially positive on the rostral side. The external potentials are considerably larger inside tube I11 than elsewhere. The potentials decrement relatively little along the tube. A subthreshold PSP is evoked in the rostral cell of tube 11. From Bennett and Grundfest (1959).

themselves is that they do. Often an eel will twitch when it discharges its main organ. Self-stimulation becomes more prominent when the fish is out of water because the voltage developed across the animal's own tissue is increased. The electric catfish will normally not move when it discharges, but if a cut is made through the organ to the body wall each discharge will cause a twitch. Evidently opening of the inner lining of the organ allows more current to flow through the body. When mormyrids are placed in air, even they may show signs of selfstimulation. Organ discharges cause twitches of tail muscles and small after-discharges of the organ, probably because of electrical stimulation of the spinal cord (Bennett and Grundfest, 1 9 6 1 ~ ) . G . Embryonic Origin and Development of Electric Organs

There is now little doubt that except in sternarchids all electric organs evolved as modifications of muscle. The muscle groups of origin can be located at any point along the body from eye muscles in Astro-

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scopus to branchial muscles in torpedinids, to pectoral muscles in Mahpterurus, to axial and tail muscles in the remainder. In electrocytes of Astroscopus, rajids, and mormyrids striations are easily seen under the light microscope. The orientation however is quite unrelated to the cell axis. Electron microscopy shows well-organized Z lines and thin filaments in both Astroscopus and rajids. Gymnurchus electrocytes have similar but less regular structures (Schwartz et al., 1971). In the mormyrid Gnethonemus there are both thick and thin filaments and Z lines. The filaments are not very well aligned, however, and the cross bridges of normal muscle are certainly reduced in number if not absent altogether. Sarcoplasmic reticulum, associated in normal muscle with control of tension, is lacking in most regions or very sparse. It is not surprising then that the cells do not move, although it would be interesting to know the biochemical “deficiency,” if any, in the contractile machinery. Electrocytes of the other three evolutionary lines of electric fish, torpedinids, gymnotids, and Mahpterurus, all have fine but disorganized filaments without Z lines (Wachtel, 1964; Schwartz et al., 1971). The origin of these tissues from muscle is less obvious in the adult, although the development of the organ from myoblast-like tissue has been demonstrated histologically (Johnels, 1956; Keynes, 1961; see also Fritsch, 18%). The physiological and pharmacological properties strongly support derivation from muscle. Some histological aspects of electric organ development have also been described for Gymnurchus, Astroscopus, and Mormyrus (Dahlgren, 1914, 1927; Szabo, 1 9 6 1 ~ ) . An interesting problem is the ontogeny of excitability in electrocytes of marine fish. Rajid electrocytes develop from rather normal appearing muscle fibers (Ewart, 1892). If they are normal, the spike generating membrane present in tail muscle of the adult (Grundfest and Bennett, 1961) would have to be lost. Embryological material is readily obtainable for this group unlike that for most other electric fish. Increase in the number of electrocytes during growth may also provide an opportunity for experimental analysis. Gymnotids regenerate their tails including electric organs (Ellis, 1913) and electric eels apparently add layers of electrocytes as they increase in length (Keynes, 1961). The existence of a neurogenic accessory organ in the chin region of Adontosternarchus (Section 11, D, 1, g ) has developmental as well as evolutionary implications. It suggests a kind of preadaptation but one not involving a purposeful change toward a structure that has survival advantage only at some later time. A probable early stage in the chin organ development was maintained oscillatory activity of a group of electroreceptors (see Chapter 11, this volume) that served as a weakly electric organ for other receptors. The afferent fibers would then have

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become modified to resemble the fibers of the main electric organ. The independent evolution of rostra1 accessory organs in other gymnotids attests to the value of these organs. Is it to be supposed that the sensory fibers of the primitive accessory organ gradually enlarged over many generations to repeat the evolutionary sequence followed in the main organ? It seems much more likely that having evolved the developmental mechanism to make large generating nerve fibers in its main organ, the fish merely evolved the ability to apply the same mechanism to the chin fibers. In molecular terms, DNA coded information required to make the main organ fibers was also present in the DNA of the sensory neurons; the fish then acquired a way of expressing this information at the different site. In brief, the argument is that there is evolution of a mechanism of turning-on in a new part of the organism a previously evolved very complex developmental sequence. This process appears much more probable than evolution of a very similar complex sequence all over again. Evolution of a structure in one part of the body and then the sudden appearance of a very similar structure in a quite different part of the body may not be uncommon in phylogenetic history. Another example from electric fish is Hunter’s organ of the eel, which appears to be evolved from a different muscle group than the main and Sachs’ organs, but which has very similar electrocytes (see Section 11, D, 1, a ) . Other examples are discussed in Bennett ( 1970). In accessory electric organs of other gymnotids and Narcine the innervation suggests that the organs developed by migration of electrocytes from the main organ rather than by development from a different and local muscle group (see Sections 11, C, 2 and 11, D, 1, d). This concept of preadaptation also raises the possibility that electric organs may have arisen in elasmobranchs only once. The argument for separate evolution is based on the muscles of origin being different in torpedinids and rajids. The organs could have originated in one muscle type in a common ancestor, then “jumped to a second muscle type, and finally have been lost at the first site. The rajids seem the more generalized of the two, and a hypothetical electric common ancestor would probably have been rajid-like. The same argument appears inapplicable to separate evolution of electric organs in different teleost groups because of their much wider evolutionary separation.

H. Electrocytes as Experimental Material Many electrocytes are very large cells that are of low resistance and easily studied by microelectrode techniques. Their greatest advantage

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over muscle is that they do not move when stimulated. However, the large size and low resistance is often a disadvantage because the membrane potential may not remain uniform over the cell when it is stinwlated by a single intracellular microelectrode; that is, the cells are not “space-clamped ( cf. Bennett, 1970). Thus, qualitative results can be obtained using microelectrodes, but quantitative characterization of membranes in terms of Hodgkin-Huxley parameters as has been carried out for other tissues may not be possible (Hodgkin and Huxley, 1952; Frankenhaeuser and Huxley, 1964). Because electrocytes have a uniform orientation, it is sometimes feasible to use external electrodes on columns of cells or even on the entire electric organ. This kind of preparation allows satisfactory impedance measurements which can be referred to the single cell (e.g., Fig. 41; see also Albe-Fessard, 1950b). Crude but useful current clamp measurements may also be possible where the properties of the individual cells are sufficiently alike that the current density through them is essentially uniform (e.g., Bennett et al., 1961). The uniform orientation also has allowed measurements of thermal and optical changes associated with activity; most important, these changes can be studied under different degrees of electrical loading of the tissue (Cohen et al., 1969; other references in Bennett, 1970). Single eel electrocytes can be isolated. The single cell can then be placed between two baths and transcellular current restricted to a limited area by pressing the innervated face of the cell against a plastic sheet with a m a l l hole through it. Current application by external electrodes is more or less uniform because the uninnervated face is inexcitable and of low resistance. This preparation has been used in voltage clamp and impedance measurements, although it is not clear that space clamping is possible even under these conditions (Nakamura et al., 1965; Morlock et al., 1969; cf. Bennett, 1970). As shown in respect to the squid axon, space clamping may fail if access resistance through the surrounding solution is too large compared to membrane resistance (K. S. Cole, 1968). It is difficult or impossible to clamp the squid axons showing the largest inward currents, and eel electrocytes pass considerably larger currents. Another problem is whether the small stalks on the innervated faces protrude far enough to be nonisopotential with neighboring membrane (as in several weakly electric gymnotids, Figs. 28 and 31). In the eel preparation the effects of membrane outside the edges of the window also must be evaluated. These factors require careful analysis as did voltage clamp of the squid giant axon, and it will be necessary to use exploring microelectrodes to verify space clamping. In spite of the possible or real shortcomings, gymnotid electrocytes allow voltage clamp-

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ing superior in respect to temporal resolution to what has been possible with skeletal muscle fibers (Adrian et al., 1970). One important measurement that has not been obtainable from muscle fibers is the high speed of onset and reversal of anomalous rectification (Bennett and Grundfest, 1966; Nakamura et al., 1965; Morlock et al., 1969). The primary factor is that the time constant of the excitable membrane is much lower in electrocytes and there is less interference from capacitative currents. Isolated eel electrocytes can be of value in flux measurements ( Higman et al., 1964), although the spatial uniformity of concentration as well as potential becomes important and the conductances are SO large that intracellular concentrations can change rapidly ( Karlin, 1967). Still the large amounts of synaptic as well as spike generating membrane and the possibility of approximately controlling transmembrane potential during drug application and flux measurements are features not readily available in muscle. Electrocytes do not appear as good as muscle for electrophysiological study of many aspects of synaptic transmission. The presynaptic fibers are no larger than those in muscle and usually do not end in a localized but accessible region. The low input resistance of the cells makes them less useful for study of miniature PSPs and actions of restricted synaptic areas. The low input resistance and wide distribution of synaptic membrane also impede studies using iontophoretic application of drugs, which remains the best method for study of kinetics of drug action. Nonetheless, through experimental simplicity, electrocytes have been and should continue to be useful in studies involving relatively gross application of drugs during current application and recording of PSPs (e.g., Karlin, 1969). The kinds of problems where electrocytes are likely to be particularly useful are in the area of biochemistry. The organs are a rich source of acetylcholinesterase ( Leuzinger and Baker, 1967), and much of the work characterizing the enzyme has been done on material of this origin. Moreover, the eel electric organ is probably the richest known source of the sodium-potassium transport ATPase and has been used in studying this enzyme ( Albers, 1967; Post et al., 1969). Eel electric organ does not have a particularly high concentration of mitochondria compared to a number of muscles, which suggests that its mean metabolic rate is not particularly high. Nevertheless, the only work it does is generation of electricity. Thus, it is reasonable that its sodium-potassium ATPase levels should be very high. From the much greater concentration of mitochondria in electrocytes of repetitively active weakly electric fish (Schwartz et al., 1971) one would expect there to be considerably higher levels of the transport ATPase. Of course, the weight of organ available

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is measured in grams rather than kilograms. When macromolecules of excitable membranes are to be isolated, electric organs are probably a good place to start. The ability to choose by choice of organ the type of excitability present in terms of PSP or spike generating membrane may be helpful in the future isolation procedures. The electric organ of Torpedo is now being used for the isolation of vesicles containing acetylcholine (Israel et al., 1968). Electric organs seem to be much better tissues than guinea pig brain in which to look for vesicles containing acetylcholine when only a very small fraction of interneuronal synapses can involve this transmitter. Another probably useful tissue for vesicle isolation is the electromotor lobe of the Torpedo brain. Available evidence indicates that synapses on the electromotor neurons are chemically transmitting, but the transmitter is unlikely to be acetylcholine (see Section 111, B ) . The electromotor lobes provide a tissue sample of up to a gram of what appear to be neurons of a single type with a single class of synaptic ending on them. While the weight of tissue is small compared to the kilograms of electric organ, the size is very large compared to other neuronal groups of comparable homogeneity.

111. NEURAL CONTROL OF ELECTRIC ORGANS

For most electric fish it is of considerable importance that the firing of individual electrocytes be synchronous. Synchrony leads to an output that is larger in terms of both voltage and power because inactive cells act as a shunt or series resistance and thus reduce the amount of current that active cells produce outside the fish. When the discharge is diphasic or triphasic, synchronization is particularly important because slightly out of phase addition leads to cancellation. As has been seen in the section on electrocyte activity, organ discharge generally involves one highly synchronized response of each cell. Only weakly electric organs of marine fish appear to have discharges that involve fused and repetitive activity of the electrocytes. There is another fundamental consideration in control of organ discharge; namely, that two spike generating membranes arranged in series tend to desynchronize each other’s activity. Inward current generated by one excitable membrane tends to hyperpolarize the corresponding membrane of the next cell in series with it and thus prevent its firing (see Fig. 2). To achieve synchronous activity each cell in series must be separately innervated and its discharge controlled centrally. Of course, cell membranes in parallel may tend to excite one another as an action

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potential propagates along a single membrane. Still separate cells are always separately innervated. One exception to the rule of central control of organ discharge is known, the chin organ of Adontosternarchus. Since this organ consists of a single layer of parallel elements, the spontaneous activity of the cells tends to be synchronized and the discharge frequency is set in the organ itself without central control (see Section 11, D, 1, g ) . As might be expected, this organ often operates at a somewhat different frequency from the main organ. Control of electric organ discharge can be divided into two problems, how the fish “decides” to discharge its organ, and, having reached the decision, how it activates the different generating elements synchronously. The “decision” to discharge the organ is reached, depending on the kind of fish, by a small group of neurons in the higher spinal cord, medulla, or perhaps midbrain ( Bennett et al., 1967a,b,c; Bennett, 1968a). These cells make up the “command nucleus,” and when they fire synchronously, the “command signal” to discharge the organ is initiated. This activity is then transmitted to the electrocytes either directly or through one or more neural relays. Neurons of the command nucleus are probably spontaneously active in the continually discharging forms; that is, they are pacemaker neurons in a manner analogous to pacemaker cells in the heart. In species that discharge only intermittently, the command neurons receive excitatory inputs and perhaps inhibitory inputs as well. When these inputs reach threshold, the neurons “decide” that the organ will be discharged. A very significant feature of the pacemaker or command cells is that they are coupled to each other by means of “electrotonic synapses.” The coupling is the basis of the highly synchronous firing that is observed in command neurons of the electric organ systems. The electrotonic synapses provide resistive pathways between cells that for this reason behave as if they are part of the same core conductor; potentials spread between the cells in the same way as they spread electrotonically along an axon. Two important properties of electrotonic synapses are relevant to their functioning in electric organ control. Current can flow in either direction across the synapses, and current begins to flow without delay when pre- and postsynaptic potentials differ. These properties differentiate electrotonic synapses from chemically transmitting synapses in which transmission is basically in one direction and postsynaptic current is delayed with respect to presynaptic impulses (by about ?h msec at room temperature). Electrotonically mediated PSPs are delayed with respect to presynaptic potentials because of the capacity of the postsynaptic cells, but generally the delay is very short compared to the delay at chemically transmitting synapses (Bennett, 1966). The electrotonic synapses mediate rapid-acting positive feedback be-

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tween cells; a relatively more depolarized cell tends to depolarize and excite its less depolarized neighbors and is itself made less depolarized and inhibited by them. Thus, the neurons tend to fire synchronously. The synapses are both excitatory and inhibitory, and it seems reasonable to call them synchronizing synapses (Bennett, 1968b). One may ask whether mutually excitatory, chemically transmitting synapses could also mediate synchronization, As will be seen below synchronization in most of the electric organ systems is so precise that the negligible delay of electrically mediated transmission is required; the delay associated with chemically mediated transmission would be too great. The experimental demonstration and several properties of electrotonic transmission are illustrated by Fig. 70. In this experiment neighboring cells in the oculomotor nucleus of the stargazer are penetrated by microelectrodes; the antidromic spikes evoked by stimulation of the oculomotor nerve are shown in Fig. 70A. Depolarizing or hyperpolarizing current applied in either cell spreads to the other cell (Fig. 70C-F). If one or both electrodes are placed in a just extracellular position the recorded voltages are very greatly reduced. They do not disappear completely because the currents develop some voltage across the volume resistance of the neural tissue. It can be concluded that the voltages recorded intracellularly involve a special, junctional relation between A

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Fig. 70. Electrotonic coupling of oculomotor neurons innervating the electric organ of the stargazer Astroscopus. Two neighboring cells are penetrated by independently mounted electrodes. Their antidromic spikes are shown in A. The upper trace shows the antidromic volley recorded at the point of exit of the oculomotor nerve from the cranial cavity. When current is passed through the electrode in the cell of the upper trace both depolarization ( C ) and hyperpolarization ( D ) spread from cell to cell (superimposed sweeps with and without a pulse; current strength shown on the lower trace). When current is passed through the other electrode, depolarization ( E ) and hyperpolarization ( F ) also spread from cell to cell. When depolarization of the first cell is adequate to evoked spikes, there are corresponding small deflections in the second cell in addition to the maintained depolarization (B, increasing stimulus strength from top to bottom). The voltage gain is the same in B-F; the sweep speed is the same in C-F. From Bennett ( 1968b ) ,

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cells and are not merely a result of proximity. Other less direct methods of demonstrating electrotonic transmission may be useful; for example, a PSP of very short latency can be assumed to be electrically mediated (cf. Bennett, 1966). The morphological basis of electrotonic coupling has been investigated in a number of systems (Pappas and Bennett, 1966; Bennett et al., 1967a,b,c; Kriebel et al., 1969; Pappas et al., 1971; these papers include other references ) . In every case close membrane appositions occur (Figs. 71 and 72) which are rare or absent in neighboring regions and which do not occur at synapses for which there is evidence that transmission is chemically mediated. These close appositions are believed to be the site of current passage between cells. They are probably what have recently been termed gap junctions (Revel and Karnovsky, 1967; Brightman and Reese, 1969). This name arises from the appearance in perpendicular sections of a 20-30 A gap between membranes following suitable fixation procedures. The gap is penetrable by marker substances applied in the extracellular space. The gap is not uniform but is made up of a more or less hexagonal lattice of channels. There is evidence that in the spaces outlined by the lattice, channels separated from extracellular space cross the junctional complex and interconnect the cell cytoplasms. The intercytoplasmic channels are perhaps 10A in diameter and provide sites for movement of ions and other small molecules between cytoplasms of the coupled cells (Payton et al., 1969; Pappas et al., 1971). This class of junctions is distinct from tight junctions, which are appositions where extracellular space appears completely occluded in perpendicular sections, and where there is no hexagonal structure in tangential sections ( Brightman and Reese, 1969). (Both types were formerly called tight junctions. The current nomenclature in this area is confusing and will probably be revised when there is more general argreement as to morphological and physiological properties of the junctions and criteria for their identification.) These latter junctions generally occur in epithelia where they form complete rings around cells (zmulae occludentes; singular, zonula occludens) that prevent transepithelial leakage through intercellular clefts ( Farquhar and Palade, 1963; Brightman and Reese, 1969). There is no evidence that zonulae occludentes form low resistance channels between cell cytoplasms because no cells are known to be both electronically coupled and joined exclusively by these junctions. Electrotonic coupling between cells of the same kind can be mediated by electrotonic synapses between somata or dendrites (Fig. 71). Alternatively, cells can be coupled by way of presynaptic fibers that form electrotonic synapses on the cells; current then spreads from one postsynaptic cell to another through the presynaptic fibers (Fig. 72). (Since

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activity can normally be conducted in either direction across some of these synapses, pre- and postsynaptic denote the usual morphological relations rather than the direction of impulse propagation. ) Coupling can also be mediated by both presynaptic fibers and dendrodendritic synapses in the same nucleus ( Pappas and Bennett, 1966). A. Pathways and Patterns of Neural Activity The circuitry of electric organ command systems in teleosts that have been studied physiologically is diagrammed in Fig. 73. The physiological analysis has generally involved microelectrode recording from the entire animal paralyzed or anesthetized. 1. THE ELECTRIC CATFISH The simplest control system is in the electric catfish (Bennett et al., 1967b). Two neurons lie in the first spinal segment, one on either side (Fig. 72A). Each neuron innervates all the electrocytes on its side and each impulse fires the electrocytes once. The two neurons are closely coupled electrotonically, and hyperpolarization produced in one cell spreads to the other (Fig. 74C ) . The coupling is so close that an impulse initiated in one cell propagates into the other (Fig. 74B). The pathway of coupling is by way of presynaptic fibers (Fig. 72). Excitatory inputs gradedly depolarize the cells (Fig. 74A,D), and when one cell is excited the other must fire also. This explains why no stimulus can be found that excites one cell without exciting the other. That the two cells comprise the command nucleus is indicated by the gradual rise in potential when organ discharge is evoked by cutaneous stimulation (Fig. 74D). When PSPs in one cell exceed threshold, both cells are depolarized since the presynaptic fibers end on both; once initiated the impulse rapidly propagates between the cells. The conduction Fig. 71. Morphological basis of electrotonic coupling: dendrodendritic junctions between electromotor neurons of the mormyrid Gnathonemus. ( A ) In a silver stained preparation ( Romanes’ method) of the medullary relay nucleus, a thick bridge appears to connect the two cell bodies without there being any intervening membrane. ( B ) With the electron microscope such cells are seen not to have cytoplasmic continuity. The cell bodies ( s ) are separated by membranes the ends of which are indicated by the arrows. Blood vessels ( b v ) and myelinated nerve fibers are seen. Axon terminals ( a ) make contact with the cells; one on the lower right shows a portion of the myelin sheath. ( C ) At a similar region of apposition between spinal electromotor neurons, higher magnification shows large regions where the membranes appear fused. In this very thin section the central dark region appears as a series of dots. Osmic acid fixation. From Bennett et al. (1967a).

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requires less than 0.2 msec, which is more rapid than could be achieved by chemically mediated transmission ( Fig. 74A). [The delay is greater when one cell is directly stimulated, for the unstimulated cell is much less depolarized when the impulse arises ( Fig. 74B) .]

2. WEAKLY ELECTRIC GYMNOTIDS In weakly electric gymnotids the command system has more than . organ frequency is set by one neural level (Bennett et al., 1 9 6 7 ~ )The what has been termed the pacemaker nucleus, which is in the medulla. (From a functional point of view it would be difficult for synchronous activity to arise within a group of neurons that are as widely separated as the spinal neurons innervating the electrocytes.) The pacemaker nucleus lies in the midline and contains some 30 to 200 cells depending on the species. It activates a relay nucleus also in the midline of the medulla, and this nucleus in turn activates the spinal neurons. There are 50 or so medullary relay neurons and some hundreds to thousands of spinal neurons. A single spike occurs at each level before each organ pulse (Fig. 75). In the pacemaker neurons there is a gradually rising depolarization between spikes (Fig. 75A,C). This depolarization is similar to pacemaker potentials in other tissues, and the cells appear to be spontaneously active. In the relay cells the potential between spikes is quite flat and the discharges arise abruptly from a level base line (Fig. 75B,D). These cells are clearly activated from a higher level which is in fact the pacemaker nucleus. Fig. 72. Morphological basis of electrotonic coupling: axosomatic and axodendritic electrotonic synapses on giant electromotor neurons of the electric catfish. ( A ) Toluidine blue stained thick section that passes through the nuclei of the two giant cells. The central canal (arrow) is just ventral to the cells. A number of small dendrites come off the somata, but there is no apparent direct connection between the cells. ( B ) Electron micrograph of two axosomatic synapses (a,a’) on the cell. At the upper one ( a ) the myelin sheath is seen to terminate in the plane of section. There is a region of close apposition of axon and soma membranes at this synapse (between arrows), but probably not a t the other one. Terminal a’ contains many vesicles, but there are relatively few in a. The former may be one of the chemically transmitting inhibitory synapses that occur on these cells. Near these endings there is a relatively large amount of extracellular space ( e ) filled with granular material. ( C ) Higher magnification of an axodendritic synapse. The axonal side is to the left and a presynaptic vesicle ( v ) is seen in it. A central dark line is formed at the close membrane apposition extending between the arrows. ( D ) An axodendritic synapse like that in C, but the close membrane apposition is cut more tangentially and striations appear with a periodicity of about 100A. ( E ) Diagram of current flow where cells are coupled by way of presynaptic fibers. The cell on the left is more depolarized. All sections of osmic acid fixed material. Modified from Bennett et al. (1967b).

468

M. V. L. BENNETT Malaplerurus Gymnot ids

Astroscopus

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A~~~~~~~~~~ )eiectrical A Axosornatic- chemical Fig. 73. Neural circuitry controlling electric organs of teleosts. The modes of transmission are diagrammed as shown in the key below. Where axosomatic synapses are indicated, axodendritic synapses are also found. The mode of transmission to the command nucleus is known only in the electric catfish, although it is indicated as chemically mediated in the others. Where there is a question mark, the cells have not been definitely localized, but several of their properties can be inferred. The command nucleus of the stargazer Astroscopus is now known to be in the medulla, not the midbrain. In Malapterurus, the gymnotids, and Astroscopus, a single command volley at each level precedes each organ discharge. In mormyrids, the activity is more complex and is diagrammed for each level. The dotted lines indicate the thresholds of the cells in the two pacemaker nuclei. The dominant pacemaker at any given time is the one receiving more excitatory inputs and firing before the other. From Bennett (1968a).

That the discharge frequency is set in the pacemaker nucleus is established by results like those in Fig. 76. If a hyperpolarizing pulse is applied in one cell, the next spike in that cell and each subsequent one is delayed (Fig. 76B,D). The descending volleys in the spinal cord are

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2 msec

10 msec

Fig. 74. Properties of the giant electromotor neurons of the electric cadish. ( A ) Upper and lower traces, recording from right and left cells, respectively. Brief stimuli of gradually increasing strength are applied to the nearby medulla (several superimposed sweeps, the stimulus artifact occurs near the beginning of the sweep). Depolarizations of successively increasing amplitude are evoked until in one sweep both cells generate spikes. ( B ) Two electrodes in the right cell, one for passing current (shown on the upper trace) and one for recording; one recording electrode in the left cell. The traces from the recording electrodes are the lower ones starting from the same base line. When an impulse is evoked in the right cell by a depolarizing pulse, the left cell also generates a spike after a short delay. ( C ) When a hyperpolarizing current is passed in the right cell, the left cell also becomes hyperpolarized, but more slowly and to a lesser degree (display as in B ) . ( D ) When organ discharge is evoked by irritating the skin, a depolarization gradually rises up to the threshold of the giant cell and initiates a burst of three spikes (lower traces, base line indicated by superimposed sweeps ). Each spike produces a response in the organ (upper trace, recorded at high gain and greatly reduced in amplitude because curare is used to prevent movement). Modified from Bennett et al. ( 1967b).

not changed in duration or desynchronized; they are reset in phase by the same amount as the spikes in the pacemaker neuron and follow the spikes at the normal interval. Thus, hyperpolarization in one cell affects the entire pacemaker nucleus and resets its phase of firing. Moreover, since hyperpolarization spreads between cells, the interaction between cells can be inferred to be electrotonic. (This has been directly demonstrated also.) The coupling between pacemaker cells obviously leads to synchronization of their firing. Each cell is spontaneously active and would get out of step with the others if there were not coupling-or positive feedback-between them. The accuracy of synchronization is so great as to require the speed of electrotonic coupling; synchronization by mutually excitatory chemically transmitting synapses would involve too great a delay. The coupling of gymnotid pacemaker neurons is not as close as in the catfish electromotor neurons and does not allow impulses to propagate

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2'UL

10 msec

2 msec

Fig. 75. Responses of pacemaker and relay cells in a weakly electric gymnotid Gymnotus. Upper traces: activity in the spinal cord and peripheral nerves leading to the electric organ (recorded by needle electrodes a t high gain in a curarized animal). Lower traces: intracellular recordings in pacemaker ( A,C) and relay (B,D) neurons. Faster sweep in C and D where the dotted lines indicate the times of firing of the cells in relation to the descending activity. From Bennett et al. ( 1 9 6 7 ~ ) .

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Fig. 76. Effect of polarization in a single pacemaker cell of a weakly electric gymnotid Gymnotus. Recording as in Fig. 75 except that current applied through the recording electrode is indicated on the lower trace. Two superimposed sweeps in each record, one with and one without applied current. The sweeps are triggered by the spike of the pacemaker cell. Faster sweep in A and B. (A,C) A depolarizing pulse that evokes a spike advances the next and subsequent spikes but does not desynchronize or itself cause any descending activity. (B,D) A hyperpolarizing pulse retards the next and subsequent spikes but does not desynchronize the descending activity. From Bennett et al. ( 1 9 6 7 ~ ) .

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from one cell to another. As shown in Fig. 76A,B an impulse in one pacemaker cell does not cause a descending volley in the spinal cord; it only advances the phase of firing. Thus, there is some spread of depolarization to the other cells but not a propagated impulse (unless the cells are very near to firing). The coupling is sufficiently close to synchronize the cells, and, when all the cells are firing in the vicinity of a cell penetrated by a microelectrode, it is extremely difficult to block that cell’s activity by hyperpolarizing current. While a spike in any one cell affects its neighbors but little, many cells firing together can produce a large and very suprathreshold depolarization in an inactive cell. As it happens, the medullary relay neurons are also electrotonically coupled in most gymnotids that have been studied. Coupling is by way of the pacemaker fibers afferent to them. At this level the coupling does not serve to keep the cells from firing out of phase, for each cell is always excited once per organ discharge by the large descending volley from the pacemaker nucleus. Presumably the coupling serves to synchronize relay cell firing, either because asynchrony was present in the initial pacemaker volley or because it has arisen in transmission from the pacemaker nucleus. The spinal relay neurons have been adequately studied only in the electric eel (Bennett et al., 1964; Bennett, 1968a). These cells, too, are electrotonically coupled and by way of the descending fibers ending on them (Pappas and Bennett, 1 W ) . Preliminary experiments indicate that the spinal relay neurons are similarly organized in Gymnotus. Again, coupling would tend to increase synchronizatio?. Sherrington (1906) called the motoneuron the final common path for muscle fiber activity. In the gymnotids the final common path for electric organ discharge extends three neurons back into the nervous system. Each level involves a number of neurons and a certain amount of signal shaping may go on in the relays, but these are only small extensions of the Sherrington concept.

3. Asmoscomrs In the stargazer the command signal is initiated in what is probably a midline nucleus in the medulla and is relayed in the very large oculo-

motor nucleus (Bennett, 1968a, and unpublished data). Neurons are electrotonically coupled in both command and relay nuclei (Fig. 70). In the relay both dendrodendritic synapses and the presynaptic fibers mediate coupling. The organ discharges are somewhat variable in size and apparently do not always involve every oculomotor neuron (Fig. 6 ) . The experimental evidence indicates that the command nucleus is not

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well synchronized. The oculomotor neurons do not simply relay the signal; they also “decide” whether or not a sufficiently large command volley is present to call for a discharge, and this decision is not always unanimous in spite of the coupling between cells. Often firing of the command neurons is in pairs of spikes, and PSPs in the oculomotor neurons show two components. Apparently, a few oculomotor neurons fire twice in normal activity and give rise to the delayed component on the falling phase of some discharges (Fig. 6 ) .

4. RAJrnS In the skate the electric organ is probably controlled by a midline nucleus in the medulla since stimuli applied in this region evoke organ discharge (Szabo, 1955, 1961b). There is no evidence as to whether this nucleus is a relay or pacemaker, or whether the neurons are electrotonically coupled. This is a particularly interesting example because the discharge is repetitive and probably asynchronous. While it seems likely that positive feedback between neurons is involved in the decision to discharge the organ, the speed of electrical transmission is apparently not required. If feedback is still mediated electrically, it would suggest that electrical coupling may be involved in other slow systems as well.

5. MORMYRIDS In the mormyrids control of organ discharge is more complex than in the fish discussed up to this point (Bennett et al., 1967a; Bennett, 1968a). There is some evidence that the pacemaker nucleus is bilateral and that and organ discharge can be initiated by a command signal arising on either side (Fig. 73). This activity is relayed through a midline nucleus in the medulla to the spinal neurons and then to the electric organ. Although each pacemaker nucleus is able to initiate a command volley independently, either because it is spontaneously active or because it receives tonic excitatory inputs, pacemaker activity on the two sides is coordinated. An impulse arising in what may be considered the dominant nucleus at that time propagates from the medullary relay “antidromically” into the other pacemaker nucleus, the subordinate one. (Transmission at the pacemaker synapses is apparently electrotonic and allows “antidromic” propagation. ) Thus both nuclei are excited, and the pacemaker potential of both nuclei is returned to the level of hyperpolarization that immediately follows spike activity; pacemaking in both nuclei is reset. The cells of the two nuclei then begin to depolarize again, but those in the dominant nucleus reach the firing level first, again resetting the activity of both nuclei.

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In the mormyrids there is not simply a single impulse at each level of the electric organ control system (Fig. 73). PSPs from what is presumably a single spike in either pacemaker nucleus initiate a two-spike discharge in the medullary relay (the second spike propagates back to the dominant pacemaker and both spikes propagate to the subordinate pacemaker). Firing in “doublets” appears to be a property intrinsic to the cell membrane rather than a result of a long-lasting PSP because the cells give a two-spike discharge in response to a brief intracellularly applied current pulse. The two medullary relay volleys descend the spinal cord and cause two PSPs in spinal neurons that innervate the electrocytes. These PSPs are chemically mediated. The first PSP initiates a three-spike discharge. Firing in triplets appears intrinsic to the cell membrane of the spinal cells as does the doublet firing of the medullary cells. However, the third spike is somewhat labile and the second PSP from the medullary relay guarantees its occurrence. The three electromotor neuron spikes are propagated out to the synapses on the stalks of the electrocytes, but only a single postsynaptic spike is produced. The first PSP is very small, the second is greatly facilitated (increased in amplitude) but still subthreshold, and the third is facilitated sufficiently to reach threshold. The PSPs are not recorded external to the fish because the synapses are on stalks far from the body of the cells and there is little or no longitudinal current associated with them. ( A similar firing pattern could be detected externally if the cells were diffusely innervated over one face.) The significance of this peculiar sequence of signal transformations is unknown. It also occurs in Mormyrus and Mormyrops in some species of which the discharge is longer lasting and the requirement for synchronization is not so great. Both medullary and spinal neurons are closely coupled electrotonically, and an impulse in one cell propagates to all the other cells of that nucleus. (An antidromic impulse in an axon generally fails to invade the cell body, at least in part because of the low input resistance resulting from the close coupling of the cells.) The neurons are connected by thick dendrodendritic bridges in both nuclei (Fig. 71), and coupling by way of presynaptic fibers probably occurs in the medullary relay as well. Interpretation of the peculiar multiple firing in the mormyrid control system is not likely to be aided by comparison to Gymnurchus. Light microscopy suggests that there are four interconnected nuclei in the medulla, but they have not been studied physiologically ( Szabo, 1961b ) . All that is known is that a single volley descends the spinal cord, and a single PSP in the electrocytes initiates each organ discharge, An interesting aspect of the mormyrid control system is that the command

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system “informs” the sensory system that an organ discharge is coming (Bennett and Steinbach, 1969; see Chapter 11, this volume). The pathways mediating this activity and the effects on afferent volleys are just being explored. 6. TORPEDINIDS

In Torpedo there is a command nucleus on each side that is located deep in the medulla (Szabo, 1954; Albe-Fessard and Buser, 19%). These nuclei activate the very large electromotor nuclei that lie on the dorsal surface of the medulla (Fig. 8). The electric organ discharges are synchronous on the two sides, and it is probable that either command nucleus can activate all the relay cells on both sides. NO data are available about interaction between the two command nuclei, but some coordinating mechanism is likely to be found. Preliminary data indicate that the electromotor cells, which comprise a relay, are not coupled to each other and that transmission from the command nucleus is chemically mediated (cf. Saito, 1966). Single volleys from the command nucleus excite single volleys in the relay cells that produce the single PSPs in the electrocytes comprising the individual organ discharges.

7. THEELECTRIC EEL The mechanism of control in the eel shows an interesting variation. All the electrocytes fire together during the large discharges, but only Sachs’ organ and posterior part of Hunter’s organ are active during the weak discharges. As in weakly electric gymnotids, the organs are controlled by a single midline relay nucleus in the medulla (Bennett et al., 1964; Bennett, 1968a). The pacemaker nucleus has not yet been found in the eel. Each command volley from the medullary relay excites all the spinal neurons that innervate the electrocytes, and the command volley passes out ventral roots to anterior as well as posterior parts of the organ. At low frequencies PSPs in the main organ and the anterior part of Hunter’s organ are small and do not excite the cells, but they are large enough to excite cells in Sachs’ organ and in the posterior part of Hunter’s organ (Albe-Fessard and Chagas, 1954). When the interval between command volleys becomes as small as several milliseconds, the PSPs in the main organ and anterior part of Hunter’s organ greatly increase in amplitude and become adequate to excite the cells of these organs. Thus the command to excite the main organ consists of high frequency activity in the same neurons that control Sachs’ organ. Only a single bulbospinal relay system is required to control both organs. This increase in simplicity is obtained at the cost of several

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milliseconds’ increase in the minimum latency at which the main organ can be activated. Probably the control system has evolved its dual functioning from an earlier stage in which there was only a single weakly electric organ. An intriguing problem is how pacemaker activity for the two organs is controlled.

B. Synchronization of Electrocyte Activity Given that the command nucleus has reached a decision to fire the electric organ, that is, has itself fired, the individual electrocytes must be excited synchronously. This is a significant problem because the different parts of the electric organ may be quite far apart. Nerve tracts running directly to the different parts of the organ could not fire the cells synchronously unless they were much more rapidly conducting than any known nerves. Two basic mechanisms are known to contribute to synchronization; both involve utilization of conduction time in nerve fibers or electrocyte stalks to equalize overall latency of the command signal in reaching the electrocytes. In one mechanism the nerve fibers run more or less directly to the more distant part of the organ, but take a more devious path to the nearer parts, thus tending to equalize path length and thereby conduction time (Fig. 77A). In the second mechanism shorter paths to nearer parts of the electric organ are of lower conduction velocity, again tending to equalize conduction time (Fig. 77B; fibers conducting more slowly are indicated as being of smaller diameter). Lower conduction velocity may also be found in only a portion of the path to the electrocytes; slowly conducting collaterals may branch off from the main rapidly conducting path to innervate the nearer parts of the organ (Fig. 77C). Equalization of path length is an obvious feature of a number of electric organs where the different nerves enter the electric organ and run for some distance before giving off branches that return to end near the point of entry. Compensatory differences in conduction velocity apparently occur in the stalk system of mormyrid electrocytes where differences in conduction distance are easily visualized and where synchronization between parts of the body of the cell is very precise (Fig. 61). As would be expected, the shorter paths involve stalks that are smaller in diameter. Probably most systems use a combination of the two mechanisms. In the electric eel differences in conduction time down the spinal cord are compensated for by both increased delay at the spinal relays

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M. V. L. BENNETT

B....

Fig. 77. Mechanisms of compensatory delay. Neural pathways are diagrammed leading to terminations in the periphery at different distances from a rostral command center. A command volley arises at the large arrow and the dotted lines represent times of arrival of impulses at equal time intervals afterward. ( A ) Equalization of path length. The paths to the nearer cells are made more devious so that all paths are of nearly equal length. ( B ) Compensatory differences in conduction velocity. The paths leading to the periphery are direct, but conduction is slower in the shorter, thinner paths. ( C ) Localized compensatory delays. Thin terminal branches in which conduction velocity is reduced are longer in the paths leading to the nearer parts of the periphery. From Bennett (1968a).

and increased delay from activity in ventral roots to spike initiation in the electrocytes ( Albe-Fessard and Martins-Ferreira, 1953). TWOlines of evidence indicate that the delay at the spinal relay arises in conduction time in collateral branches of descending axons. First, transmission from descending fibers to electromotor neurons is electrotonic and the PSPs are sufficiently rapidly rising that they can be delayed very little at the synapses themselves (Bennett, 1966). Second, action potentials at intermediate delays can be recorded in collaterals of the descending fibers. Preliminary morphological observations indicate that these collaterals are thinner at the anterior region of the spinal relay nucleus (R. M. Meszler and M. V. L. Bennett, unpublished data). The efferent axons entering the ventral roots are also thinner in the anterior regions of the spinal cord, and reduced conduction velocity probably contributes to the greater peripheral delay at the anterior of the organ. The early firing of rostral accessory organs in a number of gymnotids and of the dorsal column of electrocytes in Gymnotus presumably involves similar mechanisms to those in the eel. There is no indication of an earlier firing pacemaker or medullary relay, or of an earlier firing group of spinal neurons. Apparently the relatively delayed firing of the majority of electrocytes is achieved peripherally. It may be that early

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firing of the accessory organs really represents delayed firing of the main organ. At chemically transmitting synapses such as those on electrocytes, compensatory delays could in principle arise in “synaptic delay” [which at the neuromuscular junction at least results from time required for release of transmitter ( Katz and Miledi, 1965a,b) 1. Compensatory delays may occur at a number of chemically transmitting synapses in electromotor systems. One instance is in the spinal electromotor nucleus of the mormyrid Gnathonernus in which PSPs are synchronous at the two ends of the nucleus, although the descending volley arrives at the anterior end about 0.3 msec earlier than at the posterior end (M. V. L. Bennett and E. Aljure, unpublished data). A further instance is provided by the electric organ of the electric catfish. A single large axon branches to innervate all the electrocytes on one side of the organ. There is some equalization of path length for the axon enters the organ somewhat posteriorly and runs both anteriorly and posteriorly from the site of entry. Stimulation of the axon at its point of entry leads to a discharge that is about the same duration as the responses of single cells (Fig. 78C). Stimulation of a small branch to a piece of organ from the head end of the fish produces a response that has a longer latency than stimulation of a similar preparation from the tail of the fish (Fig. 78B,B’). This difference in latency is just sufficient to compensate for the difference in conduction

2 msec

2 msec

Fig. 78. Compensatory delays in the catfish electric organ. (A,A’) Responses of a piece of electric organ dissected from near the tail of a fish about 20 em long. The tissue has 10 em of motor nerve attached to it and is stimulated at each end of this length of nerve. ( B ) Response of a piece of organ from same fish dissected near head, stimulated via the nerve close to point of entry into tissue. (B’) Response of another piece of organ, similarly stimulated, but taken from a position 10 cni nearer tail. ( C ) Response of the electric organ on one side from another large fish, stimulated orthodromically at the central end of the nerve. ( C ) The response of the same organ, stimulated “antidroniically” at the caudal end of the nerve. From Keynes et al. ( 1961).

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time down the main trunk of the axon (A,A'). The significance of the synaptic delay can be well illustrated by recording the output of one entire side of the organ when stimulating the nerve at different sites. If the nerve is stimulated at its caudal end rather than its site of entry into the organ, the differences in delay add to the differences in conduction time and a markedly asynchronous discharge results ( Fig. 78C'). The site of these compensatory delays is unknown; it could be in presynaptic nerve branches and terminals or at the synapses themselves. In the electric catfish it could also be in the stalks of electrocytes. However, no systematic differences in length of stalks have been observed in teased preparations, and the variability of stalk length is quite great in all parts of the organ. In both mormyrids and the electric catfish, morphological investigations may reveal that terminals are longer and thinner where delays are greater. In this case one would be less inclined to consider synaptic delay as a factor in the compensatory mechanism.

C. Organization of Electromotor Systems As discussed here the control of electric organs is reasonably well described from one to several neural levels back into the nervous system. Provided one can accept the proposition that the pacemaker cells are autoactive, the analysis is reasonably complete for the very regular basal discharge frequency found in many species. Certainly one would like to know the ionic basis of the pacemaker activity, but in respect to impulse traffic, the origins and pathways are well defined. The pathways of afferents to the command nuclei in variable frequency and intermittently discharging forms are by and large unknown, although most if not all sensory modalities can be excitatory and responses can be of quite short latency (e.g., Fig. 3 B ) . The slight modifications of frequency in some high and constant frequency species also require investigation. The relative fixity of jamming avoidance responses suggests fairly direct electrosensory inputs, while the brief modulations that perhaps have a signaling function probably involve much more complex pathways (Bullock, 1970). The involvement of higher centers is also indicated by the fact that changes in organ discharge rate can be operantly and respondently conditioned ( Mandriota et al., 1965, 1968) as well as serve for signaling ( Black-Cleworth, 1970; Moller, 1971). An interesting problem is raised by the ability of Gymnorhamphichthys and some Hypopomus to maintain organ discharge frequency constant at low, high, and even intermediate levels. This ability suggests that there are neurons that maintain a tonic, asynchronous but quite constant

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excitatory input to the pacemaker nucleus. This input seems too constant to be due to casual stimulation of receptors resulting from movement, and an activating system that is itself quite stable would seem to be required to explain the data. Pathways mediating the complete inhibition of discharge in Gymnotus, Hypopomus, and Sternopygus are also not worked out. The strong excitatory drive to the medullary relay in Gymnotus and probably also Hypopomus suggests that this nucleus is not involved. Probably the inhibition operates on the pacemaker nucleus. If so, it might be considered surprising that in Gymnotus the discharge always starts and stops abruptly with only a small degree of slowing below the basal rate before and after complete cessation. However, similar observations have been made during microelectrode recording from the pacemaker nucleus in which activity at this level did indeed cease when discharge ceased and restarted when renewed discharge was evoked by spinal stimulation. These sudden changes in frequency are consistent with calculations from the Hodgkin-Huxley equations, which predict that maintained firing of the squid axon in response to a steady current can occur only at rather high frequencies ( Stein, 1967). Unlike Gymnotus, Sternopygus does not start up again by emitting full-sized organ pulses; there is a gradual recovery of pulse amplitude and synchronization. While this observation might represent a difference in the pacemaker nucleus, it might also result from properties of lower level synapses. If the spinal cord is sectioned in Gymnotus a single brief stimulus to the cord will still cause a full-sized organ discharge. However, in Sternopygus moderately prolonged stimulation at about the normal organ frequency is required to restore the discharges to their full amplitude. Granted that transmission at the spinal relays is electrotonic, the most likely site of facilitation in Sternopygus is at the nerve-electrocyte synapses. The absence of cessation of discharge in the higher frequency species Eigenmannia and sternarchids is perhaps a result of the fact that discharge does not stop following spinal section but continues asynchronously. The investigations to date have been largely restricted to elucidation of what may be considcred the final common path for organ discharge. A number of modifications to the original concept of final common path appear necessary to describe control of electric organs. A group of neurons at a particular level can fire as a single neuron, and the common path may involve several levels of neurons. The highest level determines the frequency of discharge; the lower levels act only as relays and perhaps also do some signal shaping (or as in the mormyrids, transformation of impulse number). The command path may bifurcate

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(and then cease to be common). The command to a particular organ may be expressed in terms of frequency. Several of these modifications are already required for ordinary motor systems. For example, some muscle fibers are innervated by more than one nerve fiber, which is analogous to paired command nuclei. If tension of a muscle fiber rather than any contraction at all is considered, the command may be coded in terms of frequency of firing. Furthermore, it is obvious that we do not understand all about the control of an electric organ or a muscle fiber when we know the final common path to it. We also need to know all the fibers afferent to neurons in that path. We have taken the decision to fire the organ as meaning the firing of the highest level nucleus of the final common path. The decision could also be defined as activity in any subset of fibers afferent to the highest nucleus that can cause the nucleus to fire. Similarly, the relevant output of a whole muscle, its tension, may be achieved (or coded for) by many different combinations of fibers active at different frequencies. For freshwater weakly electric fish and for the electric catfish the neural circuitry guarantees that every electrocyte receives neural excitation in every discharge. The operation of the system is essentially all-or-none and an organ discharge is present or absent. It should be recognized that amplitude can vary minorly as a result of refractoriness (and perhaps facilitation), but these changes must be secondary to changes in frequency. The situation is similar in the eel except that the amplitude normally varies over a wide range. In all these fish and to a good approximation in Torpedo and the stargazers as well, the behavior of the animal with respect to its discharge can be characterized by a single series of time intervals, a simplicity which gives the system some attractiveness for quantitative study. Two additional characteristics are frequently found in electric organ control systems. Where a group of neurons fire synchronously there is likely to be positive feedback between the cells. In command nuclei in which a highly synchronous volley arises, positive feedback must be present and must be electrically mediated because chemically mediated transmission is too slow. In relay nuclei there is no absolute requirement for feedback, but cells are often coupled electrotonically, presumably to increase synchronization. Another characteristic often found in electric organ control systems is a tendency toward reduced numbers of cells at higher levels. Apparently the decision to discharge the electric organ is always reached in a group of cells that are few in number compared to the final generating cells. The most striking example is found in the electric catfish, in which two neurons

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control the several million electrocytes. Similar but less extreme pyramids of numbers are found in other electric fish. This aspect of neural organization may be denoted the committee principle. This term is chosen in analogy with decision making by committees which tends to be more rapid the smaller the committee. One might question whether there could be a command ‘‘nucleus’’ containing a single cell, a committee of one. An example is provided by the Mauthner cells of lower vertebrates which can be considered single cell command systems for the axial musculature on either side of the body (Furukawa and Furshpan, 1963; Diamond and Yasargil, 1969). One could propose a number of reasons for the existence of control systems containing more than one cell, such as protection against loss of neurons and production of enzymes for maintenance of synaptic transmission. At lower levels progressive increase in a number of neurons may be equivalent to progressive increase in size. The two giant neurons of the electric catfish are able to support a very small synaptic area on a large number of electrocytes, but it seems likely that a large number of neurons are required to provide the vast synaptic area in the electric organs of Torpedo. Phylogenetic or ontogenetic factors may also be responsible for the presence of the relays. Evolution may yet progress to single-celled command “nuclei” although in most instances the multicelled nuclei do about as well as required by the electric organ. It is interesting to compare the function of relay nuclei to transmission of impulses along an axon. At a node of Ranvier an impulse that has been attenuated in electrotonic propagation from the preceding node triggers the generation of a new impulse that is of full amplitude. The node acts as a pulse restoring element, and decrementless conduction is thereby made possible. A relay nucleus potentially does more than this. The volley of impulses in the fibers efferent from the relay may be more synchronous than that in the afferent fibers, or all the efferents may become excited when only a fraction of the afferents are active (as appears to occur occasionally in Astroscopus) . These functions do not require coupling of the cells; all that is necessary is that a number of afferent fibers converge on each relay cell. Although midline nuclei have developed in a number of control systems, bilateral command nuclei may be fairly common because of the basically bilateral organization of the nervous system. If either of the two command nuclei can excite an entire effector system, synchronization between the two is not important. However, it would appear functional for each command to reset the phase of firing in both com-

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mand nuclei. Probably this resetting occurs in the mormyrids by actual invasion of the impulses, but mutual inhibition could also be a mechanism in other systems. For example, crossed inhibition like that between Mauthner cells (Furukawa and Furshpan, 1963; A. A. Auerbach, unpublished data) would be equally effectivc in an electric organ system. In the hatchetfish each Mauthner fiber activates the muscles depressing both pectoral fins, and these cells thus constitute a bilateral command system for the depressor muscles ( Auerbach and Bennett, 1969a,b). Contraction of these muscles causes the animals to dart upward in an escape reflex. Mutual excitation like that proposed for the mormyrid pacemaker nuclei would not work for the Mauthner cells, because each innervates the axial musculature on one side and near simultaneous excitation could lead to a counter-productive attempt to flip the tail to both sides simultaneously. Actually there is evidence that in the goldfish each Mauthner fiber excites motoneurons on one side and inhibits them on the other side but at a shorter latency (Diamond and Yasargil, 1969). Simultaneous activity of both Mauthner fibers excites no axial motoneurons. If one Mauthner fiber fires, it causes muscle contraction on one side and prevents the other Mauthner fiber from exciting the contralateral niotoneurons for about 100 msec. Recognition of the requirement for speed of transmission in synchronized systems has been useful in predicting sites where electrically mediated transmission has subsequently been found including neurons controlling sonic muscles ( Bennett, 1966; Pappas and Bcnnett, 1966) and oculoinotor neurons (Kriebel et al., 1969). In mediating activity that is not very precisely synchronized, the speed of electrotonic synapses might not be required. Examples are the bursting behavior of respiratory neurons and various invertebrate cardiac ganglia. Electrotonic coupling does occur in a number of moderately synchronized systems in both vertebrates (cf. Bennett, 1968b) and invertebrates (Hagiwara et al., 1959, Willows and Hoyle, 1969). The extent to which synchronization of slow systems is mediated by positive feedback and the degree to which the positive feedback is mediated electrically remain to be worked out. Electrotonical transmission appears to provide a simpler and perhaps more efficient means of positive feedback between cells (Bennett, 1968b). One may question whether firing of an electric organ resembles other decision processes. The final common path concept of a decision requires extension even in some very simple systems. On the other hand, it is reasonable to think that the features observed in simple effector systems havc some more general relevance. Negative feedback has been emphasized as a common property in neural control systems

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mediating homeostasis. It is also common in sensory and motor pathways where it may increase spatial resolution. Yet many phenomena involve recruitment of large numbers of cells in ways that appear to require mutual reinforcement of neuronal activities, that is, positive feedback.

IV. CONCLUSIONS AND PROSPECTS

The study of electric organs and electrocytes has illuminated many aspects of membrane physiology. Their primary usefulness is a result of evolution which has exaggerated different membrane functions in different electrocytes. For example, the concept of independent sites that pass different ions or groups of ions becomes more reasonable given that specific kinds of permeability can occur in isolation in different regions of a single cell or in different cells. Examples are the isolated occurrence of membrane generating postsynaptic potentials (in Astroscopus and torpedinids ) , of membrane exhibiting delayed rectification (in rajids), and of the electrically excitable sodium system without delayed rectification (in the electric eel), This macroscopic separation suggests microscopic separation, which in many single membranes can only be inferred from functional arguments or pharmacological data. These specializations of different kinds of membrane cannot really be said to have been responsible for major advances in electrophysiological knowledge. Yet as new morphological, biochemical, and biophysical techniques become available, electrocytes may provide the best tissues for their evaluation. Although one cannot determine electrophysiologically whether or not sodium and potassium channels of ordinary spike-generating membrane are separate, one can begin to think about isolating them biochemically. Electric organs would appear to provide a good starting material just as they are proving useful in the characterization of cholinesterase and sodium-potassium ATPase. By selection of cell type, one can select different kinds of membrane with relatively great degrees of purity compared to most other tissues. The neural systems controlling electric organs have provided a large number of examples of electrically mediated transmission, which meets the functional requirement for rapid communication between cells. This mode of transmission also proves to be able to mediate many functions often considered as restricted to chemically mediated transmission. The correlation between morphologically close apposition and electrotonic coupling was considerably strengthened by the work on electromotor

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systems. This correlation helps to validate morphological identification of electrical transmission in other systems where electrophysiological analysis is not so simple. It is not known whether there is any relevance to higher systems of the organizational principles deduced from electric organ systems. The next level of analysis of the electric organ systems may be no easier than study of less specialized systems that are of more general interest. Some knowledge is being obtained of afferent pathways from electroreceptors in weakly electric fish (see Chapter 11, this volume) which have important inputs to the electric organ control system. Both operant and respondent conditioning of the control system can be obtained and conditioned response latency can be very short. It is not unreasonable that the complete neural pathway of the conditioned response could be obtained in these cases. The central connections are minimally explored; one knows what goes in and one can go from the electric organ several synapses antidromically. The rewards for filling in the gap could be great, and prospects for at least some progress are bright. ACKNOWLEDGMENTS Some of the hitherto unpublished work was carried out in the laboratory of Neurophysiology, College of Physicians and Surgeons, Columbia University, with the support of Dr. H. Grundfest. Work on the Rio Negro expedition of the R. V. AlphaHelix was in collaboration with A. B. Steinbach and allowed considerable advance in understanding of the sternarchids. The author is greatly indebted to Dr. T. H. Bullock for his assistance in that remote place. Supported in parts by grants from the National Institutes of Health (5PO1 NB 07512 and HD-04248) and the National Science Foundation ( GB-6880).

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Szabo, T. ( 1961b). Anatomophysiologie de centres nerveux spbcifiques de quelques organes 6lectriques. In “Bioelectrogenesis” (C. Chagas and A. Paes del Carvahlo, eds.), pp. 185-2Q1. Elsevier, Amsterdam. . ontogenetiques entre l’organ Bectrique, son innervation Szabo, T. ( 1 9 6 1 ~ ) Rapports et sa commande enkphalique. Z. Zellforsch. Mikroskop. Anat. 55, 200-203. Szabo, T. (1961d). Les organes dectrique de Gymnotus carapo. Koninkl. Ned. Akad. Wetenschap., Proc. C64, 584-586. Szabo, T., and Fessard, A. (1965). Le fonctionnement des 6lectromkepteurs Btudid chez les Mormyres. J. Physiol. (Paris) 57, 343-360. Szabo, T., and Suckling, E. E. (1964). L’arrh occasionel de la dhcharge blectrique continue du Gymnarchus est-il une &action naturelle? Naturwissenshaften 51, 92-94. Takeuchi, A., and Takeuchi, N. (1959). Active phase of frog’s end-plate potential. 3. Neurophysiol. 22, 395411. Takeuchi, N. (1963). Some properties of conductance changes at the end-plate membrane during the action of acetyl choline. J. Physiol. (London) 128-140. Tasaki, I., and Freygang, W. H., Jr. (1955). The parallelism between the action potential, action current, and membrane resistance at a node of Ranvier. I . Gen. Physiol. 39, 211-223. Wachtel, A. W. (1964). The ultrastructural relationships of electric organs and muscle. I. Filamentous systems. J. Morphol. 114, 325-360. Watanabe, A., and Takeda, X. (1963). The change of discharge frequency by A. C. stimulus in a weak electric fish. J. Exptl. Biol. 40, 57-66. Willows, A. 0. D., and Hoyle, G. (1969). Neuronal network triggering a fixed action pattern. Science 166, 1549-1551.

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11 ELECTRORECEPTION M . V. L. BENNETT I. Introduction . . . . . . . . . . . . 11. Distribution of Electroreceptors . . . . . . . 111. Tonic Electroreceptors . . . . . . . . . . A. Characteristics in Gymnotids and Mormyrids . . . . B. Electrical Equivalent and Input-Output Relationships . . C. Characteristics in Catfish . . . . . . . . D. Ampullae of Lorenzini . . . . . . . . . IV. Phasic Electroreceptors . . . . . . . . . A. Large Receptors of Mormyrids . . . . . . . B. Phasic Receptors of Gymnotids . . . . . . . C . Medium Receptors of Mormyrids . . . . . . D. Phasic Receptors of Gymnarchus . . . . . . . E. Amplification and Oscillation at Phasic Receptors . . . V. Receptor Function in Electroreception , . . . . . A. Accessory Structures and Receptor Responses to Potential . . . . . . . Gradients in the Environment B. Receptor Responses to Electric Organ Discharges . . . C. Central Projections of Electroreceptor Activity . . . D. Behavioral Responses to Voltage Gradients and Conductance . . . . . . . . . . . Changes . E. Thresholds for Receptor and Behavioral Responses . . . VI. Evolution of Electrosensory Systems and Electric Organs . . VII. Implications for Receptor Function in General , . . . References . . . . . . . . . . . . .

493 496 503 503 512 515 517 520 528 533 536 540 540 544 544 550 552 555 558 561 564 56a

I. INTRODUCTION

A number of groups of fish have receptors that are specialized for the detection of electric fields (Table I ) . The existence of these electroreceptors was first clearly indicated in weakly electric fish, which continually set up low voltage electric fields around themselves by means 493

Table I Groups of Fish Possessing Electroreceptors-Types

of Receptors

Fish group

Morphological type

Physiological type

Receptor names

Elasniobranchs CatEsh Gymnotids

Ampiillarya Amprillaryc*d Ampul1aryf.o Tuber0usf.g Ampullaryh,* Tuheroiis*,t Tuberonsh,t Ampiillary m Tuberousm Tuheroiism

Toiiicb Tonic‘ Tonich I’hasich Tonich Phasich I’hasi ch Tonic? Phasic? Phasic?

Ampullae of Lorenzini Small pit, organs,c ampullary organsJ Tonich; ampullary type I,/ type I,%ampullaryg Phasic*; ampullary type 11,’ type II,i tuberousg Small,h mormyromast type I,?type Ai Medium,h mormyromast type II,? type B‘ Large,h Knollenorgane,’”organes bulbeux,z type CE Type A% Type B’ Type (2%

Mormy rids

Gymnarchus

Waltman, 1966. Murray, 1965, 1967. Mullinger, 1964. Wachtel and Szamier, 1969. e R o t h , 1969. f Lissmann and Mullinger, 1968. 0 Szamier and Wachtel, 1970.

a

Bennett, 1967. Szabo, 1965. i Cordier, 1937. IC Franz, 1921. GCrard, 1940. Mullinger, 1969.

11. ELECTRORECEPTION

495

of their electric organs.” These fish proved to be extremely sensitive to applied fields as well as to distortions in the fields they themselves generated ( Lissmann, 1958; Lissmann and Machin, 1958). Subsequently, it has become apparent that some catfish and most elasmobranchs possess electroreceptors but lack electric organs ( Dijkgraaf, 196$; Dijkgraaf and Kalmijn, 1963, 1966; Lissmann and Machin, 1963; Murray, 1967; Roth, 1968, 1969). In these instances, it is likely that the fish primarily detect signals of external origin, although the receptors may also be responsive to fields set up by the fish‘s own muscles. Other groups of fish may yet be shown to have electrosensory systems, and there are morphological suggestions of electroreceptors in several groups (Dijkgraaf, 1963). All the known electroreceptors are modified lateral line organs (see Chapter 8 by Flock, this volume). In the weakly electric freshwater fish, they are distributed along most or all of the body length as would be expected in a sensory system that detects distortions in a field set up by the fish itself. As in their mechanoreceptive precursors, the initial active transformation of the stimulus is carried out by receptor cells that are presynaptic to the innervating afferent fiber. One may classify the electrosensory systems as passive, if they detect signals of external origin, and active, if they are equipped with their own energy source, that is, an electric organ (Bennett, 1967; Machin, 1962). Familiar examples of active sensory systems are echo locating systems of bats, cetaceans, and oil birds (Griffin, 1958), and one is justified in using the term “electrolocation.” It should be emphasized that electrolocation does not involve electromagnetic waves; the system instead operates analogously to an ohmmeter or impedance measuring device. Of course, receptors in an active sensory system can also operate passively. Furthermore, freshwater weakly electric fish have two or three kinds of electroreceptor. One kind of receptor is insensitive to the high frequencies usually predominant in the organ discharge and operates passively while the remaining receptors operate actively. In the skates, which are marine weakly electric fish, the organ discharge is infrequent and the receptors probably function passively most of the time. The same consideration appears to apply to the weakly electric organ of the torpedinid Narcine. Many electric fish can detect each other’s discharges, and there is evidence that weakly electric organs can function in intraspecific com-

’Chapter 10 of this volume should be consulted for verification or elaboration of statements made in the present chapter about physiology and discharge patterns of these organs. A briefer review of electric organs and electroreception is given by Bennett (1970), and a short summary of most recent data appears in Bennett (1971b).

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munication ( Bullock, 1970; Black-Cleworth, 1970; Mohres, 1957; Moller, 1970; Westby and Box, 1970). Strongly electric organs could in principle operate in an e1ectrolocat;on system, but their use largely follows detection of prey (Bauer, 1968; Belbenoit, 1970; Pickens and McFarland, 1964). An important reason for the earlier studies of electrosensory systems was their extraordinary sensitivity. Thresholds of 0.01 and 0.1 pV/ cm have been reported in behavioral experiments (Dijkgraaf and Kalmijn, 1966; Machin and Lissmann, 1960). These sensitivities exceed those of ordinary fish by a factor of lo4 or more (Lissmann and Machin, 1958). As it turns out, the membrane processes at electroreceptors are probably similar to those at many other receptors. The sensitivity appears to be mainly a result of arrangement of external structures rather than of specialization of the receptor membranes themselves. However, the electroreceptors provide a useful model for certain stages of receptor action of much wider occurrence. This is because their specialization for detection of extracellular signals makes accessible to the experimenter a stage of action that is generally quite inaccessible.

11. DISTRIBUTION OF ELECTRORECEPTORS

Most electroreceptors fall into two classes: the tonic and the phasic receptors, each with its characteristic morphological and physiological properties. The tonic receptors are tonically and fairly rhythmically active. They give tonic or long-lasting responses to low frequency or dc stimuli although accommodation may be more or less marked. They are “ampullary” organs and have an obvious canal leading from receptor cavity to exterior. Phasic receptors are sensitive to relatively high frequencies and are insensitive to maintained or dc stimuli. They give only brief or phasic responses to step changes in stimulating voltage. Spontaneous activity if present is irregular under normal conditions. These receptors are “tuberous,’’ that is, there is no obvious channel from receptor cavity to exterior and the connection is presumably by way of intercellular clefts. There is some confusion in the literature as to nomenclature and function of electroreceptors, and a summary of types based on the most recent morphological and physiological data is given in Table I. All putative and known electroreceptors have been termed “ampullary” by Dijkgraaf (1963), but at this time it seems preferable to divide them into ampullary or tonic receptors and tuberous or phasic receptors, the choice of term depending on whether morphological or functional classification is intended.

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Both tonic and phasic receptors are found in the gymnotids and the mormyrids, and probably in Gymnurchus as well. These two groups appear to have evolved electrosensory systems independently, and the degree of convergence in the receptors is as remarkable as that in the electric organs. Phasic receptors are absent in nonelectric and marine fish. The electroreceptors in catfish are tonic as are the ampullae of Lorenzini of electric and nonelectric marine elasmobranchs. The similarities are SO great between the receptors of the same type in different species that it is more convenient to discuss the receptors in terms of function rather than phylogenetic relations. In mormyrids the electroreceptors are of three kinds (Table I ) . They have been termed large, medium, and small receptors although there is some overlap in size of the different kinds. They are found over the entire head and along the dorsal and ventral surface, but they are absent on the sides of the body and caudal peduncle. The detailed distribution of the small or tonic receptors is shown in Fig. 1 (see Harder, 1968). The distribution of the medium (phasic) receptors is similar, but there are about twice as many. Large receptors, also phasic, occur in about the same number as the small receptors but are somewhat less uniformly distributed. The receptors are innervated by either anterior or posterior lateral line nerves ( Fig. 2 ) .

Fig. 1. Distribution of small (tonic) receptors in the mormyrid, Gnathonernus petersii. The receptors as observed in the flattened skin are shown as dots. The regions containing receptors are crossed hatched in the diagram. The electric organ is just anterior to the caudal fin as indicated. From Harder ( 1968).

498

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L. BENNETT

DORSAL BRANCH OF POSTERIOR

LINE NERVE BORDER OF E LECTROREChOR REGIONS

Fig. 2. Innervation of electroreceptors in the mormyrid, Gnathonernus petersii. The head region is innervated by the anterior lateral line nerve. The dorsal strip of receptors is innervated by both medial and dorsal branches of the posterior lateral line nerve. The ventral strip of receptors is innervated by only the medial branch. Ordinary lateral line organs are found a t the level of the medial branch and are innervated by it. From Harder ( 1968).

The distribution of receptors in a representative gymnotid is shown in Fig. 3. The phasic receptors are much more numerous than the tonic receptors and most have been omitted from the figure. The density of receptors is greatest in the head region and falls off gradually toward the posterior. There are minor morphological subdivisions within the tonic and the phasic receptors, but no physiological correlations have been obtained (Lissmann and Mullinger, 1968; Szabo, 1965; Szamier and Wachtel, 1970). The electroreceptors over the entire body are innervated by the anterior lateral line nerve, a large branch of which runs posteriorly to join the posterior lateral line nerve just behind the head (Fig. 4 ) . The posterior lateral line nerve contains only mechanoreceptive fibers ( Ben-

?

S ternarchus Fig. 3. Distribution of electroreceptors in the gymnotid Sternarchus. The tonic, ampullary receptors are indicated by diamonds. The more numerous phasic, tuberous receptors are shown only in a small square in the middorsal region, but their density per square millimeter in various regions is indicated by the numbered arrows. The dashed line shows the location of the canal organs of the posterior lateral line which are indicated as squares for a short distance in the middle region. From Szabo ( 1965).

11.

499

I 1

(MECHANORECEPTIVE)

Fig. 4. Cranial nerves of the electric eel Electropholuls, which is a gymnotid. The anterior lateral line nerve innervates cranial electroreceptors (and canal organs) and sends a branch posteriorly to innervate all the caudal electroreceptors as well. The posterior lateral line nerve innervates only mechanoreceptors of the lateral line and free neuromasts if present. From De Oliveira Castro ( 1961).

nett, 1967; Suga, 196%) which come from free neuromasts and canal organs (see Chapter 8 by Flock, this volume). The electroreceptors of catfish are distributed over the entire body surface but tend to be concentrated in the head region (Fig. 5; Dijkgraaf, 1968). Receptors are even found on the fins. Innervation of the receptors

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M. V. L. BENNETT

Fig. 5. Distribution of tonic, ampullary electroreceptors of the transparent catfish, Kryptopterus bicirrhus. Each small spot represents a receptor. The direction toward which the ampulla opens is indicated in several places (see also Fig. 16). From Wachtel and Szamier (1969).

is by the lateral line nerves (Dijkgraaf, 1968; Roth, 1969). Ampullae of Lorenzini are found in most elasmobranchs (Dotterweich, 1932). The distribution varies with the species and is diagrammed for a skate in Fig. 6 and for a shark in Fig. 7 . Again, the innervation is by the lateral line nerves (Fig. 7 ) . There are differing usages as to whether the lateral line nerves are termed part of the eighth nerve, acoustico-lateralis system or whether different branches are considered part of the facial and vagal nerves ( Figs. 2, 4, and 7 ) .The central connections are those of acoustico-lateralis system, and the former terminology seems preferable (Berkelbach van der Sprenkel, 1915). In freshwater electric fish, electroreceptors are generally visible under

Fig. 6. Ampulla of Lorenzini of the skate Raja. The canals on dorsal and ventral surfaces are shown on left and right, respectively. The canal openings are indicated by dots. From Murray ( 1962).

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501

TR H Y O M A N D VII

R M A N D EX1 VENTR VII

R M A N O I N T VII

R M A N O EX1 DORS VII

(C)

Fig. 7. Distribution and innervation of ampullae of Lorenzini of the shark, Scyliorhinus cunicula. ( a ) Side view of the head indicating the course of ampullary canals and their openings (dots). ( b ) Dorsal and ventral views. ( c ) The innervation is diagrammed. The numbered areas outline the locations of groups of ampullae, the openings and canals of which are shown in ( a ) and ( b ) . Groups 1 4 comprise the supraorbital capsule, groups 5-9 comprise the infraorbital capsule, and group 10 comprises the mandibular capsule. From Dijkgraaf and Kalmijn ( 1963).

a dissecting microscope as specialized regions on the surface of the skin ( Fig. 8 ) . They are particularly clearly seen in heavily pigmented animals because the receptors themselves are unpigmented. To study individual receptors, it is convenient to isolate a single active fiber in the lateral line nerve and to establish its electroreceptive nature by its response to gross stimulation. Then a monopolar stimulating electrode can be moved over

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M. V. L. BENNETT

Fig. 8. External appearance of receptors in the gymnotid Gymnotus. From the dorsolateral quadrant near the middle of the body of a fish about 25 cm long. Rostra1 to the top, ventral to the right. Pigment-free spots of three kinds can be seen: neuromasts ( N ) , phasic electroreceptors ( P ) , and tonic eledroreceptors ( T ) . The latter occur in clusters. Scales and individual pigment cells are also visible. From Bennett ( 1967).

the surface of the body while determining changes in sensitivity with position. Inevitably in the species studied to date a maximum of sensitivity is found over a single receptor or cluster of receptors. Separate stimulating and recording electrodes can then be placed at the opening of the receptor. When current is passed between the stimulating electrode and an indifferent electrode in the bath, a potential is developed outside the receptor. Anodal and cathodal stimuli are defined as those where the stimulating electrode is positive and negative, respectively. Stimulation of this kind causes relatively little potential change inside the body (Section V, A ) . The potential recorded at the receptor opening is a good measure of the potential across the skin, and the potential across the skin (or change in it) is the stimulus parameter that affects the receptors. The role of the skin may be clarified with reference to the equivalent

11. ELECTRORECEPTION

503

circuits of Fig. 8. A receptor can be represented by a single arm of a circuit where the skin is a distributed system around it with elements T,, ri, and T, representing resistances of external medium, internal tissue, and the skin, respectively. The “access resistance” from the indifferent, distant electrode to the inside surface of the receptor is small compared to the resistance through the receptor. Thus, a locally applied stimulus results in a potential at the inside surface that is negligible with respect to the potential produced at the receptor opening. In the next part of this chapter, Sections I11 and IV, the responses of the receptors to locally applied stimuli are described. In these experiments it is generally convenient to immobilize the fish and block its electric organ discharge by curarization.

111. TONIC ELECTRORECEPTORS

A. Characteristics in Gymnotids and Mormyricls The morphology of a typical tonic receptor in the mormyrids and gymnotids is diagrammed in the upper part of Fig. 9, and light micrographs of tonic receptors from a mormyrid and a gymnotid are shown in Figs. 10 and 11. In these receptors there is a canal that opens to the exterior and usually enlarges at its base to form an ampulla. A number of receptor cells are embedded in the wall of the ampulla. Only a small part of their circumference is exposed to the lumen, although the surface in this region may be increased by microvilli (Barets and Szabo, 1965; Lissmann and Mullinger, 1968; Szamier and Wachtel, 1969, 1970). The receptor cells synapse with the single afferent fiber on their inner surface and are largely surrounded by what are presumably supporting cells. The synapses are characterized by large numbers of presynaptic vesicles and by a presynaptic ribbon or dense body (Fig. 12). Similar features are found at other receptor synapses of the acoustico-lateralis and visual systems of vertebrates ( Wersall et al., 1965; Dowling and Boycott, 1965). Over much or all of the body the epidermis contains a specialized zone that consists of many layers of flattened cells ( F in Figs. 10 and 11). Presumably, this layer is responsible for the very superficial resistive barrier that can be demonstrated in the skin (Section V, A). This layer is closely apposed to cells of the wall of the receptor canal. There are zonular tight junctions between cells of the canal walls and the supporting cells around the receptor cells. There are also tight junctions between the supporting and receptor cells (Fig. 13; Szamier and Wachtel, 1969,

M. V. L. BENNETT

504

Receptor cell

External, interna1,and skin resistances

(b)

Fig. 9. Anatomical diagrams and equivalent circuits of electroreceptors in freshwater fish: ( a ) tonic, ampullary receptor and ( b ) phasic, tuberous receptor. Cross sections through the receptors are shown with the external medium to the top. The skin and walls of the receptor cavities are represented by lines; innervation of the receptor cells is indicated. The opening to the exterior of the phasic receptor is shown as occluded by a porous mass; resistances of the external medium ( r e ) , skin ( T ~ ) and interior ( r i ) are included in the circuits. Resistances of receptor cell cytoplasm and canals are presumably small and are omitted. Electrodes for stimulating and recording with respect to a distant electrode are shown at the receptor opening. From Bennett (1967).

1970) . These junctions presumably completely occlude the extracellular space in a band around the cell apices (Farquhar and Palade, 1963). Thus, they prevent leakage of current through intercellular clefts and tend to channel current through the receptor cells (cf. Bennett and Trinkaus, 1970; Waltman, 1966). Direct measurement shows that the receptors are sensitive to the potential across the skin and are insensitive to gradients tangential to the skin (Section V, A ) , which is consistent with the morphological relations. Probably the potential across the skin spreads with little decrement down the receptor canal to appear across the receptor cells.

,

11. ELECTRORECEPTION

505

Fig. 10. Histology of a small (tonic) receptor of the mormyrid Gnathonemus. The section is perpendicular to the skin surface and the external environment is at the upper edge of the figure. The ampullary lumen ( L ) is connected to the exterior by an obvious canal. Receptor cells ( R ) are embedded in the wall of the receptor cavity. Two branches of a myelinated nerve fiber ( N ) lie at the base of the receptor. The layer of flattened cells ( F ) is seen to abut on the wall of the receptor canal. Toluidine blue stained section about 2 p thick of Epon embedded material (see Szamier and Wachtel, 1970).

506

M. V. L. BENNETT

Fig. 11. Histology of a tonic receptor of the gymnotid Eigenmannia. The lumen

( L ) of the receptor cavity is connected to the exterior by a canal. Receptor cells ( R ) are embedded in the wall of the receptor cavity. The dark oval region at the base of each receptor cell is the terminal of the afferent nerve fiber. The layer of flattened cells ( F ) abuts on the wall of the receptor canal. From Szamier and Wachtel ( 1969).

11. ELECTRORECEPTION

507

Fig. 12. Fine structure of a synapse from a tonic receptor of the gymnotid Eigenmanniu. A short process from the receptor cell ( R ) invaginates into the nerve terminal ( N ) . A presynaptic dense body ( D ) extends into the synaptic process. There are numerous presynaptic vesicles ( V ) . From Szamier and Wachtel ( 1969).

508

M. V. L. BENNETT

Fig. 13. Relations between supporting and receptor cells of a tonic receptor of the gymnotid Eigenmannia. At their apical margins bordering on the receptor lumen ( L ) , the supporting ( S ) and receptor ( R ) cells form tight junctions ( T ) . These junctions appear to completely occlude the extracellular space between the cells. A presynaptic dense body ( D ) is seen at one region of apposition between the receptor cell and afferent nerve fiber ( N ) which is filled with mitochondria. From Szamier and Wachtel ( 1969).

The operation of a tonic receptor appears to be as follows: The potential across the receptor cell is largely developed across its inner face which is of relatively higher resistance; this face is presynaptic to the innervating nerve fiber. A transmitter substance is secreted from the inner face in the absence of stimulation, and this transmitter depolarizes the

509

11. ELECTRORECEPTION

nerve fiber and causes its tonic discharge. Stimuli that depolarize the inner face of the receptor cell, i.e., make its cytoplasmic surface more positive, increase the rate of transmitter secretion and hence the discharge frequency in the nerve. Stimuli that hyperpolarize the inner face decrease the rate of transmitter secretion and thereby decrease the discharge frequency in the nerve. A typical response of a tonic receptor is shown in the inset of Fig. 14. These data are from the gymnotid Gymnotus in which the tonic receptors occur in clusters that are innervated by a single nerve fiber (Fig. 8 ) . There is a steady spontaneous nerve discharge at a frequency that in some receptors exceeds 100 per second (Fig. 14A). An anodal stimulus applied at the receptor opening (which would depolarize the inner face of the receptor cell) causes an increase in the frequency of discharge with some accommodation during the pulse (Fig. 14B). The termination of the pulse is followed by a silent period. A cathodal stimulus causes a decrease in the discharge frequency, again with some accommodation during the pulse (Fig. 14C). A period of increased discharge rate follows

I

-

\

Resting discharge level

- 0

s

-

D

-I

o

-

*

O

0

+I

Stimulus, rnV

Fig. 14. Responses of a tonic receptor of the gymnotid Gymnotus. Inset: upper trace, impulses in the afferent nerve fiber; lower trace, stimulating potential applied externally a t the receptor opening; ( A ) spontaneous discharge, ( B ) excitation by an anodal stimulus, and ( C ) inhibition by a cathodal stimulus. Positive potentials and anodal currents are indicated as upward deflections in this and subsequent figures. Graph: For the same receptor, the average impulse frequency during a stimulus of about 100 msec duration is plotted against stimulating voltage, anodal stimuli to the right, cathodal to the left. For small stimuli of either sign the input-output relationship is linear. From Bennett ( 1968a).

510

M. V. L. BENNETT

the stimulus. If a sufficiently large cathodal stimulus is given the receptor discharge is stopped completely during the pulse. The graph of Fig. 14 shows the number of impulses during the first 100 msec of the stimulus plotted against the strength of the stimulus. This represents an average frequency over a time when considerable accommodation occurs, but qualitatively the same result is obtained for shorter intervals. The change in average frequency is linearly related to the stimulus strength for small stimuli. For larger stimuli, the slope of the relation between impulse frequency and stimulus strength decreases. Stimuli of the order of 0.1 mV produce readily detectable changes in frequency. If sinusoidal stimuli are applied, the maximum sensitivity is obtained for frequencies of 30-50 Hz (R. E. Poppele and M. V. L. Bennett, unpublished data). Sensitivity falls off at both lower and higher frequencies. The falloff at low frequencies is equivalent to accommodation observed with rectangular stimuli. Accommodation reduces the dc sensitivity of the receptors, but considerable sensitivity remains at frequencies as low as 0.1 or even 0.01 Hz. Several lines of evidence indicate that transmission at the synapse between receptor cell and nerve fiber is chemically mediated. First, the morphological characteristics of the synapse are those typical of chemically mediated transmission (Fig. 12), and there are no regions of close apposition between the membranes such as are found at sites of electrically mediated transmission (see Figs. 71 and 72 of Chapter 10, this volume). A second indication of chemically mediated transmission is that if a strong brief anodal stimulus is given, the evoked responses long outlasts the stimulus (Fig. 15A). If such a stimulus is followed by a strong cathodal stimulus, one much stronger than that required to block the spontaneous discharge, there is virtually no effect on the maintained discharge (Fig. 15B). Thus, following a brief stimulus there is a maintained increase in excitation that is inaccessible to what is normally an inhibitory stimulus. It is difficult to explain this phenomenon assuming electrically mediated transmission. It is easy to explain it, if transmission is chemically mediated. The effect could, for example, be a result of transmitter accumulated in the synaptic gap. It is more likely that secretion persists beyond the depolarization required to evoke it. Possibly a brief excitatory stimulus “commits” the cell to secrete a certain amount of transmitter [as might be caused by an accumulation of Ca2+inside the cell, a process which is thought to occur at the neuromuscular junction (Katz and Miledi, 1968)l. The latter kind of explanation is supported by the fact that the high frequency impulse discharge at the end of a strong, long-lasting anodal pulse stops almost immediately upon termina-

11. ELECTRORECEPTION

511

Fig. 15. Evidence for chemically mediated transmission at tonic receptors of the gymnotid Gymnotus: (A-C) same receptor as Fig. 14 and (D-F) a different receptor. Upper traces, recording from the afferent fiber; lower traces, potential a t the external opening of the receptor. ( A ) A brief stimulus evokes an acceleration of the tonic discharge that long outlasts the stimulus. ( B ) A strong cathodal stimulus immediately following the anodal stimulus has little effect on the accelerated discharge; the number of spikes in the response is reduced by only one, although the cathodal stimulus is far stronger than required to block the resting discharge completely (cf. Fig. 14). ( C ) At high gain and with the overlying water removed, the nerve impulse can be recorded external to the receptor. The impulse at the receptor precedes each spontaneous impulse in the nerve, and a very nearly identical response is evoked by antidromic stimulation of the nerve. Two superimposed sweeps are shown of stimulation close to point of recording in the nerve a t threshold for the single fiber. When this fiber is excited, there is an antidromic response a t the receptor and the next spontaneous discharge is delayed until after the sweep. Antidromic and orthodromic conduction times are nearly identical (dotted lines). ( D ) The first response to very strong anodal stimuli arises at a constant latency of about 1.6 msec; the time of impulse initiation is obtained from the orthodromic conduction time recorded in ( F ) (dotted lines). ( E ) Still stronger stimuli cause a jump in latency to a value of less than 0.2 msec, indicating direct stimulation of the innervating nerve. ( F ) The nerve impulse is recording at high gain from the receptor opening with the overlying water removed. The orthodromic conduction time is indicated by the dotted lines. The recordings at the receptor are at the same gain in ( A ) and ( B ) and in ( D ) and ( E ). The sweep speed is the same in ( A ) and ( B ) and in (D-F). From Bennett (1968a).

tion of the pulse (Fig. 14B). If transmitter could accumulate in the synaptic gap, it would be most likely to do so under these circumstances. A third indication that transmission is chemically mediated is that there is a synaptic delay. The time of initiation of impulses can be determined because the nerve impulse can be recorded external to the recep-

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M. V. L. BENNETT

tor, particularly when the overlying water is removed (Figs. 15C and F). (This procedure increases re in Fig. 9 and thus increases the fraction of the potential produced by the nerve impulse across ri that is recorded across T , . ) This small spikelike potential is shown to be the nerve impulse by the fact that antidromic stimulation of the afferent fiber produces a spike nearly identical to the orthodromic one (Fig. 15C). Thus, one can directly measure orthodromic conduction time from receptor to the site of recording in the afferent fiber. When large stimuli obscure the impulse recorded from the receptor itself, one can still determine the time of impulse initiation from the orthodromic conduction time and recorded afferent impulse. If the strength of an anodal stimulus is increased, the time of initiation of the first impulse is soon reduced to a plateau value of about 1.5 msec which is the synaptic delay plus time for the postsynaptic potential (PSP) to excite the nerve fiber ( Fig. 15D). In view of the high frequency of h i n g the rise time of the PSP would be expected to be very small. At a stimulus strength some 104 times greater than threshold, the latency suddenly jumps to a new shorter value that is close to zero (Fig. 15E). Apparently, the shorter latency results from direct stimulation of the afferent fiber. A delay of 0 5 1 msec or more is characteristic of chemically mediated transmission [at room temperature (cf. Katz and Miledi, 1965; Bennett et al., 1967a)], and the delays at receptor synapses are reasonable for this mode of transmission. If the receptor synapses were electrically transmitting, one would expect increasing stimulus strength to cause a gradual decrease in latency down to a very small value. The responses of mormyrid tonic receptors are virtually identical to those of the gymnotid receptors. A minor difference is that a single nerve fiber innervates only a single receptor with several receptor cells rather than a cluster of receptors. The same three lines of evidence indicate that transmission is chemically mediated: (1) the synapse has a synaptic gap and presynaptic vesicles; ( 2 ) a persistent excitation follows a brief excitatory stimulus and this excitation is little affected by an inhibitory stimulus; and ( 3 ) there is an apparent synaptic delay of about 1 msec ( Bennett, 1965, 1967). Morphologically similar receptors are found in Gymnurchus but have not been studied physiologically ( Mullinger, 1969).

B. Electrical Equivalent and Input-Output Relationships If the water over a receptor opening is replaced by air, a local electrode need pass much less current to produce a given voltage change outside the receptor; the shunting by the water is reduced, that is, re in

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the equivalent circuit of Fig. 9 is greatly increased. Although the degree of residual shunting by the skin and remaining water has not been satisfactorily evaluated, the current voltage relation measured under these conditions is likely to have considerable contribution from the receptor cells because the input resistances become large, as high as several megohms. Furthermore, receptor cells of phasic receptors contribute importantly to the external potentials under these conditions (Section IV) . The receptor cells of tonic receptors appear to behave very nearly like linear electrical elements; that is, their membranes have fixed internal potentials, resistances, and capacitances. They are, in this sense, electrically inexcitable, and as shown below they differ markedly in this respect from phasic receptor cells. Of course, electric stimuli do affect the receptor cells by causing them to secrete transmitter. There is evidence that at the squid giant synapse and the neuromuscular junction, an idlux of Ca2+is a necessary step in transmitter release (Katz, 1969; Katz and Miledi, 1969a,b). Depolarization of the presynaptic terminal causes an increase in Ca2+influx which causes further depolarization. Under certain circumstances this activity can lead to an all-or-none response. A small change in Ca2+permeability, which would cause only a small electrical nonlinearity, may well occur in the cells of tonic receptors. The short latency of responses to strong stimuli indicates that stimuli act directly on the presynaptic secretory membrane or at least very close to it. The minimum synaptic delay approaches 1msec, which would leave little time for diffusion of some activating substance from a sensitive site to the secretory membrane, It is likely that the outer faces of the receptor cells are of low resistance compared to the inner, secretory faces (as has been directly shown in Kryptopterus, see Section 111,C; Bennett 1971a,b). While not required, this feature would maximize the sensitivity of the receptors to the external fields, and as seen in Fig. 14 the receptors can be very sensitive. From the foregoing data one may draw an approximate equivalent circuit of the tonic receptors in which the inner and outer faces of the receptor cells can be represented as “lumped” resistances (Fig. 9 ) . In this circuit, membrane capacities are ignored, as are the resistances of the canal lumen and receptor cell cytoplasm. If the inner faces of the receptor cells are indeed of higher resistance than the outer faces, the inner faces should have a longer time constant because their area is larger and specific capacity of cell membranes is generally about the same. The time constant of the inner faces may be one factor limiting the high frequency response of the receptors. As stimulus frequency increases the voltage drop across these faces becomes smaller, and more of the applied

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voltage is developed across the series resistances of the ampullary canal, the receptor cell cytoplasm, and the animal's interior. The impulse frequency evoked by a step excitatory stimulus often accelerates gradually over several impulses, which could represent gradual charging of the capacity of the inner faces. The decreased responsiveness of tonic receptors at high frequencies accounts for the fact that these receptors are unaffected by the electric organ in the many fish in which the organ discharge contains little or no low frequency component (Bennett, 1970; Bullock and Chichibu, 1965; Roth, 1967; Suga, 1967a; Szabo, 1970). Transmitter released by the receptor cells presumably causes inward current to flow through the subsynaptic membranes of the nerve fiber and thereby initiates nerve impulses in nearby spike-generating membrane. The nerve impulses do not appear to involve the terminals themselves since they are monophasic and positive outside the receptor opening and have a duration similar to that of the intracellularly recorded action potentials in nerve fibers (Fig. 15; Bennett, 1965). These observations suggest that the terminal lacks spike-generating membrane and that its chemosensitive membrane is electrically inexcitable like that at most other synapses ( Bennett, 1964; Grundfest, 1966). This conclusion is supported by the observation that the nerve fiber can be directly stimulated by an anodal, but not by a cathodal, stimulus applied externally (Fig. 15; Bennett, 1967). The frequency of nerve impulses should provide a reasonable measure of the transmitter concentration, although there may be some distortion because of accommodative processes such as refractoriness of the spikegenerating membrane and desensitization of the subsynaptic membrane. One can conclude that in spite of distortions the relationship between presynaptic potential and transmitter release has the same shape as the graph of Fig. 14. There is a steady release of transmitter that is accelerated or retarded by stimuli of opposite polarity, and for small signals the response is linearly related to the stimulus strength. This excitation-secretion relation is strikingly different from that at the squid giant synapse ( Katz and Miledi, 1967b), the neuromuscular junction ( Katz and Miledi, 1967a), and the Mauthner fiber, giant fiber synapse in the hatchetfish (Auerbach and Bennett, 1969). In these synapses there is little transmitter release for small stimuli, and at some approximate threshold the rate of release rises sharply. Another important characteristic of the input-output relationship of the receptor synapses is sensitivity. A very small range of stimulation, about 2 mV in Fig. 14, encompasses most of the range of transmitter secretion. The corresponding range of presynaptic potential for the squid synapse is about 50 mV. It is also likely that the postsynaptic potential

11. ELECTRORECEPTION

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at electroreceptors exceeds the stimulus in amplitude; that is, the synapse amplifies the applied potential. Verification of this point will require intracellular recording from afferent fibers, but the change in discharge frequency of the nerve appears to require a PSP considerably larger than the stimulus. Amplification would, of course, be further evidence for chemically mediated transmission. Two alternative explanations of the relationship between stimulus strength and impulse frequency should be considered. It cannot be excluded by present data that stimuli act on a tonic release of an inhibitory transmitter that alters spontaneous activity of the nerve. Another hypothesis is that the nerve is spontaneously active and that excitatory and inhibitory stimuli cause the release of excitatory and inhibitory transmitters. This hypothesis also requires a linear relationship between secretion and stimulus strength and is unlikely in view of the constancy of the slope of the impulse frequency-stimulus relationship on either side of zero stimulus strength. Exclusion of these possibilities should be feasible where it is possible to penetrate the nerve fibers close to the synapses (Section 111, D ) . The hypothesis of a single excitatory transmitter requires that resistance decreases during the postsynaptic change in potential evoked by an excitatory stimulus and that the resistance increases during the response evoked by an inhibitory stimulus. C. Characteristics in Catfish Electroreceptors in the catfish Amiurus ( IctuZurus) are the so-called small pit organs (Mullinger, 1964; Roth, 1968, 1969). These receptors are similar to tonic receptors of freshwater electric fish except that generally the ampullary canal is shorter. Similar receptors occur in the electric catfish (R. B. Szamier and M. V. L. Bennett, unpublished data). The receptor synapse and relationships between receptor and supporting cells are like those of the electric fish, but the skin lacks the multiple layers of flattened epithelial cells that are found in the gymnotids and mormyrids ( Mullinger, 1964; Wachtel and Szamier, 1969). An intriguing variation is found in the transparent catfish Kryptopterus ( Wachtel and Szamier, 1969). In this species a cluster of receptor cells are located in a small beehive-shaped cavity that has an opening to the exterior on one side. The receptor cells of these receptors, particularly of the ones on the fins ( Fig. 5 ) , are readily visible in transillumination (Fig. 16). Potentials can be recorded in the receptor cells of Kryptopterus by passing a microelectrode in through the receptor opening (Bennett, 1971a,b). The receptor cells have a small internally negative resting po-

516

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Fig. 16. A tonic electroreceptor of the transparent catfish Kryptopterus. The tissue is not fixed or dissected. The receptor is located on the caudal fin between fin rays ( F ) ; anterior is to the left. The receptor cavity opens to the exterior at the arrow. Receptor cells ( R ) can be seen in the cavity. A receptor on the other side of the fin is out of focus. Striations of muscle fibers ( M ) are visible on the left.

tential, and most of a long-lasting voltage pulse applied at the receptor opening is developed across their inner rather than their outer faces, i.e., the inner faces are of higher resistance (they also have a longer time constant). Nerve impulses can be recorded either in the receptor cells or extracellularly just deep to the receptor cells, and the responses and sensitivity are similar to those of tonic electroreceptors of gymnotids and mormyrids. Responses have also been recorded from afferent fibers of electroreceptors in Amiurus ( Roth, 1968, 1969). Adaptation appears to be somewhat more rapid than in the gymnotid and mormyrid tonic receptors, and responses to maintained stimuli are quite small.

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D. Ampullae of Lorenzini The ampullae of Lorenzini have been most completely studied in the skate, but the properties are probably the same in all elasmobranchs. The ampullae themselves tend to lie deep beneath the surface, but they are connected to the exterior by canals that may be many centimeters long (Figs. 6 and 7). It has been found that the canal wall is of very high resistance, as high as 10 M a cm2, and that the space constant of the canal for low frequency signals is long compared to their length (Waltman, 1966). The capacity of the canal wall is appreciable, and there is considerable attenuation of higher frequency signals in longer canals. As in the tonic receptors of freshwater fish, the receptor cells are embedded in the wall of the ampullary cavity with only a small portion of their surface exposed to the lumen. In the skate there may be a single cilium extending into the lumen, a feature lacking in other electroreceptors studied ultrastructurally ( Waltman, 1966). At their bases the cells form typical receptor synapses with the innervating nerve fibers, where there are presynaptic vesicles and ribbons. A minor difference from the freshwater species is that several nerve fibers innervate the receptor cells in a single ampulla. As at the other tonic receptors, nerve fibers innervating the ampullae of Lorenzini are tonically active (Fig. 17A; Murray, 1962, 1965, 1967). However, at these receptors, the sign of stimulating currents is opposite to that in the freshwater species; cathodal stimuli applied in the canal lumen increase and anodal stimuli decrease the tonic discharge (Figs. 17B-D ) . The changes are approximately linear for small signals ( Murray, 1965). The receptors are also more sensitive than those in the freshwater species. A microvolt or two has been reported to produce a perceptible change in impulse frequency (Murray, 1967). The receptors show considerable accommodation to maintained stimuli, and a stimulus causing a strong acceleration is followed by a silent period (Fig. 17). The accommodation is associated with approximately constant sensitivity in terms of incremental stimuli; that is, a small brief stimulus evokes the same change in impulse frequency if it is given alone or if it is superimposed on a long-lasting stimulus after the impulse frequency has reached a new steady state plateau level. Receptor cells of ampullae of Lorenzini appear electrically excitable ( Obara and Bennett, 1968; Waltman, 1968). In many ampullae cathodal stimuli can evoke a rather long-lasting all-or-none impulse that recorded in the canal lumen is negative-going (Fig. 17D). In other preparations only a graded response is obtained. The polarity of the impulse is what one would observe if the outer membrane of the receptor cells generated

M. V. L. BENNETT

518 , -

w

n

h

5mV

5 mV IO-*A

Fig. 17. Regenerative responses of receptor cells and afferent impulses at ampullae of Lorenzini. From the skate Raja. Upper trace: recording from the afferent nerve of the mandibular group of ampullae. Second trace: microelectrode recording of activity of a single fiber from an ampulla in which there were recording (third trace) and current passing electrodes (fourth trace). ( A ) Tonic discharge; ( B ) a cathodal stimulus of less than 2 mV causes a strong excitation of the afferent fiber; ( C ) a stronger stimulus evokes a larger number of impulses; and ( D ) a still stronger stimulus evokes a negative-going impulse recorded in the receptor lumen that is associated with a further increase of impulses in the fiber. There is also a burst response (arrow) and silent period in the entire nerve indicating that other ampullae are being stimulated by the receptor response. A calibration pulse of -5 mV, 20 msec, appears at the end of the sweep. From S. Obara and M. V. L. Bennett (unpublished data).

an impulse of conventional polarity. Threshold for alteration of the nerve discharge is well below that for the impulse, but the impulse does lead to an increased nerve discharge. Sometimes a receptor impulse leads to a burst and ensuing silent period in the entire nerve running to a group of ampullae (Fig. 17D). Evidently, the impulse in the stimulated ampulla provides an adequate stimulus to excite neighboring receptors. It is possible to visualize the nerve fibers innervating the ampullac and to penetrate them with microelectrodes fairly near the terminals. The postsynaptic changes associated with excitatory and inhibitory stimuli can then be recorded. The PSPs in response to graded excitatory stimuli are shown in Figs. 18A and B. The minimum latency of the PSP is quite long, about 8 msec. Antidromic spikes reach a lower peak value if they are evoked during such PSPs. This result suggests that the resistance is decreased during the PSP and that the reversal potential for

519

> E

n

z

E

1 0 N

2 0 mV

5 mV

Fig. 18. Postsynaptic potentials in afferent fibers of the ampulla of Lorenzini. From the skate Raja. Upper trace: intracellular recording from the afferent fiber. Second trace: potential in the ampullary lumen owing to applied rectangular current pulses. Third trace [ ( C ) and ( D ) only]: applied current. ( A ) Increasing cathodal stimuli in the ampulla evoke first a subthreshold PSP in the nerve and then firing. The impulse at the beginning of the sweep is an antidromic impulse evoked by external stimulation of the afferent nerve (several superimposed sweeps). ( B ) As in ( A ) but at different gains and faster sweep speed. The minimum latency of the PSP is about 8 msec. ( C ) A different receptor. An anodal stimulus causes a hyperpolarization of the afferent fiber. ( D ) As in ( C ) but superimposed sweeps with anodal and cathodal stimuli. From S. Obara and M. V. L. Bennett (unpublished data).

the generating process is below the peak of the spike as at other excitatory synapses (Eccles, 1964; Bennett and Grundfest, 1961). An anodal ( inhibitory) stimulus has been observed to produce a hyperpolarization of the nerve in a few experiments (Figs. 18C and D). It has not yet been determined whether this potential change is associated with an increase in resistance. A consistent if complex explanation of the responses of the ampullae of Lorenzini can be made as follows. The outer faces of individual receptor cells in an unstimulated ampulla are spontaneously generating either impulses or graded oscillations at some moderate frequency. The activity in different receptor cells is normally asynchronous because each cell produces only very small potentials in the ampulla lumen and these potentials are insufficient to synchronize receptor cell activity. The activity of the outer faces of receptor cells depolarizes the inner faces and

520

M. V. L. BENNETT

causes the release of transmitter that leads to the tonic discharge in the nerve. Each nerve fiber synapses with many receptor cells; thus, the relationship between receptor cell and nerve frequency is generally not one to one. A small cathodal stimulus increases the frequency of activity of the receptor cells. The initial effect of a cathodal stimulus on the inner faces of the receptor cells is to hyperpolarize them, but the later and predominant effect is depolarization because of increased activity of the outer faces. Thus, more transmitter is secreted and the nerve frequency is increased. Conversely an anodal stimulus decreases the frequency of firing of the receptor cells and leads to a decrease in nerve discharge. (It is the decrease of nerve response evoked by anodal stimuli that requires the hypothesis that the receptor cells are tonically active.) As strength of a cathodal stimulus is increased, more and more receptor cells are brought into activity at the onset of the stimulus and a larger negativity is evoked in the lumen of the canal. In this manner the response can become sufficiently regenerative to lead to an all-or-none impulse. This theory predicts that strong cathodal stimuli would overcome the depolarizing effect of the spikes generated in the outer faces of the receptor cells and hyperpolarize the inner faces, thus blocking transmitter release and nerve discharge. Conversely strong anodal stimuli would directly depolarize the inner faces and lead to excitation of the nerve at a shorter latency than do cathodal stimuli. Both of these predictions have been verified (S. Obara and M. V. L. Bennett, unpublished data). Microelectrode studies of the receptor cells will be required to establish the proposed mechanisms. They explain several of the peculiarities of the Lorenzinian receptors, namely, the opposite polarity of excitatory stimuli and the long synaptic delay as compared to tonic receptors of freshwater species. The action of the stimulus on an oscillating electrically excitable system rather than on secretory membrane may be a factor in the greater sensitivity of the ampullae of Lorenzini. IV. PHASIC ELECTRORECEPTORS

The morphology of phasic receptors is distinctively different from that of tonic receptors. The receptor lumen is not connected to the exterior by an obvious canal, and for this reason the phasic receptors have been called tuberous organs (Szabo, 1965). Instead of a canal there is a plug of loosely packed epithelial cells between the lumen of the ampulla and the superficial layers of the epidermis (Figs. 19-21). Electron microscopy reveals clefts between cells of the epithelial plug and super-

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Fig. 19. Histology of a large receptor of the mormyrid Gnathonemus. The section passes through the base of one receptor cell ( R ) and the side of another. The lumen ( L ) of the receptor cavity is small and no obvious canal connects it to the exterior (top of figure). A mass of loose epithial tissue ( E ) lies over the receptor; the dark staining layer of flattened cells ( F ) joins the receptor wall and does not extend over the receptor cells. The afferent nerve fiber ( N ) looses its myelin close to the base of the receptor cell. The lumenal surface of the receptor cell is greatly increased by microvilli ( V and inset which is an electron micrograph). Preparation as for Fig. 10.

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M. V. L. BENNETT

Fig. 20. Histology of a phasic receptor of the gymnotid Hypopornus. The receptoi cells ( R ) protrude into the lumen ( L ) of the receptor cavity and are attached only at their bases. No obvious canal connects the lumen and the exterior. The layer of flattened cells ( F ) is interrupted over the receptor by a plug of loose epithial tissue ( E ) . The afferent nerve fiber ( N ) looses its myelin as it enters the receptor capsule. From Szamier and Wachtel ( 1970).

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Fig. 21. Histology of a medium receptor of the mormyrid Gnathonemus. There are inner ( L i ) and outer ( L o ) receptor cavities that are connected by a canal ( C ) , but there is no clear canal to the exterior. The outer cavity extends above the dark staining layer of flattened cells and the canal between cavities penetrates this layer. Receptor cells (R,) of the outer cavity lie embedded in the wall, but receptor cells ( R i ) of the inner cavity protrude into the lumen. Afferent nerve fibers are present. Preparation as for Fig. 10.

524

M. V. L. BENNETT

ficial epidermis (Barets and Szabo, 1964; Derbin and Szabo, 1968; Lissmann and Mullinger, 1968; Szabo and Wersall, 1970; Szamier and Wachtel, 1970; Wachtel and Szamier, 1966). The layers of flattened cells that presumably increase the skin resistance do not cover the receptor, but they are closely apposed to the cells forming the receptor wall which is roughly cylindrical with ordinary epidermis at the top and the receptor cells at the bottom. Presumably there is little electrical resistance between the receptor lumen and exterior, but evidence is lacking beyond the morphological relations and receptor sensitivity to applied fields. As in the tonic receptors, zonular tight junctions are present between cells abutting on the receptor lumen including the receptor cells (Fig. 22; Lissmann and Mullinger, 1968; Szamier and Wachtel, 1970). Presumably these junctions increase current crossing the receptor cells by sealing off the intercellular clefts. The receptor walls of phasic receptors tend to have many more layers than those of tonic receptors. This feature should reduce the capacity of the wall, and perhaps it is related to the greater sensitivity of phasic receptors to high frequencies. In gymnotids the phasic receptor cells protrude' into the receptor cavity and only their bases are attached. The largest part of the surface is exposed in the cavity and this portion is greatly increased by a microvillous border ( Figs. 19 and 22). Similar characteristics are present in two out of the three kinds of phasic receptor cells in mormyrids. At the base of the receptor cells are their synapses with the innervating nerve fiber. As at tonic receptors, there are presynaptic vesicles and ribbons or dense bodies (Fig. 23; Derbin and Szabo, 1968). There is evidence that transmission is chemically mediated except in one case in which it appears to be electrically mediated. The equivalent circuit of a phasic receptor is similar to that of a tonic receptor in respect to the resistances of the skin, external medium, and internal tissue. However, the outer face of the receptor cells behaves as a series capacity while the inner face has electrically excitable as well as secretory membrane (Fig. 9). The inner face may generate graded responses or even spikes. The presence of a series capacity explains several characteristics of phasic receptors, or to put it more logically, the demonstration of these characteristics provides evidence for the existence of the series capacity. The effects of a series capacity can be described with respect to Fig. 24 and the equivalent circuit of Fig. 9. If a voltage step is applied outside the receptor, the potential across the inner face of the receptor cells is quite distorted. Initially, there is no voltage drop across the capacity and a large part of the applied voltage appears across the inner face (Figs. 24A and B). Gradually, however, the capacity of the outer face is charged

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Fig. 22. Fine structure of the attachment region of a phasic receptor cell in the gymnotid Hypopomus. The receptor cell ( R ) is attached over only a small region; its lumenal surface is greatly increased by microvilli ( V ) . The receptor cell, supporting cells ( S ) , and cells of the receptor wall ( W ) form tight junctions ( T ) at their margins bordering on the lumen of the receptor cavity. Three short processes from the receptor cell invaginate into the nerve terminal ( N ) . From R. B. Szamier and A. W. Wachtel (unpublished data).

through the resistances in the circuit. Ultimately all the applied voltage is developed across this membrane and no part of the stimulus appears across the inner face. Assuming that the inner face is not excited, the potential across this face decays exponentially from its maximum just after the onset of the stimulus. At the termination of a long-lasting pulse, the opposite sequence of potential changes occurs. Initially, a large fraction of the external potential change is developed across the inner face, and then this potential

526

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Fig. 23. Fine structure of a synapse from a phasic receptor of the gymnotid Eigenmannia. There are numerous presynaptic vesicles ( V ) . A process from the receptor cell ( R ) invaginates a short distance into the nerve terminal ( N ) , and membranes in this region are somewhat denser. A presynaptic dense body ( D ) extends into the receptor cell process. The synaptic gap appears to contain material connecting the two membranes. From R. B. Szamier and A. W. Wachtel (unpublished data).

decays away until, again, there is no stimulus across the inner face. An important fact is apparent from this discussion and Figs. 24A and B. The potential change across the inner face at the onset of a long-lasting anodal stimulus is identical to that at the termination of a long-lasting cathodal stimulus of equal amplitude. Similarly, the potential change at the onset of a long-lasting cathodal stimulus is the same as that at the termination of an equal anodal stimulus. From Fig. 24 it is seen that when a long-lasting stimulus is applied, the potential across the inner face will return to its resting value after the initial transient. If sufficient time is allowed for recovery from any response evoked by the initial transient, excitability will return to the same value as it has in the absence of the maintained stimulus. Thus, if a brief test pulse is superimposed on a long-lasting stimulus, the receptor will exhibit the same excitability as when the brief stimulus is given alone.

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V

Fig. 24. Effect of blocking capacity on potentials a t phasic receptors. Circuit in Fig. 9. Upper trace in (A-C), potential at the receptor opening, positivity upward; lower trace, potential that would be recorded across the inner face of the receptor cell, intracellular positively upward. ( A ) If a rectangular anodal voltage pulse is applied at the receptor opening, the inner face is depolarized at the onset of the stimulus, but the membrane potential then returns to its resting level. At the tennination of the stimulus, the inner face is transiently hyperpolarized. ( B ) If a rectangular cathodal voltage pulse is applied, the converse sequence is observed. Excitability of the receptor cell is neglected here. Also, the potential across the inner face would be smaller than the applied potential but is shown as larger as if recorded a t higher gain. ( C ) If the inner face generated a spike of the form shown on the lower trace, the external potential would be diphasic as indicated on the upper trace, the areas under the two phases being equal. Shown as if recorded a t the same gain. ( D ) Where the synapse between receptor cell and nerve fiber is chemically transmitting and there is a gap, nerve action currents (arrows) that make the exterior positive will tend to hyperpolarize the inner, excitable face of the receptor cell. ( E ) Where there is an electrotonic synapse between receptor cell and nerve fiber, action currents ( arrows ) making the exterior positive will depolarize nonjunctional membrane of the inner face. From Bennett ( 1967 ) .

This property is responsible for the phasic character of these receptors, that is, the absence of maintained impulse discharge in response to longlasting stimuli. In terms of receptor function, there is complete accommodation to maintained stimuli. A further property resulting from a series capacity is that impulses generated by the inner face of the receptor cell cannot cause a net current to flow in the external circuit. If the inner face generates a positive-going monophasic spike, a biphasic positive-negative potential will be generated externally ( Fig. 24C). During the rising phase of the impulse, current flows to make the charge on the capacity more inside positive; during the falling phase, the charge returns to its initial value. The total current flow during each phase is equal since no net charge passes through the capacity. Therefore if resistances in the current path around the receptor

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cell remain constant, the voltag+time integral of the externally recorded response will be zero, or in graphical terms the area under the positive phase of the externally recorded impulse will equal the area under the negative phase. In summary, three properties resulting from the presence of a series capacity have been described: (1) equivalence of onset and termination of long-lasting stimuli of opposite polarity; ( 2 ) absence of excitability change during maintained stimuli; and ( 3 ) absence of net current flow during externally recorded responses.

A. Large Receptors of Mormyrids These receptors contain 1-8 large receptor cells that are all innervated by a single fiber (Harder, 1968; Szabo, 1965). Those containing the larger numbers are the ones that are visually recognizable in vivo as belonging to this class of receptor. A cross section through the base of one receptor cell and tangential to another is shown in Fig. 19. Each receptor cell is covered by microvilli on its lumenal surface and largely fills the cavity that it occupies. The response of a large receptor in the mormyrids is illustrated in Fig. 25. The first trace shows activity in the single fiber that innervates the receptor, the second trace shows the potential recorded outside the receptor, and the third trace shows the stimulating current. Figure 25A and E give the threshold responses to anodal and cathodal stimuli which are long lasting in that lengthening them has no effect on the responses. At the onset of the anodal stimulus, a brief all-or-none impulse is recorded outside the receptor. The response to a cathodal pulse is identical but occurs following the termination of the stimulus at a threshold amplitude identical to that for an anodal stimulus. The externally recorded impulse is always followed by an impulse in the innervating fiber. In Fig. 25 the externally recorded response is about 5 mV in amplitude. When the preparation is immersed in water the spikes are 1-2 mV in amplitude. When the skin is allowed to dry, the amplitude can approach 20 mV. The maximum potential is always obtained when the recording electrode is directly on the receptor. The thresholds of the large receptors, that is, the stimulus strengths required to evoke firing about 50%of the time, are usually 0.2-0.5 mV. Often the receptors are spontaneously active, and with sufficient data processing it is probable that they could be shown to be affected by much smaller voltages. When the stimulus strength is increased, the response changes little, except that it occurs in every sweep (Figs. 25B, C, F, and

11. ELECTRORECEPTION

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Fig. 25. Responses of a large receptor of the mormyrid Gnathonemas. Upper trace: recording from the afferent nerve fiber. Middle trace: recording from the receptor opening by means of a small, Ringer-filled electrode. Lower trace: polarizing current applied by means of a large water-filled electrode a t the receptor opening. The skin was somewhat dry, but spontaneous activity was reduced by connecting the large and indifferent electrodes by a relatively low resistance shunt (cf. Fig. 33). Several superimposed sweeps in ( A ) , ( D ) , ( E ) , and ( H ) . ( A ) Threshold anodal stimuli. (B,C) Increasing anodal stimuli. ( D ) Response to anodal stimuli of intermediate strength under conditions of increased spontaneous activity. A delayed spike sometimes occurs following termination of the stimulus. ( E ) Threshold cathodal stimuli. (F,G) Increasing anodal stimuli. ( H ) Recorded under the same conditions as in ( D ) , a delayed response sometimes follows the onset of cathodal stimuli. From Bennett (1967).

G). There continues to be only a single impulse in the nerve following each impulse outside the receptor. The shape of the externally recorded responses is virtually identical comparing onsets and terminations of stimuli of equal amplitude and opposite polarity (Figs. 25A-C and E-G) . Under some conditions that are not readily controlled, there can be somewhat delayed responses following the termination of anodal stimuli and onset of cathodal stimuli. As may be seen from Fig. 24, there should be at these times a transient hyperpolarization of the inner faces of the receptor cells. Presumably the delayed responses represent anode break responses as a result of the hyperpolarization. Again, each externally recorded impulse is followed by a single impulse in the innervating fiber. Very strong stimuli can cause repetitive firing. These responses require

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M. V. L. BENNETT

further investigation; they may be of physiological significance because the electric organ can provide stimuli in this range. Several lines of evidence suggest that the receptor cells generate the externally recorded impulse and that it is not generated by the afferent fiber. First, denervation of the skin leads to degeneration of the receptors in a few days to weeks. The externally recorded impulses and receptor cells disappear at about the same time and somewhat more slowly than the nerve fibers, although there is considerable overlap in time and time of block of nerve action potentials is unknown (Roth and Szabo, 1969).* The second line of evidence for a receptor cell-nerve synapse is that there is a point of low safety factor for transmission from the receptor to the innervating nerve (Bennett, 1967). If a pair of stimuli are given, the second nerve response can fail at a time when there is still a full-sized response outside the receptor. The point of failure is close to the receptor because a stronger testing stimulus can evoke a second nerve response after a shorter interval. The most reasonable place for the point of failure to be is at the junction between receptor cell and nerve fiber. A third line of evidence is pharmacological. Tetrodotoxin blocks nerve activity without affecting the externally recorded impulses (Zipser, 1971). A further but less satisfactory argument for receptor cell potentials at the large receptors is comparative; receptor cells of other phasic receptors generate responses that can be recorded external to the receptor opening. The data in Fig. 25 illustrate the first property described as resulting from a series capacity. The second property, absence of excitability change during maintained stimuli, is illustrated in Fig. 26. A brief stimulus evokes an externally recorded response and nerve impulse at the same threshold in the presence or absence of a maintained stimulus, whether the maintained stimulus is anodal or cathodal. The third property, absence of net current flow during an externally recorded response, is shown in Fig. 27. The impulse recorded outside the receptor is diphasic, and it can be seen particularly clearly in spontaneous discharges that the area under the positive phase is very nearly equal to that under the negative phase. It can be concluded that the large receptors do have a capacity in series with their responsive membranes. The polarity of the responses is consistent with generation of a conventional impulse by the inner faces of the receptor cells. Furthermore, the morphology suggests that the capacity is located in the outer face of the receptor cells. Calculations indicate that the series capacity of the receptor is compatible with the area * Earlier I stated that the receptor cells survive 1-2 months after denervation (Bennett, 1965, 1967). This is an error because I did not know that small branches from the main posterior lateral line nerve innervate some receptors in the dorsal group (Fig. 2 ) , and I had sectioned only the dorsal branch of the nerve.

11. ELECTRORECEPTION

531

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N

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Fig. 26. Insensitivity of a large receptor to maintained stimuli. From the mormyrid Gnathonemus. Same receptor, recording, and display as in Fig. 25; several superimposed sweeps in each record. ( A ) Threshold stimulation using a short pulse. ( B ) Superposition of the same threshold stimulus on a maintained anodal stimulus about 10 times stronger had no effect on the externally recorded response or nerve impulse. ( C ) A similar result was obtained when the test stimulus was superimposed on a large cathodal stimulus. Slow potential changes during the prolonged pulses were probably a result of capacity of the skin and neighboring receptors. From Bennett ( 1967).

of the outer faces and a specific capacitance of about 1 pF/cm2 which is typical of plasma membranes in general (Bennett, 1967). There is evidence-that transmission between receptor cells and nerve fiber is mediated electrically. The delay between excitation of the receptor and initiation of an impulse in the innervating fiber is very short, about 0.2-0.3 msec (Bennett, 1967; Steinbach and Bennett, 1971). This delay is shorter than the synaptic delay at chemically transmitting synapses at the same temperature, but it is quite reasonable for electrically mediated transmission (Bennett et al., 1967a). If the innervating fiber is antidromically stimulated, a small positive-negative potential is recorded outside the receptor. Occasionally this potential excites the full-sized external impulse, which arises near the positive peak of the externally recorded antidromic response. In most known cases, electrically mediated transmission occurs at low

532

M. V. L. BENNETT

. .

. ’

. *

&

.*.

:1

N

0.2 msec

Fig. 27. Absence of net current flow during externally recorded responses at a large receptor. From the mormyrid Gnathonemus. ( A ) A spontaneous impulse (several superimposed sweeps to show base line). The areas under 6rst and second phases are equal to within 10% as measured by planimeter. ( B ) When the receptor is oscillating, areas under the positive and negative phases are also equal to within 10%. The horizontal line shows the zero level of potential obtained by placing the electrode on the nearby skin. Voltage calibration in (A), 2 mV. From Bennett ( 1967).

resistance junctions between cells where current flows as diagramed in Fig. 24E (Bennett, 1968a). This arrangement means that if the nerve impulse generates a positive-going potential outside the receptor, there is simultaneously depolarization of the nonjunctional part of inner face of the receptor cell. If, on the other hand, transmission is chemically mediated, there is likely to be a gap between receptor cell and nerve fiber. A current producing an external positivity tends to hyperpolarize the inner faces of the receptor cells ( Fig. 24D). Excitation of the receptor cell by antidromic activity of the nerve, if it occurs, should be delayed until after the external positivity. Thus, the phase at which the full-sized antidromic spikes occur supports the hypothesis of electrically mediated transmission at the receptor synapse. At gymnotid phasic receptors, where there is convincing evidence of chemically mediated transmission, the receptor cell response to antidromic excitation of the nerve occurs as would be expected from the relationships in Fig. 24D. A further indication of electrically mediated transmission is that the threshold for nerve impulses from large receptors is virtually unaffected by high magnesium solutions ( Steinbach and Bennett, 1971). In contrast, chemically mediated transmission at medium receptors is profoundly depressed ( Section IVYC ) .

11. ELECXRORECEPTION

533

Ultrastructural studies of the large receptors are in progress and should provide further evidence as to mode of synaptic transmission. Available data indicate that small branches of the nerve form some receptor synapses typical of those where transmission is chemically mediated (Derbin and Szabo, 1968). Morphological evidence of electrically mediated transmission has not been found but could have been missed. It should be recalled that morphological features associated with chemically mediated transmission often occur at electrically transmitting synapses, even when there is little or no chemically mediated component in the PSPs ( Bennett et al., 1967a,b). Transmission at least at one other receptor synapse appears to be electrically mediated. At calyx synapses of the vestibular system the cuplike shape of the nerve terminal would greatly impede postsynaptic currents through chemically sensitive membrane on the inner surface of the calyx, and on this basis alone electrically mediated transmission appears likely ( Bennett, 1964). Recently, close appositions between receptor cell and afferent nerve have been found that resemble electrotonic synapses at other sites (Spoendlin, 1966; see Figs. 71 and 72 in Chapter 10, this volume).

B. Phasic Receptors of Gymnotids The phasic receptors of gymnotids generally contain 10 or more receptor cells in a single receptor cavity (Fig. 20). The cells protrude into the cavity, and the lumenal surface is further increased by microvilli (Fig. 22). All the cells are innervated by a single nerve fiber, and in some species (Eigenmunnia and Sterrwpygus) a group of receptors lying close together are also innervated by the same fiber. The response of a phasic receptor in the gymnotid Gymnotus is shown in Fig. 28. The first trace shows stimulating current, the second trace shows the response recorded outside the receptor, and the third trace shows the impulses in the innervating nerve. All but the largest stimulus (Fig. 28H) evoke damped oscillations outside the receptor. These responses occur at onset and termination of both anodal and cathodal stimuli. As the stimulus strength is increased, the responses to onset of anodal and termination of cathodal stimuli gradedly increase in amplitude and finally become spikelike (except in Fig. BH, see below). These are responses where the stimuli depolarize the inner face of the receptor cells. The responses to termination of anodal and onset of cathodal stimuli increase in amplitude as stimulus strength is increased, but they are progressively delayed. These are responses where the stimuli hyperpolarize the inner face of the receptor cells and presumably are analogous to

M. V. L. BENNETT

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Fig. 28. Responses of a phasic receptor of the gymnotid Gymnotus. Upper trace: Current applied at receptor opening. Middle trace: potential at the receptor opening. Lower trace: recording from the afFerent fiber. Slower sweep speed in (I-L), Description in text. From Bennett ( 1967).

anode break responses. In Figs. 28D and H there is apparently no response at these times. Other experiments demonstrate that stimuli causing a large hyperpolarization of the inner face of the receptor cells put them into an unresponsive state for a period of several hundred milliseconds (Bennett, 1967). The nature of the unresponsiveness is unknown, but its termination is signaled by a burst of impulses in the nerve and often an impulse-like response external to the receptor. The initiation of the unresponsive state explains the absence of responses to termination of the stimuli in Figs. 28D and H. Aside from these, there is good correspondence between the externally recorded responses to onset of anodal and termination of cathodal stimuli and between the responses to termination of cathodal and onset of anodal stimuli. Provided the stimuli are sufficiently strong and long lasting, there are multiple discharges in the nerve at both onset and termination of anodal and cathodal stimuli. For long-lasting stimuli of equal amplitude, the numbers of impulses correspond for onset of anodal and termination of cathodal stimuli and vice versa (Figs. 281-L). The correspondence does not hold for the shorter stimuli in Figs. 28A-C and E-G, presumably be-

535

11. ELECTRORECEPTION

cause the system had been left somewhat depressed by the responses to the onset of the stimuli. The externally recorded response of phasic receptors of Gymnotus shows no excitability change during maintained stimuli as illustrated in Fig. 29, an experiment identical in procedure to that of Fig. 26. This property also holds for the nerve discharge if the testing stimulus is given long enough after the onset of the maintained stimulus. Otherwise the nerve discharge evoked by the test stimulus is reduced as in Fig. 29B. In the gymnotids it is difficult to establish the absence of net current flow during a response because there are ordinarily no spontaneous impulses external to the receptors. The damped oscillations such as those following the stimuli in Figs. 28B and C do appear to be symmetrical around the line representing passive decay of the applied voltage, and in

(C)

-

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;I

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5 0 msec

Fig. 29. Insensitivity of a phasic receptor to maintained stimuli. From the gymnotid Gymnotus, recording and display as in Fig. 28. ( A ) Response to a brief test stimulus well above threshold, ( B ) When the test stimulus is superimposed on a strong anodal stimulus about 60 msec after its onset, the externally recorded response is unchanged, but there is a slight reduction in the number of afferent impulses ascribable to depression following the response to onset of the stimulus. ( C ) When the test stimulus is superimposed on a strong cathodal stimulus, both externally recorded and nerve response are unchanged, From Bennett ( 1967).

536

M. V. L. BENNETT

other experiments similar results are obtained with oscillations following much smaller and briefer stimuli (Fig. 32). All-or-none impulses can be evoked at the end of the unresponsive state induced by strong cathodal stimuli, and these responses show little net current flow. It can be concluded that these phasic receptors also exhibit the properties indicating the presence of a series capacity. As in the mormyrids the morphological relationships suggest that the capacity is in the outer faces of the receptor cells. The polarity of the externally recorded responses is consistent with a conventional polarity of responsiveness in the inner faces. Although the tendency to oscillate is unusually great, similar graded subthreshold oscillations are predicted by the Hodgkin-Huxley equations (Mauro et al., 1970). In gymnotid phasic receptors the response in the nerve is poorly correlated with the response recorded outside the receptor. There may be oscillations outside the receptor with no nerve discharge, and the nerve discharge can outlast the externally recorded oscillations. Also, when the external resistance is increased by removing overlying water, the small and brief nerve impulse can be recorded external to the receptor in the same way as at tonic receptors (Fig. 15; Bennett, 1967). These data by themselves suggest that the externally recorded oscillations are generated by the receptor cells and that transmission to the nerve is chemically mediated. Confirmation of chemically mediated transmission was obtained by experiments similar to those of Fig. 15 where it was found that strong cathodal stimuli do not block responses initiated by brief anodal stimuli and that there is a synaptic delay. Furthermore, the external response at phasic receptors can be obtained at least one month following denervation which is after externally recorded nerve impulses have disappeared at both phasic and tonic receptors (Bennett, 1967). Finally, tetrodotoxin blocks the nerve responses, but not the externally recorded oscillations ( Zipser, 1971).

C. Medium Receptors of Morrnyrids The medium receptors are morphologically more complex than other electroreceptors. There is an outer and an inner receptor cavity connected by a small channel (Fig. 21). The outer cavity is, as in other phasic receptors, covered over by loose epithelial tissue and the outer layer of epidermis but not by the layers of flattened epidermal cells which are closely apposed to the wall of the outer receptor cavity. Receptor cells occur in both cavities. In the outer cavity the cells are embedded in its wall; in the inner cavity the cells protrude into the cavity and are covered by microvilli on their lumenal surfaces. Three nerve fibers innervate each

11. ELECTRORECEPTION

537

receptor, but whether particular fibers end on one or both kinds of receptor cells has not been determined. The thresholds of medium receptors can be quite high, 10 mV or more, but in the range of stimulation provided by the electric organ (Section V, B). The response of a medium receptor is illustrated in Fig. 30. The middle traces show the external potential recorded at low gain. The receptor response is generally small compared to the stimulus required to evoke it, and in order to observe the response more clearly it may be convenient to use a bridge circuit that subtracts from the response a voltage step proportional to stimulus strength. The upper traces of Fig. 30 were recorded in this way. The lower traces show the response in the innervating fiber. Stimulating current and responses recorded externally by means of a bridge circuit are shown for another receptor in Fig. 31.

Fig. 30. Responses of a medium receptor from the mormyrid Gnathonernus. Upper trace: high gain recording at the receptor using a bridge circuit; rectangular current pulses were applied (input resistance of the receptor and skin, 1.4 MO). Middle trace: low gain recording from another electrode at the receptor opening. Lower trace: recording from the afferent nerve fiber. ( A-D ) Responses to increasing strengths of anodal stimuli. (E-H) Responses to increasing cathodal stimuli. In ( H ) four nerve impulses were evoked in the nerve, but the last occurred after the end of the sweep. The response at the termination of anodal and onset of cathodal stimuli is indicated by an arrow in ( Dj and ( H j. From Bennett ( 1967).

538

M. V. L. BENNETT

Fig. 31. Responses recorded external to a medium receptor of the mormyrid Gnathonemu. Upper trace: current applied at the receptor opening. Lower trace:

responses externally recorded by a bridge circuit. ( A ) Anodal stimulation. ( B ) Cathodal stimulation. Several superimposed sweeps at different stimulus strengths in each record. From Bennett ( 1965).

The externally recorded responses are graded in amplitude and increase with increasing strength of stimulation. At the onset of an anodal stimulus and termination of a long-lasting cathodal stimulus, the response is diphasic initially positive. There are corresponding impulses in the afferent fiber that decrease in latency and increase in number as stimulus strength is increased. Up to four impulses are present in the responses of Fig. 30. Somewhat larger numbers have been observed from other receptors. There is a small and brief external response at the onset of cathodal stimuli and termination of anodal stimuli. In some but not all afferent fibers there are impulses corresponding to this phase of stimulation (Bennett, 1965). These responses are of higher threshold than the other responses, and they also decrease in latency and increase in number as stimulus strength is increased. As would be expected, the voltage threshold for afferent impulses increases as stimulus duration is decreased. Surprisingly the threshold for sufficiently brief stimuli is lower for cathodal than for anodal stimuli (Bennett, 1965). The responses of medium receptors show equivalence of onset and termination of long-lasting stimuli of opposite polarity (Figs. 30 and 31). Also, their excitability is unchanged during maintained stimuli, These properties indicate the presence of a blocking capacity as at the other phasic receptors. It is difficult to determine whether externally recorded responses involve net current flow because of their small size and high threshold.

11. ELECTROFECEPTION

539

It is tempting to ascribe the two kinds of external response to the two kinds of receptor cell. The responses at onset of anodal and termination of cathodal stimuli probably represent either delayed rectification or graded regenerative responses of ordinary polarity in the inner faces of one kind of receptor cell. The responses at onset of cathodal and termination of anodal stimuli are unusual in that their latency changes little as stimulus strength is increased. They do not appear to be anode break responses and may represent increases in resistance during hyperpolarization similar to those observed in a number of other cells (Bennett and Grundfest, 1966). The brevity of these responses as compared to the others suggests that the blocking capacity in series with the generating membrane is different for the two response types. This observation is consistent with the responses being generated in the two kinds of receptor cell and with location of the blocking capacity in the outer faces of the cells. The briefer response would be most likely to arise in the outer receptor cells, the external surface of which is smaller and presumably of lower capacity. Nerve fibers that innervate both classes of receptor would then respond to either direction of change in stimulating voltage; those innervating only the inner receptors would respond only to onset of anodal and termination of cathodal stimuli. The relatively small outer face of the outer receptor cells would presumably not be important because of the low sensitivity of these cells. The lack of correspondence between external response and afferent impulses suggests that transmission from receptor cell to innervating nerve is chemically mediated as at the phasic receptors of gymnotids. This inference is supported by the fine structure of the receptor synapses (Barets and Szabo, 1964) and by the presence of a synaptic delay of about 1msec (Steinbach and Bennett, 1971). A limited amount of pharmacological data has been obtained from medium receptors ( Steinbach and Bennett, 1971). High magnesium solutions reduce transmitter secretion at neuromuscular junctions and interneuronal synapses, presumably by competing with Ca2+at some stage of the release process (cf. Katz, 1969). These solutions greatly increase the stimulus strength required to evoke afferent impulses from medium receptors. Nerve responses are not eliminated completely; at least a single impulse always remains. A tentative explanation is that Mg2+can, to a small extent, substitute for Ca2+in the release process. Transmission at medium receptors is not cholinergic; curare, atropine, and acetycholine have no effect. The levorotatory form of glutamate has been implicated as a transmitter at a number of synapses (Beranek and Miller, 1968; Usherwood and Machili, 1968). The medium receptors are excited by L-glutamate and by the related L-aspartate, but they are not affected by D-glutamate. In procedures to date the concentrations

540

M. V. L. BENNETT

required are large, but the data are nevertheless suggestive of glutaminergic transmission.

D. Phasic Receptors in Gymnarchus Two types of tuberous receptor are found in Gymnarchus. These may correspond to large and medium receptors of mormyrids, but they have not yet been adequately studied physiologically. In each receptor type there is the peculiarity that the receptor cavity invaginates into the receptor cell. However, numerous microvilli increase the lumenal surface far beyond that of the other surface (Mullinger, 1969), and action of the lumenal face as a blocking capacity is not unreasonable. One type of tuberous receptor (Szabo’s type B, Szabo, 1965) contains a single receptor cell but occurs in clusters innervated by a single fiber. The other kind (Szabo’s type C) contains one large and one small receptor cell innervated by the same fiber, which may also innervate several neighboring receptors of the same kind. It is tempting to homologize the latter receptors with the medium receptors of mormyrids (Szabo’s type B) because of the presence of two kinds of receptor cell. However, this type of receptor is supposed to generate the spontaneous potentials on the skin that resemble the potentials external to large receptors of mormyrids ( Szabo, 1962). The correspondence between receptor types in mormyrids and Gymnarchus requires further study.

E. Amplification and Oscillation at Phasic Receptors The external responses at phasic receptors of gymnotids show an interesting correlation with the organ discharge of the particular species. The period of evoked oscillations at the receptors is similar to the duration of each organ discharge (Bennett, 1967). The correlation holds whether organ pulses are emitted at high or low frequency. The responsiveness of the receptors appears to represent tuned amplification of the main frequency components present in the discharge. Most phasic receptors in the eel appear not to have regenerative electrical responses (Bennett, 1967). Perhaps the large voltages produced by even the weakly electric organ do not require the amplification present in phasic receptor cells of other gymnotids. Given a regenerative responsiveness like that of phasic receptor cells, it would not be surprising if maintained oscillations could be obtained under suitable conditions. In fact, in a number of gymnotids, phasic re-

541

11. ELECTRORECEPTION

ceptors begin to oscillate continually if the skin is allowed to dry (Figs. 32E and F). These oscillations cease immediately when the skin is wet; they result from reduced electrical loading of the receptor and are not a result of injury through dessication, The effect of loading is illustrated in Fig. 32 where a low resistance, saline-filled electrode over the receptor is connected to the indifferent electrode by a variable resistance. If the loading is small ( a high resistance connection to the indifferent electrode) a brief stimulus evokes an oscillatory potential that is only slowly damped (Figs. 32A and C). If the loading is substantially increased, the oscillation is much more rapidly damped; it is also slightly reduced in amplitude (Figs. 32B and D ) . Spontaneous activity of large receptors of mormyrids ordinarily is not so great as to obscure threshold measurements. If, however, the skin is allowed to dry the receptors will often oscillate at high frequencies (Figs. 33A and F). Depending on the species and receptor, the frequency can be in the range of 1OO0, 2000, or even 3000 impulses/sec (Harder, 1968).

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50msec

Fig. 32. “Ringing” of phasic receptors of the gymnotid Gymnotus. ( A-D) A large electrode (upper trace ) filled with aquarium water in agar and a smaller Ringer-agar electrode (lower trace) were placed over the receptor, the water was drained away, and the skin was allowed to dry. Stimulating currents were applied through the large electrode using a bridge circuit. (A, C ) The large electrode was connected to the stimulator by means of a 10 M a resistance. A cathodal stimulus of approximately 4 mV, measured by the small electrode, evoked a slowly damped oscillation. Well after the stimulus, there appeared to be little net current flow during the oscillation, which was nearly symmetrical around the base line. (B, D ) The large electrode was connected to the indifferent electrode by a resistance of 100 kn. The same voltage stimulus as measured by the small electrode evoked a smaller and more rapidly damped oscillation. The change from ( A ) to ( B ) was immediately reversed by changing the shunt resistance to its former value. Same sweep speed in ( A ) and ( B ) and in ( C ) and ( D ) . Lower trace gain is the same in ( A-D). Upper trace gain is higher in ( B ) and ( D ) than in ( A ) and ( C ) . ( E-F) A different experiment, in which the receptor was oscillating continually. Periods in which the nerve impulses tended to follow the oscillation one-to-one alternated with periods in which impulses failed altogether. From Bennett ( 1967).

542

M. V. L. BENNETT

(B)

J+

0

102

~

2msec

Fig. 33. Effect of shunting on spontaneous activity of a large receptor. From the morniyrid Gnathonemus. Recording by means of a small, Ringer-filled electrode. A large (about 1 mm diam) electrode filled with aquarium water, with a resistance of about 50 kf? cm is also placed over the receptor. This electrode is connected to the indifferent electrode by various resistances. ( A-F) From left to right: sample records from the small electrode, value of shunting resistance, and rate of spontaneous impulses. The sequence of changes in shunting resistance is from ( A-F). From Bennett ( 1967 )

.

Again, the oscillations result from reduced loading, for if the load is varied as in the experiment of Fig. 32, the rate of spontaneous activity is changed accordingly (Figs. 33B-E). As would be expected, the effect of loading is immediate (within 1 msec), which may be conveniently shown by using a high-speed relay to connect and disconnect the loading resistance ( Bennett, 1967). During maintained oscillations the nerve discharges cease to follow the receptor cell frequency (Figs. 32E and F ) and may cease altogether. The receptor thus becomes insensitive to stimuli other than the very strong ones required to cause a significant pause in the maintained oscil-

11.

ELECTRORECEPTION

543

lations. Although oscillations have been recorded from some mormyrid large receptors immersed in water (Harder, 1968), it is to be expected that these responses are infrequent under normal conditions and represent malfunctioning of the system. An important factor in the oscillations of submerged receptors is the conductivity of the water. The rate of spontaneous discharge of receptors submerged in low conductivity water is substantially reduced when a small quantity of salts is added (M. V. L. Bennett, unpublished data). It would be of interest in this respect to know the conductivity of the natural waters in which mormyrids are found. The input-output relationship of synaptic membrane of phasic receptor cells differs from that of tonic receptor cells in that there is little resting release of transmitter. It is difficult to be confident of the shape of the potential-secretion relationship because of active processes in the receptor cells. It is likely but uncertain that the secretory membrane of phasic receptors does have an input-output relationship which has a much greater slope than that at known interneuronal and neuromuscular synapses. At some phasic receptors the number of impulses in response to a stimulus rises sharply after a certain threshold is reached and then saturates ( Hagiwara and Morita, 1963; Szabo and Hagiwara, 1967). Since these receptors are activated by the electric organ and presumably operate in electrolocation, it makes biological sense for them to have a high threshold that lies in the range of stimuli provided by the organ and to be very sensitive to changes above this threshold, that is, to have a low incremental threshold once absolute threshold has been reached. These receptors would then be best able to signal small influences of objects in distorting the field of the electric organ. It is difficult to be confident of the amplitudes of the membrane responses of the receptor cells. The maximum values of the externally recorded responses are 10-20 mV when the skin is dry, but significant loading may still be present. Nonetheless, on the reasonable assumption that permeability changes underlie the responses, a small voltage must produce a fairly large change in permeability, and an impulse of 10-20 mV amplitude might be sufficient to move a sensitive receptor cell through the entire cycle of permeability changes associated with the response. Since loading blocks receptor oscillations, it probably reduces the amplitude of the transmembrane potentials, and at large receptors of mormyrids there is marked shortening of the externally recorded response (Fig. 3 3 ) . The large receptors of mormyrids and ampullae of Lorenzini are apparently comparable in terms of the energy required to stimulate, about

544

M. V. L. BENNETT

W sec. The threshold currents are similar for the two kinds of receptor, about 1@l0 A. The threshold voltage is about 100-fold greater for the mormyrid receptors, but the duration of stimulus required is at least 100-fold shorter (Bennett, 1965; Murray, 1967). The mormyrid receptors do not appear to be limited by electrical noise of the Johnson type under normal conditions ( Bennett, 1965), and the ampullae of Lorenzini should be even farther from this limitation. The mormyrid large receptors in particular are remarkable in that they are poised very close to threshold, generally without excessive spontaneous activity. Other nerve cells presumably can be held as close to threshold for short times, but subthreshold responses and accommodation would be expected to follow within a few milliseconds. V. RECEPTOR FUNCTION IN ELECTRORECEPTION

A. Accessory Structures and Receptor Responses to Potential Gradients in the Environment As the middle ear and lens are important to their sense organs, so are accessory structures important to electroreceptors. It has already been suggested that current is channeled through the receptor cells by the flattened cells in the skin and receptor canal and by the circumferential tight junctions between receptor and supporting cells. The morphological relationships as well as our general knowledge of synaptic transmission indicate that the receptors should be sensitive to the potential across the skin and insensitive to the gradients along it. Experimental verification of this point is shown in Fig. 34. In this experiment one recording electrode is placed external to a tonic receptor and a microelectrode is pushed through the skin to record the potential beneath the receptor. When stimuli are applied externally fairly close to the opening, the potential inside the fish is small and the positioning of the internal electrode is not very critical. When a stimulus is applied directly over the receptor, the sensitivity is greatest; the minimum current is required to generate a given potential across the skin and to evoke a given response in the nerve ( Fig. 34D ) . When the stimulating electrode is moved 2 mm away either rostrally (Fig. 34A), dorsally (Fig. 34B), ventrally (Fig. 34C), or caudally, a considerably larger current is required to produce the same potential across the skin, but the corresponding neural response is very nearly the same. (There is some slowing of the time course of the potential in Figs. 34A-C compared to Fig. 34D because of skin capacity and separation of the electrodes.)

11,

545

ELECTRORECEPTION

-+ I r

IP

Fig. 34. Sensitivity to potentials across the skin and independence of tangential gradients: a tonic receptor of the gymnotid Gymnotus. Upper trace: recording from an afferent fiber. Middle traces starting from the same base line: potential at the receptor opening and potential recorded by a microelectrode pushed through the outer layers of nearby skin; the former potential is the larger in each record. The potential across the skin is given by the difference between the two. Lower trace: stimulating current through a separate electrode. The stimulating electrode is 2 mm anterior in ( A ) , 2 mm dorsal in ( B ) , and 2 mm ventral in ( C ) . The same current pulse produces about the same potential change across the receptor and the same nerve response. ( D ) Stimulation over the receptor. A much smaller current than in ( A-C) is required to produce the same potential across the skin, which evokes nearly the same nerve response. The potential across the skin is less slowed by the capacity of the skin than in (A-C) because the recording and stimulating electrodes are closer together. Lower current gain in (A-C). From Bennett (1967).

An important aspect of this experiment is that it contradicts the hypothesis of Lissmann and Machin (1958) that the receptors are sensitive to the second spatial derivative of the potential over the surface of the skin. The ingenious feature of their hypothesis is that it predicts that the sensitivity to applied stimuli should be the same as that to distortions of the electric organ discharge, which is in fact approximately true. Since different receptors are apparently involved in the two modes of operation, the agreement may be fortuitous. In mormyrids the skin resistivity is about 50 kn cm2 which is some hundred times greater than that of goldfish or hatchetfish (the latter is a South American freshwater fish). The skin resistivity is much smaller in gymnotids, 1-3 kn cm', but it is still greater than in the other freshwater fish (Bennett, 1965, 1967). It is not clear to what extent these values are affected by current passing through tonic receptors in the area of measurement.

546

M. V. L. BENNETT

The resistive barrier is localized to the skin surface as shown in Fig. 35, which also illustrates the importance of the skin in determining the potentials across the receptor cells. Electrodes are placed as in Fig. 34D, and anodal and cathodal stimuli are applied that accelerate and retard the nerve discharge (Figs. 35A and B ) . Then the current electrode is moved 2 mm caudally and pressed against a superficial scratch in the skin; the polarity of sensitivity is now reversed, although the electrode does not penetrate the pigment layer of the dermis (Figs. 35C and D ) . The same trans-epidermal potential causes the same responses as in Figs. 35A and B, but the current required to produce the potential is greatly increased as well as altered in sign. Moving the electrode away from the skin a short distance reestablishes the same polarity as in Figs. 35A and B, but the sensitivity remains low. Evidently the current passes through the receptor cells in opposite directions depending on whether the electrode is just internal or external to a resistive barrier localized in the skin. Similar experiments indicate that the electroreceptors of catfish are sensitive to the potential across the skin and insensitve to the gradient along it ( M . V. L. Bennett, unpublished data; Roth, 1969). When a fish is placed in a uniform potential gradient, the sensitivity of a particular receptor is greatly dependent on accessory structures. It turns out that the maximum potential difference across the fish is a more

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+

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1

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Fig. 35. Insulating characteristic of the skin. Same receptor and display as in Fig. 34. (A, B ) Anodal and cathodal stimulation at the receptor opening; the external potential is the larger. (C, D ) The stimulating electrode is pressed against a superficial scratch in the skin 2 mm caudal to the receptor. A cathodal stimulus evokes acceleration and an anodal stimulus evokes deceleration; the potential internal to the receptor is the larger. Potentials across the skin associated with a given neural response are the same as in ( A ) and ( B ) . From Bennett ( 1967).

11. ELECTRORECEPTION

547

meaningful measure of stimulus strength than is the potential gradient, that is, voltage per unit length along the fish. Because of accessory structures, an applied voltage gradient multiplied by the diameter of a receptor cell gives a very great underestimate of the actual potential across the receptors. The simplest example is provided by the skate, the skin of which is of relatively low resistance. When the fish is placed in a uniform field, the field is not greatly distorted within the fish, although the body is of somewhat higher resistivity than seawater (Murray, 1967). As noted above, canals of the ampullae of Lorenzini have walls of very high resistance, and the space constants are quite long compared to the lengths ( Waltman, 1966). As far as dc potentials are concerned, the potential in the ampulla itself must be very close to that at the external opening of the canal. The receptor thus detects the difference in potential between the canal opening and the serosal side of the receptor cell. The receptor is like a voltmeter with its terminals at these points. If one assumes a uniform field, the stimulus to the receptor is given by the field (in volts per unit length) times the distance between receptor cells and canal opening times the cosine of the angle between the field and the axis of the canal. The stimulus is greatest if the canal is oriented in the direction of the field; the stimulus is zero if the canal is oriented perpendicular to the field. The fish obtains directional information by having many receptors with canals oriented in different directions (Fig. 6 ) . Canals are of different lengths, and receptors with long canals are sensitive to smaller uniform gradients but provide less spatial resolution. Receptors with long canals are also less sensitive to higher frequencies because of the capacity of the canal walls (Waltman, 1966). There may be a sacrifice of frequency response for sensitivity in these receptors, which could represent a useful adaptation to overcome noise. However, response of the receptor cells drops off at high frequencies in any case (Murray, 1965) and the more important limiting factor is undetermined. When uniform gradients are applied to mormyrids and gymnotids the situation is more complex. The skin is of high resistance and the inside of the fish is of much lower resistance than the external medium. The voltage gradients within the fish are thus much smaller than those in the external medium, although they have not been determined in detail. The individual receptors detect the difference between the external and the internal potential, where the latter is some average of the potential over the whole fish. In a uniform gradient the stimulus to the receptors depends on the maximum voltage across the animal. If the skin resistance is uniformly high, about one-half the maximum voltage across the animal is developed across the skin at either end (or side) in the direc-

548

M. V. L. BENNETT

tion of the field. For longitudinally oriented stimuli the receptors at the rostral and caudal ends of the fish are most affected. There is a smaller potential across the skin near the center of the fish, and receptors in this region are less affected. Since current enters the fish at one end and leaves at the other, the polarity of stimulation is opposite at the two ends. Near the center of the fish there is a point or rather a line around the fish where current is parallel to the body surface and there is no potential across the skin. Most fish are considerably longer than they are wide, and the maximum voltage across the animal is greater for longitudinally oriented fields than for transversely oriented fields. Thus, it would be expected that the fish's sensitivity is considerably greater for a field oriented in the longitudinal direction. The effect of receptor location on sensitivity to longitudinally oriented stimuli is illustrated for large receptors of a mormyrid in Fig. 36. There is no consistent variation along the body of thresholds to locally applied stimuli. However, longitudinally oriented stimuli excite receptors at the ends of the fish at much lower thresholds than they excite the receptors in the center of the body. Moreover, there is a sharply localized transition region anterior to which the receptors respond to onset of head-positive stimuli and caudal to which the receptors respond to onset of headnegative stimuli. These data indicate that when head-positive stimuli are applied, current enters the fish anterior to the transition region and exits posterior to it. The lower sensitivity near the center of the body indicates that a much smaller fraction of the applied voltage is developed across the skin in this region. The greater sensitivity of the receptors in the head region as compared to the most caudal ones may mean that the skin is of higher resistance rostrally; alternatively, the most caudal receptors are located well rostral to the tail (Fig. l ) , which also could account for their lower sensitivity. In freshwater electric fish the arrangement of the receptors and accessory structures probably represents a compromise between the requirements of detecting weak fields of extrinsic origin, of establishing the field distribution, and of setting up and measuring the field of the fish's own organ. The distribution of skin capacity may be important in receptor responses, particularly since tonic and phasic receptors are sensitive to different frequency ranges. Accurate measurements must be obtained to distinguish the resistance and capacity of the receptors from those of surrounding skin, and more complete studies of regional differences are required. The flattened epidermal cells of mormyrids ( F in Fig. 10) are present only in the receptor regions; the skin resistance is not much lower on the sides of the fish (Bennett, 1965),but presumably the capacity is greater. In Torpedo the skin resistance over the electric organs is lower than that over the remainder of the body, which tends to maximize cur-

11. ELECTRORECEPTION

549 Distance (cm)

I

O

Head-tail stimulation

Dorsal Ventral

..... . 0

0

0

00

Fig. 36. Responses of receptors to longitudinally oriented potential gradients. The diagram of the fish is at the same scale as the abscissa and shows the approximat6 positions of the receptors on the longitudinal axis of the fish; the stippling indicates the regions in which the receptors are located. In the graph points from the receptors in dorsal and ventral regions are indicated by filled and open circles, respectively. The points below the horizontal dashed line are voltage thresholds for stimulation by an electrode at the receptor opening. The points above the dashed line are thresholds for head-tail stimulation measured as the potential difference between head and tail. These thresholds have been normalized by multiplying by the ratio of 0.4 mV t o the threshold for local stimulation of that receptor. The vertical dashed line separates receptors that respond at make of head-positive stimuli from those that respond at make of head-negative stimuli. From Bennett (1965).

rent in the external medium (Bennett et al., 1961). Analogous differences may be found in weakly electric fish. The effects of body openings should be evaluated but may not be very important. The accessory organs of Steatogenys make the inside of the mouth positive with respect to the outside even when the mouth is open. The skin of freshwater catfish is probably of high resistance similar to the situation in freshwater weakly electric fish; the canals of the small pit organs are quite short; and a high skin resistance would be required to achieve a high receptor sensitivity. Although the skin lacks the layers of flattened cells found in the weakly electric fish (Wachtel and Szamier, 1969), the resistance could still be high because a single epithelial layer can have a very high resistance; however, the capacitance would be expected to be large (Waltman, 1966). Probably a high skin capacity would not be important to catfish because of lack of receptor sensitivity to high frequencies. In contrast to freshwater catfish the ampullary receptors of the marine

550

M. V. L. BENNETT

catfish Plotosus have long canals like the receptors of marine elasmobranchs ( Gkrard, 1947). Conversely ampullae of Lorenzini of the freshwater stingray have very short canals (R. B. Szamier and M. V. L. Bennett, unpublished data). It seems likely that the difference in canal lengths is an adaptation to the relative resistivities of fresh- and saltwater. The tissue of a marine fish is of higher resistivity than the water surrounding it, and even if its surface resistance is low the tissue will not “load’ the current pathway through the seawater surrounding the fish. In contrast the tissue of a freshwater fish is of considerably lower resistivity than is the water in its environment. If there were not a high surface resistance, the resistance of the environment would be large compared to the resistance of the fish and the voltage across the animal would be reduced. Because of its high surface resistance the freshwater electric fish presents less of a load to the environment. Actually, there seems to be some impedance matching in freshwater electric fish, at least as far as dc voltages are concerned. These fish do load their environment to some extent, as they should for maximum power dissipation across the receptors.

B. Receptor Responses to Electric Organ Discharges Many of the studies of afferent impulses evoked by organ discharge have not involved identification of receptor type, although it is often possible to infer from the response what kind of receptor was being stimulated. Because tonic receptors have low sensitivity to higher frequency (sinusoidal) stimuli, they do not respond to the electric organ discharge in species in which the discharge has little low frequency dc component (Bennett, 1970; Bullock and Chichibu, 1965; Roth, 1967; Suga, 1967a; Szabo, 1970). It appears then that in most electric fish tonic receptors act largely passively and detect low frequency signals of extrinsic origin. What are probably tonic receptors in the eel do respond to discharges of the weak electric organ (Hagiwara et al., 1965a); these pulses are monophasic and therefore have a significant low frequency component. The tonic receptors could probably also be activated by the electric organ discharge in the gymnotid species of Hypopoinus that has a monophasic discharge and in the mormyrid Mormyrw. The rostra1 accessory organs of several gymnotids also emit monophasic pulses. There may be some stimulation of tonic receptors by muscle action potentials and dc potentials such as those resulting from injuries of skin or muscle (Bennett, 1967; Bullock and Chichibu, 1965). In the dogfish, ampullae of Lorenzini apparently respond to potentials associated with the animal’s own res-

11.

ELECTRORECEPTION

551

piratory movements ( Dijkgraaf and Kalmijn, 1966). Conceivably, changes in these responses could signal the presence of objects. In many afferent fibers the presence of insulators and conductors alters the number of impulses evoked by the electric organ discharge (Bullock and Chichibu, 1965; Hagiwara and Morita, 1963; Hagiwara et al., 1965a,b; Szabo and Fessard, 1965). Except for the stimulation of tonic receptors noted above, all of these responses are presumably from phasic receptors, as has been confirmed by direct identification in Gymnotus (Suga, 1967a) and mormyrids (Roth, 1967; Szabo and Hagiwara, 1967). Although both medium and large receptors of mormyrids are activated by organ discharge, changes in afferent responses have been fully reported only for medium receptors. Because of the great sensitivity of large receptors, they may be maximally activated by the organ and transmit negligible information about distortions of its field. If so, their primary function may be detection of weak high frequency signals from other mormyrids, which is perhaps of significance in communication ( cf. Moller, 1970). The effects of objects on afferent discharges, presumably from a phasic receptor, are illustrated for the gymnotid Steatogenys in Fig. 37. On the left are shown oscilloscope records with histograms of the number of impulses after each organ discharge when a silver plate was in the indicated positions. On the right are graphs of mean number of impulses in two fibers for successive positions of paraffin blocks as well as silver plates. The monopolar threshold measurements indicate that for the upper graph, at least, the receptor lies at about the point where the minimum response is obtained with the silver plate and the maximum response is obtained with the paraffin block. A variety of coding mechanisms have been described whereby changes in afferent impulses evoked by organ discharge may signal distortions of the organ fields (Bullock and Chichibu, 1965; Hagiwara and Morita, 1963; Hagiwara et al., 196%; Szabo and Hagiawara, 1967). In fish emitting discharges at a low frequency, a fiber may carry several impulses following each discharge; changes in the field may alter the number of impulses per discharge as in Fig. 37. Latency may or may not be changed concurrently. Particularly in fish emitting discharges at high frequencies, the impulses may fail to follow the organ discharge frequency one to one, and changes in the field may alter the probability of a single impulse’s occurrence as well as its latency. Latency changes are very pronounced in discharges from some medium receptors of the mormyrid Gnathonemus. Other medium receptors show less change in latency but greater changes in number of spikes. Phasic receptors continue to give an altered response in the presence of a stationary stimulating object, although there may be some adaptation. The continued responding has sometimes been

552

M. V. L. BENNETT

I Threshold

10 2 3 4 5 6

F E O C B A

7 8 91011 121314 cm

F E D C B A

cm

Fig. 37. Afferent volleys evoked by electric organ discharges are altered by the presence of conductors and insulators. Left: Oscilloscope records of afferent impulses, organ discharges appear as a small deflection preceding each nerve volley. The diagram indicates the center positions of a 1.5 by 0.5 cm silver plate for corresponding oscilloscope records. Histograms by each record show the distribution of number of impulses in 100 successive volleys. Right: Graphs of mean number of impulses in volleys evoked by organ discharge as affected by a silver plate or paraffin block. Same fiber on the left as in lower graph. The open circles in the upper graph are thresholds for monopolar stimulation and indicate that this receptor lay at a point nearly corresponding to 7 cm along the abscissa. From Hagiwara and Morita ( 1963).

called tonic (Hagiwara et al., 1965b), although it is not owing to a maintained electric stimulus but rather to repeated stimulation of the receptor by an organ discharge of altered size. There results are important in that they establish the possibility that electroreceptors operate in electrolocation. Unfortunately, little has been done to determine how the presence of insulators and conductors changes the potentials across the skin resulting from the organ discharge. Further investigations along these lines are required to correlate the responses to objects with the responses of the receptors to local stimulation. C. Central Projections of Electroreceptor Activity

A noteworthy characteristic of fish with electrosensory systems is the large size of the cerebellum compared to that of other fish. In Fig. 38 are

11, ELECTRORECEPTION

553

diagrams of the brains of several fish with and without electrosensory systems. Neuroanatomy of the cerebellum of fish has recently been reviewed by Nieuwenhuys ( 1967) and Schnitzlein and Faucette ( 1969). The trout, Salmo fario, may be taken as a generalized teleost (Fig. 38A). The buffalo fish Curpiodes is a sucker, a bottom feeding relative of the carp. It has a very developed gustatory system and correspondingly the vagal lobes are very enlarged, but otherwise its brain resembles that of the trout (Figs. 38B and C). The squirrelfish has very large eyes, and its optic tecta are quite enlarged (Fig. 38D). The stargazer has an electric organ but is not known to have electroreceptors. Its brain is quite similar to that of the trout except for the smaller cerebellum and the very large oculomotor nerves that innervate the electric organ (Fig. 38E). The cerebellum is also implicated in the control of movement, and the small size in the stargazer correlates with its sedentary mode of life. It generally lies buried in the sand with only its small eyes protruding and waits until prey comes near enough to gulp down. The elasmobranchs are divided into two main lines, the sharks and the batoids including skates, electric rays, stingrays, and guitarfish. A wide but similar range of cerebellar development is seen within each line ( Ariens-Kappers et ul., 1936; Schnitzlein and Faucette, 1969). The brain of a ray that has a simple cerebellum is shown in Fig. 38F. This cerebellum is still considerably larger than that of the trout. In the guitarfish and stingray, the corpus is highly convoluted and greatly enlarged. No data are available for correlation of electrosensory function with cerebellar enlargement within the elasmobranchs. There is some tendency for more elaborate cerebella to be found in more actively swimming species, but whether this increased development represents increased sensory or control requirements is not yet clear. The cerebellum of the catfish Amiurus is greatly enlarged compared to that of the teleosts without an electrosensory system (Fig. 38G). The cerebellum of gymnotids is similarly enlarged ( Fig. 38H). The extreme case of enlargment of the cerebellum is in the mormyrids in which it constitutes most of the brain (Figs. 381 and J ) . The cerebellum in mormyrids is extraordinarily specialized cytologically ( Nieuwenhuys and Nicholson, 1969a,b; Kaiserman-Abramoff and Palay, 1969). Just as increase in optic tecta or vagal lobes implies a role in increased visual or gustatory sensitivity, hypertrophy of the cerebellum in fish with electrosensory systems implies a role in electroreception. There are corresponding increases in the afferent pathways from lateral line inputs to at least the valvulae of the cerebellum (Berkelbach van der Sprenkel, 1915; Nieuwenhuys and Nicholson, 1969a; Szabo, 1967). There is limited physiological evidence for involvement of the cerebel-

554

M. V. L. BENNETT

L

Fig. 38. Brains of fish with and without electrosensory systems. ( A ) Trout, Salrno fario (from Nieuwenhuys, 1967); ( B , C ) dorsal and lateral views, buffalo fish, Carpiodes (from Herrick, 1905); ( D ) squirrelfish Holocentrus (from Meader, 1934); ( E ) stargazer Astroscopus. ( F ) thornback ray, Platyrhinoides triseriata (from Nicholson et al., 1969); ( G ) catfish Leptops (from Herrick, 1905); ( H ) electric eel Ekxtrophorus (from Couceiro and Fessard, 1953); ( I ) mormyrid Gnathonernus (from Nieuwenhuys and Nicholson, 1969a); and ( J ) mormyrid Mormyrops (from Stendall, 1914). Abbreviations: C, cerebellum; L, lateral line nerve; 01, olfactory bulb; Op, optic tectum; V, valvula of the cerebellum; VL, vagal lobe; and 111, oculomotor nerve.

lum in electroreception. Volleys from electroreceptors have been observed to project to the lateral line lobes, midbrain, and valvulae of the cerebellum in mormyrids (Bennett and Steinbach, 1969; B. Zipser and M. V. L. Bennett, unpublished data). Morphological studies indicate the cited order is the actual pathway, but projections of cerebellar outputs have not yet been identified (Nieuwenhuys and Nicholson, 1969a). Physio-

11. ELECTRORECEPTION

555

logical data establish projections of electroreceptors to the eminentia granulosa in gymnotids (Enger and Szabo, 1965) and to the lateral line lobes in gymnotids and elasmobranchs (Enger and Szabo, 1965; Nicholson et al., 1969). Single units in the lateral line lobe of gymnotids can signal the presence of objects by an alteration in the number of spikes following an electric organ discharge (Enger and Szabo, 1965). No responses to electrosensory inputs have been found in the corpus cerebelli of elasmobranchs, and the suggestion has been made that this part of the cerebellum is involved in control of movement rather than electroreception ( Nicholson et al., 1969). An interesting aspect of the mormyrid electrosensory system is that the neural command signal that fires the electric organ also projects to many parts of the cerebellum and hind brain. This signal presumably prepares the afferent pathways for incoming signals from the electroreceptors. Both inhibitory and facilitatory effects on central responses to afferent volleys can be observed when electroreceptors are activated in specific time relation to the command signal to excite the organ (Bennett and Steinbach, 1969; B. Zipser and M. V. L. Bennett, unpublished data). These effects are independent of actual organ discharge and occur in the curarized animal. They indicate that the fish “knows” when it is going to discharge its electric organ. At least in principle, the fish can measure the latency of the receptor response. In a passive sensory system only differences in latency between receptors can be determined.

D. Behavioral Responses to Voltage Gradients and Conductance Changes

A number of different kinds of behavioral responses that apparently involve electroreception have been observed in electric fish. Many gymnotids move away from a metal rod that is brought close to them, whereas they tend to ignore visually similar insulators. Individuals of more aggressive species such as Gymnotus and Electrophorus may attack metal objects, A number of catfish and sharks also respond to metal rods, much more strongly than to insulators (M. V. L. Bennett, unpublished data; Dijkgraaf, 1963, 1968). In electric fish the differential responsiveness may involve active electroreception, and as already noted alterations in the nerve volleys evoked by organ discharge can be produced by metallic objects (Fig. 37). In addition inhomogeneities in metals cause eddy currents in surrounding water, and these fields can be adequate to stimulate tonic receptors as has been observed in gymnotids and dogfish (Fig. 39). Presumably the latter mode of action is

556

M. V. L. BENNETT

0 . 5 sec

Fig. 39. Response of a tonic receptor to a metallic rod. From the gymnotid Gymnotus. Recording from the afferent fiber in a curarized animal. ( A ) Resting discharge. ( B ) Responses to passing a submerged screwdriver back and forth over the receptor. The discharge is alternately retarded and accelerated. ( C ) The response to slowly placing the tip over the receptor is a gradual block of discharge. Upon removal there is a long-lasting burst of impulses. The changes in base line in ( B ) and ( C ) result from ac coupling of the amplifier. From Bennett ( 1967).

responsible for the detection of metals by animals with passive electrosensory systems. (Because of eddy currents the finding of responses to metals or of discrimination between metallic objects and insulators does not demonstrate the existence of an active electrosensory system.) Lissmann (1958) noted an interesting correlation between the occurrence of weakly electric organs and mode of swimming. All weakly electric fish tend to swim with the body held relatively rigid. In gymnotids propulsion for ordinary swimming is provided by the anal fin. The dorsal fin of Gymnarchus serves a similar function (see Fig. 1of Chapter 10, this volume). Most of the body musculature does not act in ordinary swimming and is probably used primarily for escape movements. The suggestion is that the body is held rigid to simplify the functioning of the electrolocation system, just as in many animals the eye can be rotated around all three axes to stabilize the retinal image. A further behavioral feature of freshwater weakly electric fish is their tendency to back into a strange environment and to investigate it by moving the tail around. Although the electroreceptors are concentrated in the head region, the external field strength is greatest just outside the tail, and sensitivity may be reasonably great using the backward approach. It would also seem faster to escape from an unpleasant situation by swimming forward rather than by backing out and turning around.

11. ELECTRORECEPTION

557

Many gymnotids caught in the field have partially regenerated tails. This common history of injury could be a result of their peculiar mode of investigation. It is also true that the tip of the tail is the most salient part of an electric fish to another fish that is detecting it electrically, and when electric fish fight in aquaria, the tails are often the primary target of attack. Several kinds of unconditional responses in addition to avoidance of metallic rods have been used in exploration of electric sensitivity. For example, heart rate in the skate or dogfish may accelerate when a weak electric stimulus is presented ( Dijkgraaf and Kalmijn, 1966). These fish will also burrow into sand over electrodes emitting signals similar to the respiratory potentials of flatfish on which they feed, a dramatic demonstration of a function of passive electrosensory systems. Mormyrids and variable frequency gymnotids accelerate their organ discharge rate when weak electric stimuli are given or when the conductance is altered between a pair of electrodes at head and tail (Bennett, 1965; Bennett and Grundfest, 1959; Enger and Szabo, 1965; Hagiwara et al., 1965a; Larimer and MacDonald, 1968; Moller, 1970; Szabo and Fessard, 1965). In the second, fifth, and seventh of these examples the case for active electroreception is strong because the response to a conductance change occurs only if an organ discharge occurs during the change; no response is obtained to changes restricted to periods between discharges. An interesting form of unconditional response can be evoked in higher frequency fish ( Watanabe and Takeda, 1963; Larimer and MacDonald, 1968; Bullock, 1970). If the animal is presented with a stimulus close in frequency to its own discharge, it will shift its discharge frequency away from the interfering frequency. The experimenter can slowly “chase” the fish‘s frequency up or down within a certain range; beyond this range the fish will fairly rapidly change its frequency to the other side of that of the interfering signal. The response appears to represent avoidance of “jamming” of the sensory system by the closely neighboring frequency. A related kind of response is evoked in Gymnotus and Gymnurchus. A weak electric stimulus near the fish‘s own frequency can cause the fish to cease discharging entirely for a period of a fraction of a second up to minutes (references in Section 111 of Chapter 10, this volume). The longer cessations may represent a kind of hiding in these fish which are highly aggressive toward other electric fish including members of their own species. Similar cessations can be evoked in mormyrids by weak electric stimuli over a fairly wide range of frequencies (Moller, 1970). There are interactions between mormyrids evidenced by other changes in discharge rate that must be mediated by the electrosensory system

558

M. V. L. BENNETT

( Black-Cleworth, 1970; Mohres, 1957; Moller, 1970). In Gymnotus cessations appear to be involved in aggressive interactions between individuals ( Black-Cleworth, 1970; Westby and Box, 1970). Recent field studies show that changes in organ discharge frequency are involved in courtship behavior of several gymnotids ( C. Hopkins, unpublished data). Positive reinforcement by feeding has been used by Lissmann and Machin to establish sensitivity to conductivity differences and electric stimuli in Gymnarchus (1958) and to electric stimuli in the catfish Clarias ( 1963). Avoidance conditioning in which acceleration of organ discharge is the conditioned response has been carried out using a conductance change as the conditional stimulus and strong electric shock as the aversive stimulus (Bennett, 1968b; cf. Mandriota et al., 1968). A few experiments indicate that as would be expected the components of the electrosensory system mediate the behavioral responses to electric stimuli. In gymnotids section of the posterior branch of the anterior lateral line that innervates the electroreceptors in the posterior of the body abolishes the response to conductors placed posteriorly, but conductors near the head region are still avoided (Bennett and Grundfest, 1959). Unilateral section of this nerve reduces sensitivity on the affected side and causes all responses to be toward that side, indicating that the responses that do occur are mediated by the intact contralateral nerve. Section of the posterior lateral line nerves, which contain only mechanoreceptive fibers, has no effect on the avoidance response. Similar results have been obtained by cutting the appropriate nerves in catfish and dogfish ( Dijkgraaf, 1968; Dijkgraaf and Kalmijn, 1963). Simple energy considerations indicate that the electric organ discharge is necessary for the detection of conductance changes. As noted above, electric fish do not give the unconditional response of acceleration of discharge rate if a conductance change is restricted to the periods between organ discharges.

E. Thresholds for Receptor and Behavioral Responses Granted that the electroreceptors mediate the behavioral responses, it may then be asked whether the observed behavioral thresholds can be explained in terms of the receptor thresholds. The comparison is difficult to make because of differences in the methods by which the two types of experiment have been carried out. Nonetheless, from considerations of receptor thresholds and the effects of accessory structures, a preliminary evaluation can be made (Table 11). In the skate the threshold field for cardiac acceleration is about 0.01

559

11. ELECTRORECEPTION Table I1 Behavioral and Receptor Thresholds"

Behavioral response

Fish Skates, dogfish, sharks Gymnarchus Mormyrids Catfish Clarias K ryptopterus Gymnotids

Threshold gradient (rV/cm)

Computed Measured receptor receptor stimulus threshold (PV) (PV)

Heart rate acceleration Feeding Change in organ discharge rate

0.01 0.15 50

4

Feeding

1

10

Jamming avoidance

3-100

15-500

0.2

500

2 1006 20w

100 100

References in text. Mormyrid tonic receptors. Large receptors.

pV/cm, and the minimum receptor threshold is about 2 pV (Dijkgraaf and Kalmijn, 1966; Murray, 1967). Since the longest ampullary canals were probably 20 cm or more in length in the behavioral experiments, an applied field of 0.01 pV/cm would result in a stimulus to these receptors of 0.2 pV or more. The Gymnurchus studied by Machin and Lissmann (1960 ) was sensitive to fields of about 0.15 pV/cm, and a slightly higher threshold was calculated for distortions of fields produced by objects. Since the animal was about 50 cm in length, the field strength would result in a receptor stimulus of about 4 pV. The thresholds of receptors in Gymnurchus have not been well studied, but the required sensitivity is only 10-20 times greater than is observed in small (tonic) receptors in mormyrids. In Gymnarchus the threshold for high frequency rectangular pulses is given by the dc component of the stimuli. This observation indicates that the phasic receptors are no more sensitive to the ac component of this form of stimulus than the tonic receptors are to the dc component ( granted that these classes of receptor exist). The catfish Clarias has been shown to be sensitive to a gradient of about 1 pV/cm in training using food reinforcement (Lissmann and Machin, 1963). For a 20-cm animal this would correspond to a receptor stimulus of about 10 pV assuming that the skin is of high resistance. Receptor thresholds 5-10 times this value have been observed in the transparent catfish Kryptopterus ( Bennett, 1971a,b). In mormyrids, threshold gradients along the entire fish for electric pulses to evoke changes in organ discharge frequency are about 0.05

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M. V. L. BENNETT

mV/cm (Moller, 1970). These gradients would give a receptor stimulus of about 0.5 mV in a 20-cm long fish which is above threshold for most large receptors ( Bennett, 1965, 1967). Thresholds for locally applied stimuli to evoke an acceleration of organ discharge are similar to the thresholds of large receptors (Harder et al., 1967). The jamming avoidance response of gymnotids has a sensitivity of 3100 pV/cm (Watanabe and Takeda, 1963). In these experiments the receptor stimulus would have been about five times the applied voltage per centimeter. The larger values easily exceed the absolute thresholds of the receptors measured directly ( Bennett, 1967). The relevant parameter in the jamming avoidance response is the incremental threshold of the receptors when the applied stimulus is superimposed on the stimulus provided by the organ discharge. This property has not been studied in fish emitting discharges at a fairly constant frequency. In Gyrnnotus incremental thresholds are not much different from absolute thresholds ( Suga, 1967a). However, in phasic receptors of Hypopornus, the stimulus required to evoke a single spike is much greater than the increase in stimulus required to add a spike to a train evoked by a moderately suprathreshold stimulus (Hagiwara and Morita, 1963). Correspondingly, the animal responds to a weaker applied pulse when it sums with the electric organ discharge than when it is presented between discharges (Bullock, 1970; Larimer and MacDonald, 1968). In the mormyrid Gnathonemus the behavioral responses to electric pulses have a threshold at least 10-fold higher when the pulses are superimposed on the organ discharge compared to when they are given between discharges ( Moller, 1970). Although for some medium receptors incremental thresholds are lower than absolute thresholds (Szabo and Hagiwara, 1967), the large receptors no doubt mediate the lower threshold behavioral responses, and their sensitivity to changes in the organ discharge is not yet clear. The “significance” to the fish may also differ in that pulses between organ discharges could represent another electric fish while pulses superimposed on organ discharges could represent resistance changes. The conclusion at this time is that there is a factor of approximately ten between observed behavioral and receptor thresholds. Direct measurements of the stimuli applied to the receptors under the conditions of the behavioral experiments are still required and may alter this factor somewhat. At least part of the discrepancy must lie in the difficulties in electrophysiological experiments of finding the lowest threshold receptors and of maintaining the animal in optimal condition. A further aspect of the discrepancy may be correlation by the central nervous system of inputs from a number of receptors. In the presence of

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561

spontaneous activity the central nervous system may also discriminate changes in the responses of a single receptor more sensitively than does the experimenter. Although it can be accepted that the electroreceptors are sensitive to electric fields and thereby provide perceptually useful information, there is a possibility that these receptors also convey other kinds of information to the central nervous system. Phasic receptors are quite insensitive to mechanical, thermal, or other modes of stimulation, but tonic receptors may be rather sensitive to temperature and salinity changes and also have some sensitivity to mechanical stimulation (Murray, 1967; Suga, 1967a; Szabo, 1970). The effects of salinity changes are ascribable to changes in diffusion potentials (Murray, 1967). In the ampullae of Lorenzini the location deep in the body would reduce sensitivity to thermal and mechanical stimuli. The sensitivity to pressure differences between inside and outside of the canal is not very great but might become significant during rapid swimming. Pressure changes with depth would have little effect being communicated equally to inner and outer faces of the receptor cells. It is an open question whether activation of electroreceptors by nonelectric stimuli is interpreted by the animal as electric stimuli in the same way that we see lights when our eyes are electrically stimulated. Extensive conditioning experiments would be required to resolve this question,

VI. EVOLUTION OF ELECTROSENSORY SYSTEMS AND ELECTRIC ORGANS

In a chapter of The Origin of Species entitled “Difficulties of the Theory,” Darwin raised the problem of evolution of electric organs. It was difficult to see by what small intermediate steps the strongly electric organs of the eel and Torpedo could have arisen, when the early stages could have no value in defense or offense. Furthermore, the highly specialized but weakly electric organ of the ray could have no value in these functions. He wrote, “. . . it would be extremely bold to maintain that no serviceable transitions are possible by which these organs might have been gradually developed.” Only relatively recently did Lissmann (1958) provide evidence for a transition stage by demonstration of the electrosensory system of the weakly electric fish. The explanation became that early stages of the weakly electric organs were selected for their electrosensory function. Once a weakly electric organ reached a certain size, it then began to be adaptive in defensive-offensive actions. In view

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M. V. L. BENNETT

of the ineffectiveness of electric organs in offensive roles it seems likely that the defensive functions were more important in initial stages of conversion from purely electrosensory to defensiveoffensive organs. In agreement with Lissmann’s hypothesis the eel retains a weakly electric organ and has many weakly electric relatives. Some torpedinids also have a weakly electric organ, and some of the little known benthic forms are blind and may use their organs in an active electrosensory system. No evidence for weakly electric organs has been found in catfish, and the intermediate stages leading to the electric catfish are presumably lost. Evidence for active or passive electroreception is lacking for the stargazers. Lissmann further suggested, although less explicitly, that electroreceptors and electric organs developed concurrently in the evolution of the weakly electric fish. As lateral line receptors became modified to detect electric signals, muscles became modified to generate electric signals. In light of subsequent knowledge it seems more reasonable to believe that passive electrosensory systems were evolved initially and that electric organs were added subsequently. Most elasmobranches have ampullae of Lorenzini indicating that the primitive forms giving rise to rajids and torpedinids had passive electrosensory systems. Many catfish have electrosensory systems so that the hypothesized weakly electric ancestor of the electric catfish probably originally had a passive electrosensory system. All known mormyrids and gymnotids have electric organs, but the presence of the tonic electroreceptors is consistent with a prior stage possessing a passive electrosensory system. It does seem possible that the phasic receptors in these groups developed concurrently with the electric organs and they are certainly adapted to each other in different fish (Section IV, D ) . The similarities between receptors of mormyrids and Gymnarchus suggest that Gymnurchus already had an electric organ when it diverged from the mormyrid group. No data concerning electroreceptors are available from the stargazers, nor is the cerebellum at all enlarged unlike known electrosensory fish (Section V, C ) . This group remains a possible anomaly. In the initial stages of evolution of passive electrosensory systems, lateral line receptors presumably lost mechanical sensitivity and developed electrical sensitivity. This transformation probably required little more than an alteration of the outer face of the receptor cell. It would be expected that the inner face would already have developed a high electrical sensitivity for detection of sinall microphonic potentials generated by the outer face (see Section VII). The arrangement of the receptor cells in an epithelium is similar in all lateral line receptors and their homologs, and the major change leading to tonic electroreceptors may

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well be increased skin resistance, at least in freshwater fish. Further insight into these questions should be gained when membrane properties of receptor cells of ordinary lateral line organs are determined. Mechanoreceptors in weakly electric fish are 20 (Roth, 1968) to 400 times (Suga, 1967b) less sensitive to electric stimuli than are the electroreceptors. This difference is considerably less than the difference between electric and nonelectric fish ( Machin and Lissmann, 1960). Probably the increased skin resistance in freshwater fish with electroreceptors increases the electric sensitivity of the mechanoreceptors as well. Many electric fish live in turbid waters or, if they live in clear waters, are nocturnal. The electrosensory system which is basically quite short range would appear to be most useful under these conditions of restricted use of vision. Some electric fish have quite reduced eyes and the retinae of the electric eel apparently degenerates with age (see discussion by Keynes following de Oliveira Castro, 1961). But a number of electric fish have what appear to be quite good eyes, and many elasmobranchs at least are diurnal. In addition to the parallel evolution of electric organs, electroreceptors, and a peculiar mode of swimming, long snouts have been developed in both mormyrids and gymnotids (and probably more than once in each group; see Figs. 16 and 60 in Chapter 10). The snouts are certainly used in poking about muddy bottoms, but are most likely a later development than the electrosensory systems. It is not clear that development of electrosensory systems was always in compensation for poor visual conditions, although it seems likely to have been so a number of groups (Lissmann, 1958, 1961). In any case, development of lateral line organs with electric sensitivity has not led to any great decline of the ordinary lateral line system, which like the electrosensory systems should be particularly useful under poor visual conditions (Dijkgraaf, 1963). It would be useful to know more about electric fields present in the ordinary fish's environment and the extent to which these stimulate ordinary lateral line receptors. Muscle potentials up to tens of millivolts can be produced by synchronously contracting muscles. Examples are sonic muscles where the fundamental frequency is set by the frequency of muscle contraction and axial muscles when excited by Mauthner fibers (unpublished, cf. Lissmann, 1958). These potentials conceivably could activate mechanoreceptors electrically. Many catfish have sonic muscles and their innervation is similar to that of the electric organ of the electric catfish, which lacks a sonic muscle. Perhaps this organ was modified from a sonic muscle, a suggestion which could be strengthened by further morphological and embryological study of the catfish group (see Johnels, 1956).

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A final question that has been raised by the evolution of electrosensory systems is their absence in invertebrates and aquatic amphibia (Grundfest and Bennett, 1961). There are too few of the latter to make their difference from the fishes significant, and perhaps also too few invertebrates of sufficient size to make an electrosensory system useful. The cutaneous receptors of invertebrates are generally peripherally located neurons without separate receptor cells, and receptors of this kind may not well adapt to an electroreceptive function. VII. IMPLICATIONS FOR RECEPTOR FUNCTION IN GENERAL

The electroreceptor can be looked upon as a model of a secondary receptor, that is, one where the primary transduction is done by a receptor cell which then transmits synaptically to an afferent fiber. Functionally, a receptor cell can be divided into three parts: (1) the outer face which is passive in electroreceptors; ( 2 ) the "sides," which can be passive in tonic electroreceptors or electrically excitable in phasic electroreceptors; and ( 3 ) the presynaptic membrane, which secretes transmitter if the synapse is chemically transmitting (Fig. 40). The sides and presynaptic membrane comprise the inner face of electroreceptors (and some others); this face is separated from the outer face by a circumferential tight junction with neighboring cells. The three functional parts of a receptor cell correspond to dendritic (receptive), axonal (conductive), and terminal (secretory) portions of a generalized nerve cell ( Grundfest, 1966). In electroreceptors of freshwater fish, the outer face is unaffected by the stimulus, but the stimulus can be modified by the outer face, either

T

SUPPORTING C f l l

OUTER FACE: PASSIVE, M K H A N O SENSITIVE, CHEMOSENSITIVE OR PHOTOSENSITIVE

PI

SIDE: PASSIVE OR ELKTRKALLY EXCITABLE

PRESYNAPTIC MEMBRANE: SECETORY OR FORMING A N ELKTROTONIC SYNAPSE

Fig. 40. Diagram of a generalized receptor cell.

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as a series capacity distorts it or as a resting potential changes its dc level. The sides of the receptor cell may have no active influence on the (electrical) stimulus as in tonic receptors or they may alter it by responding as in most phasic receptors. The response may be regenerative, either graded or all-or-none, or it may be only degenerative, as produced by delayed rectification. The stimulus thus transformed (not really transduced) is transmitted electrotonically to excite the nerve, or it causes the presynaptic membrane to increase or decrease the release of transmitter. There may or may not be sufficient resting secretion to cause tonic activity in the nerve. The operation of other receptor cells is probably analogous. There is considerable evidence that the outer faces of mechanoreceptor cells generate a potential by changing their resistance and/or potential in response to a deformation (cf. Davis, 1965; Furukawa and Ishii, 1967), and carotid body receptors may operatc similarly (Eyzaguirre et al., 1965, 1970). This potential change is (electronically) conducted to the secretory face of the receptor cell and acts on the presynaptic membrane to cause release of transmitter ( Furukawa and Ishii, 1967). In one kind of vestibular receptor, morphological evidence indicates that the receptor potential is directly transmitted to the postsynaptic nerve by low resistance pathways ( Spoendlin, 1966). There are little data except in electroreceptors indicating whether receptor responses are modified by electrically excitable membrane in the sides of receptor cells. The occurrence of such membrane in one kind of receptor cell suggests that it will be found in others as well. Of course, axons of primary sensory neurons must conduct action potentials, if the axon is long compared to its space constant. What have been termed the outer face, sides, and presynaptic membrane may not be spatially segregated. Depending on how the stimulus can reach the cell and the mode of innervation, the three idealized regions could be intermixed with each other to a greater or lesser extent. In most electroreceptors the sides and presynaptic membrane are probably comingled in the inner face. Nonethcless, one can distinguish three separate functions and it is probable that the membranes mediating them are distinguishable, at least on the molecular level. The restriction of function to particular regions is only required where membranes of different function must be oriented differently with respect to the cell's environment. Generally, exteroceptors will have an outer face that is indeed directed outward, but sides and presynaptic membrane can be intermixed. Because the receptor cell is so short, it can be considered analogous to a presynaptic terminal. In this respect it is interesting that the elec-

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trically excitable responses of receptor cells of electroreceptors are insensitive to tetrodotoxin (Zipser, 1971). In the same species, and in most other forms, the ordinary Na channels mediating propagated spikes are blocked by tetrodotoxin. However, tetrodotoxin resistant spikes can be obtained in prcsynaptic terminals of the squid giant synapse and apparently of the neuromuscular junction as well (Katz and Miledi, 1969a,b). These neuronal responses are dependent on divalent ions and presumably mediate the normal Ca influx required for transmitter secretion. Although the ionic dependence of the receptor cell responses has yet to be studied, it is tempting to suppose that their spike generating mechanism represents evolution of an increased number of electrically excitable channels that carry divalent ions. Smaller numbers of these channels would presumably be present in receptor cells lacking an obvious electrical response where they would mediate transmitter release. Divalent ions certainly affect transmitter release in electroreceptors, although there is not the clear-cut antagonism between Ca and Mg that has been observed at a number of interneuronal synapses ( Steinbach and Bennett, 1971). An interesting question about receptor synapses is whether transmitter is released in small packets or quanta as has been so elegantly documented for the neuromuscular junction ( see Katz, 1969). The presence of presynaptic vesicles at receptor synapses suggests that release is quantized, and a certain amount of indirect confirmatory evidence involving analysis of intervals between afferent impulses has been obtained for lateral line receptors ( Harris and Flock, 1967). The irregularity of intervals between impulses from tonic receptors could be a result of quanta1 variations in the PSP, and intracellular recording from nerve terminals should provide more direct evidence ( Fig. 18). It is likely that an input-output relationship similar to that of tonic receptors will be found in other receptor cells, where more or less linearly graded information about stimulus amplitude is transmitted centripetally. The absolute sensitivity is also likely to be similar to that in electroreceptors, at least in the most sensitive receptors for a given modality. In all the sensitive receptors there should be strong selection pressure for evolution of a presynaptic face that can detect very small signals generated by the outer face. The cochlear microphonic is estimated to have a value of 0.01 pV at threshold for hearing (Machin and Lissmann, 1960). The potcntial across the inner face of the hair cells is undoubtedly much larger since the receptor should have evolved so that most of the voltage drop is developed across the inner face. A hundred- to a thousandfold larger potential across the secretory membrane is not unreasonable and brings the actual potential across the secretory face into the range where elec-

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troreceptors are capable of operating. The cochlear microphonic would not cause much electrical interference between cells since the external current from a single receptor is divided into the low resistance of the external path around the cells and the parallel paths provided by all the other cells. Intracellular recording from receptor cells of lateral line receptors of an amphibian indicates that a quite small microphonic, as low as 100 pV across the hair cell inner membrane, can be associated with a stimulus well suprathreshold for excitation of the postsynaptic fiber (Harris et al., 1970). This amplitude of potential would be adequate to excite tonic electroreceptors of freshwater fish. Thus, it appears that at least the most sensitive mechanoreceptors of the acoustico-lateralis system are of similar sensitivity to electroreceptors in terms of the potentials generated across their secretory faces. (An unanswered question is the mechanism whereby high frequency sinusoidal microphonics still cause release of transmitter. Possibly the secretory membrane of tonic electroreceptors would respond to high frequencies, but the potential across it at these frequencies is limited by its large time constant. ) The sensitive input-output relationship of electroreceptor synapses may be found in some neurons as well as receptor cells. In short axon cells such as the bipolar cells of the retina (Dowling and Boycott, 1965) and at reciprocal synapses such as those of the olfactory bulb (Rall et d, 1966), the propagated action potential probably is not required to transmit potentials between receptive and output parts of the cells. Slow and small PSPs generated at one site could cause release of transmitter by secretory membrane without intervention of large regenerative responses. In this connection it is interesting that the bipolar cell synapses have presynaptic ribbons like those found otherwise only in receptor cells. The experimental advantage of the tonic electroreceptors of freshwater fish is that they are specialized to detect low frequency voltages across the skin. The specializations that channel current through the receptor cells and the electrical linearity of the cells allow the experimenter considerable control over the presynaptic potential and has made possible the determination of the input-output relationship. The presynaptic POtential cannot be well controlled at phasic receptors, which do however demonstrate the possibility of electrical excitability and regenerative responses in receptor cells. A series capacity is an elegant way to achieve accommodation, but no other receptors are likely to have it. The only other cells where a membrane is known to act as a series capacity are electrocytes of several electric organs. The mechanisms of central analysis in electrosensory systems are largely unknown and may not turn out to be easier to analyze than other

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systems of more widespread occurrence. Nonetheless, the ease of experimental preparation and of stimulating known numbers of individual receptors makes the system worthy of further exploration. The macroscopic and cytological specialization of the underlying brain structures ( Section V, C ) also makes them highly intriguing subjects for physiological investigation. One can conclude that the electrosensory systems have provided information of considerable general as well as comparative physiological interest. It can be anticipated that further study will continue to be rewarding. ACKNOWLEDGMENTS

I am indebted to Dr. R. B. Szamier for many of the morphological figures. Supported in part by grants from the National Institutes of Health ( 5 PO1 NB 07512 and HD-04248) and the National Science Foundation (GB-6880).

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11. ELECTRORECEPTION

573

Pickens, P. E., and McFarland, W. N. (1964). Electric discharge and associated behaviour in the stargazer. Animal Behav. 12, 362-367. Rall, W., Shepherd, G. M., Reese, T. S., and Brightman, M. W. (1966). Dendrodendritic synaptic pathway for inhibition in the olfactory bulb. Exptl. Neurol. 14, 44-56. Roth, A. ( 1967). Propriktks fonctionnelles et morphologique des diffkrents organes de la ligne laterale de Mormyrides. J. Physiol. (Paris) 59, 486. Roth, A. (1968). Electroreception in the catfish, Amiurus nebulosus. Z. Vergleich. Physiol. 61, 196-202. Roth, A. ( 1969). Elektrische Sinnesorgane beim Zwergwels Ictalurus nebulosus ( Amiurus nebulosus) Z. Vergleich. Physiol. 65, 368-388. Roth, A., and Szabo, T. (1969). The effect of sensory nerve transection on the sensory cells and on the receptor potential of the tuberous (Knollen) organ in mormyrid fish (Gnathonemus sp.). Z. Vergleich. Physiol. 62, 395-410. Schnitzlein, H. N., and Faucette, J. R. (1969). General morphology of the fish cerebellum. In “Neurobiology of Cerebellar Evolution of Development” ( R. Llinb, ed. ), pp. 77-105. Am. Med. Assoc., Chicago, Illinois. Spoendlin, H. (1966). Some morphofunctional and pathological aspects of the vestibular sensory epithelia. In “2nd Symposium on the Role of Vestibular Organs in Space Exploration,” pp. 99-115. NASA, Washington, D. C. Steinbach, A. B., and Bennett, M. V. L. (1971). Effects of divalent ions and drugs on synaptic transmission in phasic electroreceptors in a mormyrid fish. J. Gen. Physiol. ( I n press.) Stendell, W. ( 1914). Morphologische Studieren an Morniyriden. Verhandl. Deut. Zool. Ges. 24, 254-261. Suga, N. (1967a). Coding in tuberous and ampullary organs of a gymnotid electric fish. I. Comp. Neurol. 131, 437452. Suga, N. (196713). Electrosensitivity of canal and free neuromast organs in a gymnotid electric fish. J. Comp. Neurol. 131, 453-458. Szabo, T. (1962). The activity of cutaneous sensory organs in Gymnarchus niloticus. Life Sci. 7, 285-286. Szabo, T. (1965). Sense organs of the lateral line system in some electric fish of the Gymnotidae, Mormyridae, and Gymnarchidae. J. Morphol. 117, 229-250. Szabo, T. ( 1967). Activity in peripheral and central neurons involved in electroreception. In “Lateral Line Detectors” (P. Cahn, ed.), pp. 295-312. Indiana Univ. Press, Bloomington, Indiana. Szabo, T. (1970). Uber eine bisher unbekannte funktion der sog. ampullaren organe bei Gnathonemus petersii. Z. Verleich. Physiol. 66, 164-175. Szabo, T., and Fessard, A. (1965). Le fonctionnement des Blectrortkepteurs Ctudik chez les Mormyres. J. Physiol. (Paris) 57, 343-360. Szabo, T., and Hagiwara, S. (1967). A latency change mechanism involved in sensory coding of electric fish (mormyrids) Physiol. Behav. 2, 331-335. Szabo, T., and Wersall, J. ( 1970). Ultrastructure of an electroreceptor (niormyromast) in the mormyrid fish, Gnathonemuy petersii. J. Ultrastruct. Res. 30, 47-90. Szamier, R. B., and Wachtel, A. W. (1969). Special cutaneous receptor organs of fish. 111. The ampullary organs of Eigenmannia. J. Morphol. 128, 261-290. Szamier, R. B., and Wachtel, A. W. (1970). Special cutaneous receptor organs of fish. VI. The tuberous and ampullary organs of Hypopomus. J. Ultrastruct. Res. 30, 450-471.

574

M. V. L. BENNETT

Usherwood, P. N. R., and Machili, P. (1968). Pharmacological properties of excitatory neuromuscular synapses in the locust. J. Exptl. Biol. 49, 341-361. Wachtel, A. W., and Szamier, R. B. (1966). Special cutaneous receptor organs of fish: The tuberous organs of Eigenrnannia. J. Morphol. 119, 51-80. Wachtel, A. W., and Szamier, R. B. (1969). Special cutaneous receptor organs of fish. IV. Ampullary organs of the non-electric catfish, Kryptopterus. J. Morphol. 128, 291-308. Waltman, B. (1966). Electrical properties and fine structure of the ampullary canals of Lorenzini. Acta Physiol. Scand. Suppl. 264, 1-60. Waltman, B. ( 1968). Electrical excitability of the ampullae of Lorenzini in the ray. Acta Physiol. Scund. 74, 29A-30A. Watanabe, A,, and Takeda, K. (1963). The change of discharge frequency by A.C. stimulus in a weak electric fish. J. Exptl. Bwl. 40, 57-66. Wersall, J., Flock, A., and Lindquist, P. G. (1965). Structural basis for directional sensitivity in cochlear and vestibular sensory receptors. Cold Spring Harbor S y m p . Quant. Biol. 30, 115-132. Westby, G. W. M., and Box, H. 0. ( 1970). Prediction of dominence in social groups of the electric fish, Gyrn!iotus curupo. Psychon. Sci. 21, 181-183. Zipser, B. ( 1971 ). Tetrodotoxin resistant electiically excitable responses of receptor cells. Biophys. SOC. Abstr. 15th Ann. Meeting, 44a.

AUTHOR INDEX Numbers in italics refer to the pages on which the complete references are listed. Banner, A., 181, 186, 192, 199, 232, 236 Abrahamson, E. W., 15, 27 Bannister, L. H., 84, 85, 86, 114 Adler, J., 95, 114 Barber, S . B., 151, 192 Adrian, E. D., 34, 35, 53, 94, 98, 114, Bardach, J. E., 87, 88, 89, 102, 103, 163, 192 106, 113, 114, 115, 120, 122, 124, Adrian, R. H., 359, 459, 484 13.0, 133 Agassiz, J. L., 136, 192 Barets, A., 300, 306, 344, 503, 524, 539, Akerman, M., 385, 487 568 Albe-Fessard, D., 351, 357, 373, 385, Barnett, R., 450, 453, 486 449, 458, 474, 476, 484, 485 Baron, J., 4, 27 Albers, R. W., 352, 459, 485 Barron, S. E., 343, 345 Albers, V. M., 138, 155, 192 Bartelmez, G. W., 266, 274, 325, 344 Alderdice, D. F., 109, 114 Bartels, E., 389, 459, 488 Alexander, R. M., 152, 192 Bauer, R., 496, 568 Alford, R. S., 137, 143, 144, 197 Baylor, E. R., 2, 6, 7, 27 Ali, M. A., 10, 11, 27 Beatty, D. D., 18, 21, 22, 27, 30 Aljure, E., 461, 463, 465, 486 Beccari, N., 274, 300, 344 Allen, W. R., 383, 487 Beidler, L. M., 87, 118 Allison, A. C., 90, 91, 98, 114 Belbenoit, P., 496, 568 Altamirano, M., 349, 383, 386, 388, 448, Benamy, D. A., 391, 415, 458, 459, 489 485, 486 Benjamins, C. E., 229, 236 Amatniek, E., 373, 489 Bennett, M. V. L., 257, 262, 340, 342, Andersen, R., 166, 194 344, 348, 349, 351, 354, 357, 359, Andrews, C. W., 124, 132 361, 362, 363, 364, 366, 371, 372, Arden, G. B., 43, 53 373, 375, 376, 377, 378, 379, 380, Ariens-Kappers, C. U., 553, 568 390, 391, 392, 393, 395, 396, 397, Aronov, M. I., 187, 200 398, 399, 401, 402, 406, 408, 412, Aronson, L. R., 89, 98, 114, 267, 340, 413, 414, 415, 416, 417, 421, 424, 344 427, 428, 429, 433, 436, 437, 441, Asada, Y., 463, 490 443, 444, 445, 446, 447, 448, 449, Atema, J., 106, 120 450, 452, 453, 454, 455, 456, 457, Auerbach, A. A., 340, 342, 344, 482, 485, 514, 568 458, 459, 461, 462, 463, 464, 465, Autrum, H., 170, 192 466, 467, 468, 469, 470, 471, 472, 474, 476, 477, 478, 482, 485, 486 B 488, 489, 490, 494, 495, 499, 503, 504, 509, 511, 512, 513, 514, 515, Backus, R. H., 137, 138, 143, 177, 189, 519, 529, 530, 531, 532, 533, 534, 190, 192, 198, 200 535, 536, 537, 538, 539, 540, 541, Bailey, S. E. R., 129, 132 542, 544, 545, 546, 548, 549, 550, Baker, A. L., 459, 489 575

A

576 554, 556, 557, 558, 559, 564, 566, 568, 569, 570, 572, 573 Beranek, R., 539, 569 Berkelbach van der Sprenkel, H., 500, 553, 569 Berkowitz, E. C., 342, 344 Bernhard, C. G., 38, 53 Bernstein, J. J., 26, 29, 60, 76 Best, A. C. G., 11, 27 Bigelow, H. B., 162, 192, 229, 236, 374, 486 Bjorklund, R. G., 122, 133 Black-Cleworth, P., 354, 391, 395, 478, 486, 496, 558, 569 Blaxter, J. H . S., 10, 27 Bloom, F. E., 450, 453, 486 Bodenheimer, T. S., 317, 319, 346 Bodian, D., 274, 275, 300, 320, 344 Bogatyrev, P. B., 8, 27 Bondesen, P., 155, 192 Boudreau, J. C., 98, 114 Box, H. O., 496, 558, 574 Boycott, B. B., 503, 567, 570 Brahy, B. D., 138, 143, 188, 193, 201 Brauer, A., 5, 11, 12, 13, 14, 27 Brawn, V . M., 158, 187, 192 Breder, C. M., 106, 114 Breder, C. M., Jr., 188, 192 Brett, J. R., 1, 7, 9, 10, 27, 109, 114 Bridge, T. W., 136, 147, 163, 192 Bridges, C. D. B., 4, 18, 21, 22, 24, 27, 28, 36, 39, 54 Brightman, M. W., 463, 486, 567, 573 Brindley, G. S., 35, 43, 53 Brock, L. G., 378, 486 Bronghton, W. B., 155, 192 Brown, K. T., 39, 43, 50, 53 Brown, P. K., 25, 31 Brown, P. S., 25, 31 Brunner, G., 60, 76 Buerkle, U., 176, 192 Bull, H. O., 122, 133, 162, 167, 193, 228, 236 Bullock, T. H., 354, 385, 395, 402, 409, 417, 432, 478, 486, 496, 514, 550, 551, 557, 560, 569 Burkenroad, M. D., 137, 142, 143, 144, 145, 186, 193 Burkhardt, D. A., 36, 53 Burne, R. H., 82, 83, 114

AUTHOR INDEX

Buser, P., 474, 484 Busnel, R.-G., 155, 189, 193 Byzov, A. L., 40, 49, 53, 56

C Cahn, P. H., 135, 142, 168, 173, 193, 262 Caldwell, D. K., 144, 187, 193 Caldwell, M. C., 144, 187, 193 Carregal, E. J. A., 87, 119 Case, J., 87, 89, 103, 114 Cass, A., 358, 486 Chagas, C., 385, 474, 485 Chagnon, E. C., 171, 180, 197, 227, 229, 230, 234, 237 Chandler, W. K., 359, 459, 484 Chapman, C. J., 158, 176, 187, 196, 199 Charlton, T., 303, 344 Chichibu, S., 514, 550, 551, 569 Churchill, J. A., 257, 262 Clark, E., 69, 77 Clarke, W. D., 13, 28, 190, 192 Coates, C. W., 349, 383, 386, 388, 485, 486 Cohen, L. B., 449, 458, 487 Cohen, M. J., 129, 133, 150, 158, 175, 188, 193 Cole, K. S., 391, 458, 487 Cone, R. A,, 43, 53, 55 Conti, F., 536, 572 Cordier, R., 87, 114, 494, 569 Couceiro, A., 385, 487, 554, 569 Couteaux, R., 430, 487 Craddock, J. E., 190, 192 Crescitelli, F., 19, 21, 28 Creutzberg, F., 113, 114, 115 Crickmer, R., 113, 114 Cronly-Dillon, J. R., 26, 30, 60, 63, 68, 76, 77 Crosby, E. C., 98, 117, 553, 568 Cumniings, W. C., 138, 143, 188, 193, 201 Curtis, H. J., 391, 487 Cushing, D. H., 189, 193

D Dalilgren, U., 362, 364, 456, 487 Dann, R., 138, 190, 197 Darlington, P. J., Jr., 20, 28

577

AUTHOR INDEX

Dartnall, H. J. A,, 15, 18, 19, 21, 23, 24, 25, 28 Davies, D. H., 177, 193 Davis, H., 565, 570 Davis, L. I., 155, 192 Daw, N. W., 43, 54 de Burlet, H. M., 163, 193, 211, 225, 234, 236 Deelder, C. L., 113, 115 del Castillo, J., 402, 487 Delco, E. A., Jr., 187, 193 Denker, A., 229, 236 Denton, E. J., 3, 11, 12, 13, 19, 21, 25, 28 De Oliveira Castro, G., 417, 487, 499, 563, 570 Derbin, C., 524, 533, 570 Desgranges, J. C., 87, 115 Detweiler, S. R., 34, 54 de Vries, H., 169, 170, 196 Diamond, J., 276, 278, 285, 290, 291, 297, 301, 302, 306, 312, 316, 319, 343, 344, 346, 481, 482, 487 Diesselhorst, G., 170, 174, 180, 193, 228, 229, 230, 236 Dietrich, G., 183, 193 Dijkgraaf, S., 122, 123, 122, 129, 133, 136, 146, 163, 165, 167, 168, 169, 170, 174, 178, 179, 181, 187, 193, 194, 203, 228, 229, 230, 231, 234, 236, 239, 242, 262, 495, 496, 499, 500, 501, 555, 557, 558, 559, 563, 570 Disler, N. N., 168, 194 Dixon, R. H., 166, 181, 198, 338, 345 Djahanparwar, B., 98, 118 Dobrin, M. B., 137, 143, 194 Dodge, F. A., 449, 487, 536, 572 Doving, K. B., 84, 90, 97, 98, 99, 115 Dohlman, G., 215, 236 Dorai Raj, B. S., 143, 170, 194 Doran, R., 257, 262 Dotterweich, H., 500, 570 DBtu, Y., 147, 149, 194 Dowling, J. E., 33, 47, 51, 54, 56, 503, 567, 570 Drujan, B., 47, 54 Dudok van Heel, W. H., 179, 194 Dufosst., M., 136, 137, 146, 149, 194

E Eccles, J. C., 519, 570 Eccles, R. M., 378, 486 Edstrom, A., 343, 344 Eigenmann, C. H., 383, 487 Eimer, T., 218, 236 Eisenberg, F. A., 449, 487 Eisenberg, J. F., 144, 199 Eisenberg, R. S., 361, 487 Ellis, M. M., 381, 383, 384, 456, 487 Emling, J. W., 137, 143, 144, 197 Enger, P. S., 165, 166, 167, 171, 172, 173, 180, 194, 227, 231, 232, 235, 236, 550, 551, 557, 570, 571 Entine, G., 26, 29 Evans, H. M., 162, 194 Ewart, J. C., 375, 456, 487 Eyzaguime, C., 565, 570

F Fange, R., 150,194 Fagerlund, U. H. M., 109, 113, 114, 115, 116 Farkas, B., 163, 164, 174, 194, 195, 228, 236, 237 Farquhar, M. G., 463, 487, 504, 570 Fatehchand, R., 47, 54, 55, 56 Faucette, J. R., 553, 573 Fawcett, D. W., 151, 195 Fessard, A., 441, 491, 551, 554, 557, 569, 573 Ffowcs-Williams, J. E., 142, 195 Fidone, S., 565, 570 Finkelstein, A., 358, 486 Fish, M. P., 137, 143, 144, 145, 146, 149, 150, 154, 156, 157, 158, 186, 187, 195 Fletcher, H., 173, 180, 195 Flock, A., 165, 195, 221, 222, 237, 249, 250, 253, 254, 257, 262, 503, 566, 567, 571, 574 Fox, H., 343, 345 Frankenhaeuser, B., 389, 449, 458, 487 Franz, V., 34, 54, 494, 570 Freygang, W. H., Jr., 391, 491 Frings, H., 184, 195 Frings, M., 184, 195 Frishkopf, L. S., 249, 250, 262, 557, 571

578

AUTHOR INDEX

Fritsch, G., 487 Froese, H., 163, 167, 195 Froloff, J. P., lG2, 195, 228, 229, 237 Fujimoto, K., 26, 29 Fujiya, M., 102, 103, 114, 115 Furakawa, T., 166, 195 Furshpan, E. J., 271, 273, 275, 276, 288, 315, 316, 317, 322, 324, 325, 327, 333, 337, 344, 345, 481, 482, 487 Furukawa, T., 258, 262, 271, 273, 275, 276, 315, 320, 322, 324, 325, 327, 333, 334, 337, 341, 342, 345, 481, 482, 487, 565, 570

Grosse, J. P., 442, 448, 488 Gruber, S. H., 4, 28, 177, 178, 181, 186, 199, 204, 232, 238 Grundfest, H., 349, 351, 357, 3591, 362, 363, 364, 366, 367, 371, 372, 373, 374, 377, 383, 386, 388, 389, 390, 391, 392, 393, 395, 396, 415, 427, 428, 429, 430, 437, 441, 443, 444, 445, 446, 447, 448, 449, 450, 452, 453, 454, 456, 458, 459, 477, 482, 485, 486, 488, 489, 490, 514, 519, 539, 549, 557, 558, 564, 569, 570

G

Haddle, G. P., 154, 160, 201 Haddon, A. C., 136, 147, 163, 192 Haedrich, R. L., 190, 192 Haempel, O., 229, 237 Hardig, J., 101, 116 Hafen, G., 229, 237 Hager, H. J., 63, 7 6 Hagiwara, S., 129, 133, 273, 346, 482, 488, 543, 550, 551, 552, 557, 560, 571, 573 Hahn, W. E., 107, 110, 118 Haller, H., 300, 345 Hama, K., 257, 262 Hamasaki, D. H., 4, 28 Hamasaki, D. I., 4, 28, 36, 39, 54 Hanaoka, T., 26, 29 Hanyu, I., 3, 29 Hara, T. J., 96, 97, 98, 99, 106, 110, 111, 112, 113, 115, 116, 120 Hardenberg, J. D. F., 137, 196 Harden Jones, R. R., 63, 76 Harder, W., 432, 488, 497, 498, 528, 541, 543, 560, 571 Harris, A. J., 73, 76 Harris, G. G., 141, 153, 169, 170, 183, 196, 242, 249, 250, 258, 261, 262, 566, 567, 571 Hartig, G. M., 174, 204 Hartline, H. K., 35, 40, 41, 54 Hashimoto, H., 39, 44, 50, 51, 54 Hashimoto, T., 189, 190, 196 Hashimoto, Y., 47, 56 Hasler, A. D., 80, 92, 105, 110, 116, 119, 146, 152, 187, 200 Hawkins, A. D., 158, 176, 187, 196, 199 Hawkins, J. E., 180, 196

Gage, P. W., 361, 449, 487 Gainer, H., 151, 152, 195 Galler, S. R., 189, 195 Gardner-Medwin, A. R., 43, 53 Gasser, H. S., 90, 96, 115 Gautron, J., 460, 488 Gaze, R. M., 63, 64, 76 Gemne, G., 84, 90, 98, 99, 115 Geoffroy St. Hilaire, I., 138, 195 GBrard, P., 494, 550, 570 GBry, J., 383, 487 Gilbert, P. W., 9, 12, 29, 105, 115 GimBnez, M., 412, 461, 463, 4 6 5 467, 469, 470, 471, 474, 486, 533, 569 Glaser, D., 93, 115 Gleisner, L., 213, 222, 239 Comer, P., 242, 259, 262 GO,, H., 106, 115 Gorbman, A., 96, 97, 98, 99, 106, 107, 110, 111, 112, 115, 116, 118, 120 Goronowitsch, W., 26~3,345 Grangaud, R., 19, 28 Granit, R., 35, 36, 37, 38, 39, 40, 54 Grass&,P.-P., 164, 167, 195 Gray, E. G., 302, 303, 304, 305, 306, 344, 345 Gray, G.-A., 187, 188, 195 Green, W. C., 138, 190, 197 Greene, C . W., 136, 137, 149, 150, 158, 195 Greenwood, P. H., 383, 437, 487, 488 Griffin, D. R., 166, 170, 189, 196, 495, 570 Grimm, R. J., 105, 115 Groen, J. J., 216, 237

H

579

AUTHOR INDEX

Hays, E. E., 138, 139, 203 Hazlett, B. A., 146, 147, 151, 158, 187, 188, 196, 204 Heinecke, P., 187, 199 Held, R., 72, 76 Hemmings, C. C., 107, 112, 116, 176, 199 Hemmings, G., 73, 76 Hensel, H., 131, 132, 133 Hering, E., 52, 54 Herrick, C. J., 87, 116, 164, 196, 554, 571 Herrniknd, W. F., 138, 188, 193 Hersey, J. B., 138, 200 Herter, K., 60, 76 Hester, F. J., 189, 196 Hibbard, E., 343, 345 Hidaka, I., 100, 101, 116, 117 Higman, H. B., 389, 459, 488 Hille, B., 358, 362, 449, 454, 458, 487, 488 Hirata, Y., 87, 88, 116 Hoagland, H., 100, 116, 123, 125, 132, 133 Hodgkin, A. L., 349, 352, 358, 359, 458, 459, 484, 488 Hodgson, E. S., 105, 115 Hoglund, L. B., 101, 116 Hoff, I., 166, 198 Holl, A., 83, 84, 102, 103, 114, 116 Holmgren, F., 54 Hopkins, A. E., 84, 85, 86, 116 Horton, J. W., 166, 196 Hoyle, G., 482, 491 Hubel, D. H., 61, 76 Huber, G. C., 98, 117, 553, 568 Hudson, R. C. L., 343, 345 Hiittel, R., 108, 116 Humbach, I., 94, 116 Huxley, A. F., 389, 449, 458, 487, 488 Hyvarinen, J., 99, 115

I Idler, D. R., 105, 109, 114, 116, 117 Ikeda, H., 43, 53 Ingle, D., 62, 63, G4, 65, 67, 68, 69, 70, 72, 73, 74, 76 Inouye, K., 8, 29 Ishii, Y., 166, 195, 258, 262, 320, 334, 342, 345, 565, 570

Ishikawa, T., 33, 49, 57 Ishiyama, R., 375, 488 Israel, M., 372, 460, 488 Iversen, R. T. B., 175, 176, 196 Iwai, T., 87, 116, 168, 196

J Jacobs, D. W., 171, 173, 179, 180, J96 Jacobson, M., 63, 64,75, 76 Jagodowski, K. P., 84, 85, 86, 116 Jakubowski, M., 243, 262 Jasinski, A., 98, 116 Jielof, R., 169, 170, 196 John, K. R., 11, 29 Johnels, A. G., 427, 456, 488, 563, 571 Johnson, M. W., 137, 196 Jonas, R. E. E., 105, 109, 116, 117 Jones, F. R. H., 117, 166, 196 Jones, M. P., 10,27 Jurand, A., 343, 345

K Kahmann, H., 4, 29 Kaiserman-Abramof, I. R., 553, 571 Kalmijn, A. J., 495, 496, 501, 551, 558, 559, 570 Kandel, E. R., 98, 117 Kaneko, A., 26, 31, 39, 40, 44, 45, 46, 50, 51, 52, 54, 55, 56 Kappers, C. U. A., 98, 117 Karlin, A., 459, 488 Karnovsky, M. J., 463, 490 Katsuki, Y., 103, 117 Katz, B., 257, 262, 288, 345, 402, 477, 487, 488, 510, 512, 513, 514, 539, 566, 571 Keenleyside, M. H. A., 107, 117 Kellaway, P., 348, 349, 488 Kellogg, W. N., 158, 196 Kelsey, A. S., Jr., 137, 156, 195 Kendall, J. I., 103, 117 Kennedy, D., 3, 29 Keynes, R. D., 349, 383, 389, 427, 428, 429, 449, 456, 458, 477, 487, 489 Kilarski, W., 152, 197 Kinzer, J., 161, 197 Rlancher, J. E., 152, 195 Klausewitz, W., 145, 197

AUTHOR INDEX

Kleerekoper, H., 80, 86, 107, 108, 112, 117, 166, 169, 171, 180, 181, 197, 227, 229, 230, 231, 234, 237 Kluver, H., 65, 76 Knudsen, V. O., 137, 143, 144, 197 Kobayaslii, H., 37, 54 Koczy, F. F., 138, 201 Kolster, R., 300, 345 Konislii, J., 100, 101, 102, 117 Koynno, H., 565, 570 Kramer, E., 187, 199 Krausse, A., 229, 237 Kreidl, A., 162, 197 Krespi, V., 358, 486 Kriebel, M. E., 463, 482, 489 Krinner, M., 93, 117 Kritzler, H., 167, 170, 177, 178, 197, 232, 237 Kronengo!d, M., 138, 190, 197, 201 Kruger, L., 8, 31 Kubo, I., 218, 237 Kuchnow, K. P., 9, 12, 29 Kuffler, S. W., 40, 41, 54 Kuiper, J. W., 169, 170, 197 Kume, S., 459, 490 Kuroki, T., 181, 197 Kusano, K., 151, 195

1 Lafite-Dupont, J., 162, 197 Lander, M. R., 18, 21, 28 Lanyon, W. E., 155, 197 Larimer, J. L., 417, 489, 557, 560, 571 Laufer, M., 47, 54, 55, 56 Leghissa, S., 300, 345 Leitner, L. M., 565, 570 Lele, P. P., 123, 129, 133 Lesbats, B., 460, 488 Leuzinger, W., 459, 489 Licklider, J. C. R., 179, 197 Liebnian, P. A,, 26, 29 Lindeman, V. F., 3, 29 Lindquist, P. G., 503, 574 Lissmann, H. W., 349, 374, 391, 408, 432, 437, 489, 494, 495, 496, 498, 503, 524, 545, 556, 558, 559, 561, 563, 566, 572, 573 Locliner, 1. P. A., 177, 193 Loewenstein, J. M., 138, 190, 197 Losey, G. S., Jr., 109, 119

Lowenstein, O., 130, 133, 162, 165, 197, 208, 210, 211, 212, 213, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 233, 234, 235, 237, 238 Lowrey, A., 405, 489 Loye, D. F., 137, 197 Ludwig, C., 94, 98, 114 Luft, J. H., 2.44, 262, 385, 489 Lundquist, P.-G., 213, 222, 239 Lyon, E. T., 218, 238 Lythgoe, J. N., 15, 18, 21, 23, 24, 25, 28, 29

M MacBain (Spires), J. Y., 138, 201 McBride, J. R., 105, 109, 113, 115, 116, 117 McCleary, R. A., 26, 29, 60, 69, 76 McDonald, H. E., 162, 197, 229, 238 MacDonald, J. A,, 417, 489, 557, 560, 571 McFarland, W. N., 23, 29, 364, 490, 496, 572 Machili, P., 539, 573 hlachin, K. E., 495, 496, 558, 559, 563, 566, 571, 572 hhckenzie, K. V., 138, 197 MacKinnon, D., 109, 114 Mackintosh, J., 74, 77 Mackintosh, N. J., 61, 62, 73, 74, 77 McNally, W. J., 219, 221, 226, 238 MacNaughton, I. P. J., 219, 221, 226, 238 MacNichol, E. F., 26, 29, 41, 42, 43, 44, 47, 48, 49, 53, 54, 55, 56, 57 Mahajan, C . L., 144, 198 Maier, H. N., 229, 238 Maksimova, E. M., 52, 55 Malar, T., 181, 197 Maliukina, G. A., 137, 170, 174, 198 Mandriota, F. J., 441, 478, 489, 496, 536, 558, 559, 566, 572 Maniwa, Y., 189, 190, 196 Mann, H., 102, 117 Manning, F. B., 163, 164, 198, 218, 229, 238 Zlansueti, R. J., 143, 202 Marage, M., 162, 198 Marcstrom, A., 92, 118

581

AUTHOR INDEX

Marks, W. B., 26, 29, 44, 51, 52, 55, 60,

77 Marler, P., 184, 198 Marhall, J. A., 137, 143, 147, 151, 152, 158, 160, 187, 188, 198, 204 Marshall, N. B., 5, 13, 29, 138, 142, 148, 149, 156, 166, 198 Martins-Ferreira, H., 383, 389, 449, 476, 485, 489 Massonet, R., 19, 28 Mathews, R. D., 109, 119 Mathewson, R. F., 105, 115, 151, 195, 364, 367, 373, 374, 388, 430, 450, 453, 454, 489 Matsuda, H., 101, 117 Matthews, R., 34, 35, 53 Matthews, W. A., 62, 77 Mattocks, J. E., 207, 240 Mauro, A., 349, 373, 489, 536, 572 Mauthner, L., 266, 345 Maxwell, S. S., 215, 218, 238 Mayoh, H., 109, 116 Mayser, P., 266, 345 Mead, G. W., 190, 192 Meader, R. G., 554, 572 Meder, E., 137, 198 Meesters, A., 63, 77 Melzack, R., 129, 133, 339, 346 Meyer, E., 152, 166, 198 Meyer, S. L., 13, 30 Midttun, L., 166, 198 Miesner, H.-J., 92, 118 Miledi, R., 257, 262, 345, 477, 488, 510, 512, 513, 514, 539, 566, 571 Miles, S. G., 113, 118 Milkman, R. D., 3, 29 Miller, P. L., 539, 569 Milne, D. C., 258, 262 Mitarai, G., 47, 49, 54, 55, 56 Mittelstaedt, H., 129, 133 Moatti, J.-P., 19, 28 Mohres, F. P., 496, 558, 572 Mogensen, J. A., 108, 117 Moller, P., 354, 441, 469, 478, 489, 551, 557, 558, 559, 560, 572 Moncrieff, R. W., 87, 118 Moorhouse, V. H. K., 162, 198, 228, 238 Moreau, A., 136, 150, 198 Mori, Y., 102, 117 Morita, H., 543, 551, 552, 5f30, 571

Morlock, N. L., 391, 415, 458, 459, 489 Morton, R. A,, 15, 29 Motais, R., 3, 29 Motokawa, K., 38, 39, 41, 47, 55 Moulton, D. G., 87, 95, 118 Moulton, J. M., 135, 136, 138, 143, 144, 145, 150, 153, 156, 157, 158, 160, 162, 164, 166, 181, 187, 188, 189, 190, 198, 338, 343, 345 Mowbray, W. M., 137, 151, 156, 187, 192, 195 Miiller, J., 136, 147, 148, 198, 199 Mullinger, A. M., 494, 496, 498, 503, 512, 515, 524, 540, 551, 557, 558, 559, 560, 572 Munk, O., 5, 11, 13, 14, 29, 30 Munson, W. A., 173, 180, 195 Muntz, W. R. A., 26, 30, 60, 77 Munz, F. W., 15, 18, 21, 22, 23, 24, 25, 28, 29, 30 Murakami, M., 26, 31, 39, 40, 43, 45, 46, 51, 52, 53, 55, 56 Murray, R. W., 124, 130, 131, 132, 133, 247, 262, 494, 495, 500, 517, 544, 547, 559, 561, 572 Myers, G. S., 383, 488 Myrberg, A. A., Jr., 186, 187, 199

N Nachmansohn, D., 383, 386, 485 Naito, K., 19, 22, 30 Naka, K. I., 52, 55 Nakajima, S., 389, 415, 448, 449, 458, 490 Nakajima, Y., 412, 461, 463, 465, 467, 469, 470, 471, 472, 474, 486, 512, 531, 533, 569 Nakamura, Y., 389, 415, 448, 449, 458, 490 Nanba, R., 98, 118 Negishi, K., 47, 54 Nelson, D. R., 167, 177, 178, 180, 181, 186, 199, 204, 232, 236, 238 Nelson, E. M., 167, 199 Nelson, K., 146, 187, 199 Neurath, H., 92, 118 Nicholson, C., 553, 554, 572 Nicol, J. A. C., 1, 6, 9, 11, 12, 13, 19, 27, 28, 30

582

AUTHOR INDEX

Nieuwenhuys, R., 300, 345, 553, 554, 572 Nishi, K., 565, 570 Nishimura, M., 190, 196 Niwa, H., 4, 26, 31, 49, 56, 101, 117 Noell, W. K., 39, 55 Nosaki, H., 46, 50, 56 Noto, S., 8, 29 Nursall, J. R., 154, 199

0 Ohara, S., 519, 572 O’Connell, C. P., 4, 30 Ogawa, T., 38, 39, 41, 47, 55 Ohtsu, K., 22, 30 Oikawa, T., 38, 39, 47, 55 Olmsted, J. M. D., 104, 118 Orcutt, B., 459, 490 Orlov, 0. Yu., 52, 55 Oshorn, C. M., 9, 32 Osborne, M. P., 208, 210, 211, 212, 213, 221, 223, 224, 238 Oshima, K., 96, 97, 98, 106, 107, 110, 112, 118 Ostroy, S. E., 15, 27 Otsuka, N., 343, 346 Ottoson, D., 95, 96, 118

P Packard, A., 151, 199 Pak, W. L., 43, 55 Palade, G. E., 463, 487, 504, 570 Palay, S. L., 87, 89, 119, 553, 571 Palmer, E., 123, 133 Pappas, G. D., 392, 401, 402, 406, 412, 416, 433, 444, 450, 453, 454, 456, 459, 461, 463, 465, 467, 469, 470, 471, 472, 474, 482, 489, 490, 512, 531, 533, 569 Parker, G. H., 80, 104, 118, 162, 163, 177, 199, 218, 219, 227, 229, 238 Parkhurst, R. M., 109, 119 Parrish, B. B., 176, 199 Parvulescu, A., 142, 155, 173, 199 Pautler, E. L., 26, 31, 45, 46, 51, 52, 56 Payton, B. W., 463, 490 Pearcy, W. G., 13, 30 Pearson, A . A,, 163, 164, 199 Pellegrin, J., 437, 490 Pfeiffer, W., 83, 108, 109, 119, 144, 199

Piatt, J., 343, 346 Pickens, P. E., 364, 490, 496, 572 Piper, H., 227, 238 Pipping, M., 82, 119 Pitt, G. A. J., 15, 29 Plack, P. A., 19, 30 Poggendorf, D., 170, 171, 174, 192, 199, 227, 231, 238 Poll, M., 437, 439, 490 Polleski, T. R., 389, 459, 488 Popper, A. N., 171, 199 Post, R. L., 459, 490 Prince, J. H., 1, 4, 30 Protasov, V. R., 135, 137, 138, 161, 187, 198, 200 Proudfoot, D. A., 137, 197 Pumphrey, R. J., 5, 6, 7, 31, 166, 182, 200

R Rall, W., 567, 573 Ramsay, D. A,, 110, 119 Rashcheperin, V. K., 161, 200 Rauther, M., 149, 200 Reed, J. R., 108, 119 Reese, T. S., 86, 119, 463, 486, 567, 573 Reickel, A,, 137, 200 Reinhardt, F., 163, 181, 200, 234, 238 Remmler, W., 490 Retzius, G., 208, 210, 239 Retzlaff, E., 279, 280, 320, 346 Revel, J. P., 151, 195, 463, 490 Rhodin, J., 244, 262 Richard, J. D., 177, 178, 181, 186, 190, 199, 200, 204 Richardson, E. G., 155, 200 Riddell, L. A., 36, 37, 54 Roberts, T. D. M., 165, 197, 233, 234, 235, 237 Robertson, J. D., 317, 319, 346 Rochon-Duvigneaud, A., 1, 4, 31 Rode, P., 163, 200 Rodgers, W. L., 339, 346 Roggenkamp, P. A., 166, 169, 171, 197, 227, 231, 237 Ronianenko, E. V., 161, 200 Romanes, G. H., 218, 239 Roper, S., 343, 346 Rosen, D. E., 383, 488 Rosenberg, H., 490

583

AUTHOR INDEX

Rosenblatt, R. H., 109, 119 Roth, A., 494, 495, 500, 514, 515, 516, 530, 563, 564, 573 Rovainen, C. M., 342, 346 Ruhin, M. A., 123, 124, 133 Rushton, W. A. H., 52, 55 Russell, I. j., 257, 261, 262

S Safriel-jorne, O., 74, 77 Saito, N., 474, 482, 488, 490 Salmon, M., 143, 145, 152, 187, 200 Sand, A,, 130, 131, 132, 133, 163, 200, 215, 216, 217, 237, 238, 258, 262 Sasaki, Y., 39, 55 Sato, Y., 47, 56 Saxena, A., 63, 65, 66, 77 Scharf, B., 180, 200 Scharrer, E., 87, 89, 119 Schevill, W. E., 138, 200 Schief, A., 560, 571 Schneider, H., 138, 144, 145, 146, 147, 149, 151, 152, 155, 157, 158, 166, 174, 180, 187, ZOO, 228, 230, 239 Schneider, J. E., 72, 77 Schneirla, T. C., 184, 191, 200, 201 Schnitzlein, H. N., 90, 91, 119, 553, 573 Schoen, L., 219, 226, 239 Schone, H., 226, 239 Schor, R., 536, 572 Schriever, H., 163, 201 Schroeder, W. C., 374, 486 Schrodinger, E., 52, 55 Schukneckt, H. F., 257, 262 Schulte, A., 65, 66, 69, 70, 77 Schutz, F., 108, 119 Schwanzara, S. A., 15, 16, 17, 18, 20, 21, 23, 24, 30, 31 Schwartz, E., 242, 263 Schwartz, I. R., 392, 401, 402, 406, 416, 433, 444, 450, 453, 454, 456, 459, 490 Schwasqman, H. O., 8, 31, 408, 489 Sebeok, T. A., 184, 201 Segall, M., 11, 29 Seliger, H. H., 9, 31 Sen, A. K., 459, 490 Shapiio, S. M., 70, 77 Shaw, E., 6, 7, 27, 168, 191, 193, 201 Shaw, T. I., 21, 28

Sheldon, R. E., 90, 98, 104, 119 Shepherd, G. M., 95, 118, 567, 573 Sheridan, M. N., 372, 450, 453, 490 Sherrington, C., 471, 490 Shihuya, T., 94, 95, 119 Shimada, H., 112, 118 Shishkova, E. V., 153, 201 Shores, D. L., 190, 192 Siler, W., 173, 193 Silvester, C. F., 362, 364, 487 Simpson, G. G., 21, 31 Sims, R. T., 337, 346 Sinclair, D. C., 129, 133 Sjostrand, F. S., 33, 55 Sjostrand, J., 343, 344 Skinner, W. A., 109, 119 Skoglund, C. R., 38, 53, 151, 201 Skudrzyk, E. j., 154, 160, 201 Smith, E. D., 177, 193 Smith, H. M., 136, 201 Smith, I. C., 271, 346 Smith, M., 113, 115 Smith, S. W., 87, 89, 119 SGrensen, W., 136, 137, 144, 148, 201 Sorgente, N., 143, 145, 152, 187, 200 Spath, M., 125, 126, 127, 128, 133 Sperry, R. W., 69, 77 Spires, J. Y., 138, 143, 193 Spoor, A., 169, 170, 196 Spoendlin, H., 533, 565, 573 Sprengling, G., 108, 116 Stage, D. E., 317, 319, 346 Stampehl, H., 137, 201 Stefanelli, A., 267, 340, 343, 346 Stein, R. B., 479, 490 Steinhach, A. B., 417, 421, 424, 474, 486, 490, 531, 532, 539, 554, 566, 569, 573 Steinherg, J. C., 138, 201 Steinhausen, W., 215, 239 Stell, W. K., 33, 47, 56 Stendell, W., 573 Sten\io, E., 208, 239 Stetter, H., 162, 163, 164, 170, 179, 201, 203, 219, 226, 228, 229, 230, 231, 239 Stevens, D. M., 107, 119 Stevens, S. S., 180, 196 Stipetii., E., 163, 174, 179, 201, 228, 230, 239

584

AUTHOR INDEX

Stone, H., 87, 119 Stout, J. F., 187, 201, 204 Strieck, F., 91, 93, 119 Strother, W. F., 174, 204 Suckling, E. E., 169, 170, 202, 432, 491 Suckling, J. A,, 169, 170, 202 Suga, N., 499, 503, 550, 551, 560, 561, 563, 573 Sullivan, C. M., 123, 124, 133 Sutherland, N. S., 61, 62, 65, 66, 67, 68, 74, 76, 77 Svaetichin, G., 35, 47, 48, 49, 54, 55, 56 Swanson, R. T., 23, 30 Szabo, T., 392, 430, 432, 441, 442, 443, 447, 448, 456, 472, 473, 474, 487, 488, 490, 491, 494, 498, 503, 514, 520, 524, 528, 530, 533, 539, 540, 543, 550, 551, 553, 557, 560, 561, 568, 570, 571, 573 Szaniier, R. B., 494, 498, 500, 503, 505, 506, 507, 508, 515, 522, 524, 549, 573, 574

T Tagliani, G., 300, 346 Takeda, K., 354, 409, 491, 557, 560, 574 Takeuchi, A., 448, 491 Takeuchi, N., 359, 365, 448, 491 Tamura, T., 4, 26, 31, 49, 56 Tarrant, R. M., Jr., 92, 119 Tasaki, I., 273, 346, 391, 491 Tasaki, K., 38, 39, 55 Tateda, H., 102, 120 Tavolga, W. N., 106, 120, 135, 136, 138, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 155, 156, 157, 158, 159, 160, 161, 164, 165, 167, 171, 173, 174, 175, 179, 180, 184, 185, 186, 187, 188, 190, 196, 197, 202, 204, 231, 239 Taylor, J. R., 565, 570 Taylor, M., 143, 202 Teal, J. M., 190, 192 Teichniann, H., 80, 82, 83, 92, 120 Tesch, F. W., 113, 115, 120 Tester, A. L., 103, 117 Thompson, R. L., 441, 478, 489, 546, 547, 558, 563, 566, 572 Thornhill, R. A., 208, 210, 211, 223, 224, 225, 238

Thurm, U., 263 Tiegs, 0. W., 300, 302, 346 Tobin, T., 459, 490 Todd, J. H., 106, 113, 114, 120 Tomaschek, H., 163, 202 Tomita, T., 26, 31, 35, 39, 44, 45, 46, 47, 50, 51, 52, 56 Tomlinson, N., 105, 109, 113, 115, 116, 117 Tosaka, T., 47, 56 Tower, R. W., 136, 146, 149, 150, 203 Toyoda, J., 46, 50, 56 Toyota, M., 101, 117 Tracy, S. C., 167, 203 Trevarthen, C. B., 63, 72, 73, 77 Trifonov, Yu. A., 49, 53, 56 Trinkaus, J. P., 436, 486, 504, 569 Trudel, P. J., 93, 120 Trujillo-Cen6z, O., 84, 87, 120 Tschiegg, C. E., 138, 139, 203 Tsvietkov, V. I., 161, 200 Tucker, D., 95, 118

U Uchida, M., 102, 117 Uchihashi, K., 90, 91, 120 Uchizono, K., 304, 346 Ueda, K., 110, 111, 116, 120 Uhlemann, H., 432, 488, 560, 571 Ulrich, H., 218, 239 Usherwood, P. N. R., 539, 573

V Vallecalle, E., 47, 54, 55, 56 van Bergeijk, W. A., 141, 164, 169, 173, 181, 182, 183, 196, 203, 261, 262, 263 Vanderwalker, J. G., 189, 203 van Heusen, A. P., 162, 199, 229, Vendrik, A. J. H., 216, 237 Verheijen, F. J., 109, 120, 179, 194, 236 Verrier, M.-L., 4, 27 Versteegh, C., 211, 225, 236 Vigonrenx, P., 138, 203 Villegas, G. M., 33, 56 Villegas, J., 47, 54, 55, 56 Villegas, R., 33, 56

170, 242, 238 230,

585

AUTHOR INDEX

Vilter, V., 5, 13, 31 Vincent, F., 158, 162, 203 Vinnikov, J. A., 86, 95, 120 von Baumgarten, R., 92, 98, 118 von BAkAsy, G., 180, 203 von Boutteville, K. F., 170, 171, 203, 229, 231, 239 von Buddenbrock, W., 89, 120 von Frisch, K., 89, 107, 120, 162, 163, 164, 165, 171, 181, 203, 208, 219, 226, 227, 228, 229, 234, 239, 320, 321, 346 von Holst, E., 129, 133, 219, 226, 239 von Ihering, R., 136, 203 von Kries, J., 52, 56 Vu-TBn-Tu&,383, 487

W Wachtel, A. W., 364, 367, 374, 375, 388, 430, 450, 453, 454, 456, 489, 491, 494, 498, 500, 503, 505, 506, 507, 508, 515, 522, 524, 549, 573, 574 Wagner, H. G., 26, 29, 41, 42, 43, 53, 55. 56. 57 Wald, G., 15, 19, 25, 31 Walker, M. A., 19, 28 Walker, T. J., 168, 203 Wall, P. D., 129, 133 Walls, G. L., 1, 2, 4, 5, 7, 8, 9, 11, 13, 31 Walters, V., 149, 203 Waltman, B., 494, 504, 517, 547, 549, 574 Warner, L. H., 162, 204 Warren, F. J., 19, 25, 28 Warwick, R. T . T., 90, 114 Watanabe, A., 273, 346, 354, 409, 482, 488, 491, 557, 560, 574 Watanabe, K., 47, 56 Watkins, W. A., 155, 158, 190, 204 Waxman, S. G., 463, 482, 489 Weale, R. A., 13, 31 Weber, E. H., 162, 163, 204 Weddell, G., 123, 129, 133 Weiler, I. J., 60, 77 Weiss, B. A., 171, 172, 173, 174, 204 Weitzman, S. H., 383, 488 Welsh, J. H., 9, 32 Wenz, G. M., 140, 141, 155, 204

Werblin, F. S., 33, 51, 54, 56 Werner, C. F., 219, 239 Werner, H., 77 Wersa11, J., 165, 195, 212, 213, 221, 222, 238, 239, 253, 262, 503, 52.4, 573, 574 Westby, G. W. M., 496, 558, 574 Westerfield, F., 180, 204, 228, 230, 240 Westerman, R. A,, 61, 77, 85, 86, 90, 98, 120 Weston, D. E., 189, 204 Whitear, M., 88, 120 Whittaker, V. P., 372, 491 Wiesel, T. N., 39, 50, 53, 61, 76 Williams, B., 87, 119 Willmer, E. N., 21, 32 Willows, A. 0. D., 482, 491 Wilson, D. M., 342, 346 Wilson, H. V., 207, 240 Wilson, J . A. F., 85, 86, 90, 98, 120 Wilt, F. H., 19, 22, 30, 32 Wing, A. S., 190, 192 Winn, H. E., 143, 145, 146, 147, 150, 151, 152, 156, 158, 160, 175, 187, 188, 193, 195, 196, 2.00, 204 Wisby, W. J., 92, 116, 177, 178, 181, 204 Witkovsky, P., 36, 52, 53, 57 Wittenberg, B. A., 3, 32 Wittenberg, J. B., 3, 32, 150, 194 Wodinsky, J., 153, 167, 171, 173, 174, 175, 193, 202, 204, 231, 239 Wohlfahrt, T. A., 163, 167, 179, 204, 205, 209, 226, 230, 234, 240 Wolbarsht, M. L., 26, 29, 41, 42, 43, 53, 55, 56, 57 Wolfe, J., 68, 76 Wolff, D. L., 174, 176, 177, 189, 205 Wood, L., 167, 170, 177, 178, 197, 232, 237 Wrede, W. L., 106, 120 Wiirzel, M., 351, 357, 371, 372, 373, 449, 452, 454, 486, 549, 569 Wyllie, J. H., 19, 28 Y

Yager, D., 26, 32, 61, 77 Yamada, E., 33, 49, 57 Yamashita, E., 39, 41, 55 Yanagisawa, K., 103, 117

AUTHOR INDEX

586 Yasargil, G. M., 276, 278, 285, 290, 291,

Yokota, S., 100, 116 Young, J. Z., 9, 32 Young, T., 51, 57

Z

Zipser, B., 530, 536, 566, 574 Zotterman, I., 165, 205 Zotterman, Y., 100, 117, 129, 133

SYSTEMATIC INDEX Note: Names listed are those used by the authors of the various chapters. No attempt has been made to provide the current nomenclature where taxonomic changes have occurred. 366, 373, 389, 451, 462, 468, 471, A 480,481, 483, 553, 554, 562 Abramis, 98 Astyanax mexicanus, 171 Acerina cernua, 177, 229 Atherinops asinis, 109 Acipenser fdvescens, 88 Adontosternarchus, 383, 423-425, 456, Auks, 339 461 B Aequidens pulcher, 62 Bagre marinus, 144, 148, 151, 159, 160 Agnatha, 208 Balistes, 144, 145 Alburnus lucidus ( A . alburnus), 229 Barracuda, see Sphyraena Alosa, 6 Ameiurus, 9, 87, 104, 106, 234, 515, 516, Bathygobius, 106 Bathylychnops, 13, 14 553 Bathystoma rimator, 107 A . nzelas, 102 Bathytrocetes, 5 A. nebulosus, 122, 229, 231 Batrachoididae, 158, 188 Amia, 3 Amphibia, 75, 96, 106, 154, 266, 267 Belonesox, 21 Beta splendens, 228 Amphioxus, 123 Bittern, 340 Amphiprion, 144 Blennius, 161, 167, 187 Anabantidae, 228, 230, 231 B. pholis, 122 Anabas scandens ( A . testudineus), 228 Blenny, see Blennius Anableps, 8, 14 Blue acara, see Aequidens pulcher Anacanthini, 98 Botia hymenophysa, 145 Anchovies, 4, 160 Brotulidae, 148, 149 Anglefish, 3 Bullhead, see Ictalurus Anguilla, 9, 82-84, 90, 91, 174, 180 Yellow bullhead, see Ictalurus natalis A. anguilla, 81, 83, 92, 109, 113, 228, Burbot, see Lota 230 A . iaponica, 94 C A . rostrata, 113 Caranx, 156, 160, 161 A. vulgaris, 113 Carassius, 11, 90, 98 Anguillidae, 174, 228 C. auratus, 92, 96, 110, 122, 171, 172, Anoptichthys iordani, 93, 94, 108 179, 218, 229 Apeltes quadracus, 144 C . auratus L., 276, 283, 304, 305, 322, Aplodinotus, 146, 187 323, 338 Apteronotus, 383, 448, 455, 456 C. carassius, 85, 90 Argyropelecus, 13 Carcharinus, 4 Astronotus ocellatus, 60, 108 C. leucas, 177, 232 Astroscopus, 349, 351, 362, 363, 365, Carp, 26, 35, 36, 38, 39, 4446, 49-51, 587

SYSTEMATIC INDEX

53, 66, 69, 70, 90, 91, 94, 95, 100, 101, 103, 181, 553, see also Carassius European, see Carassius carassius Japanese, 100 Swedish, 100 Carpiodes, 553, 554 Catfish, 91, 94, 95, 98, 100, 102, 136, 146, 147, 150-152, 158, 231, 494, 495, 497, 549, 562, 563, see also Ameiurus, Ictalurus, Clarias, Leptops, Parasilurus Gafftopsail, see Bagre marinus Japanese, 273 Marine, 10, 157, 186, 188, see also Galeichthys felis, Plotosus anguillariS Centrarchid, 22 Centronotus gunnellus, 122 Centropomidae, 48, 49 Cetacea, 138, 143, 495 Channa, 94 C . argus, 95, 100 Characidae, 20, 146, 187 Characinidae, 229, 231 Cichlasoma seuerum, 108 Cichlidae, 187 Clarias, 558, 559 Clown fish, see Amphiprion Clupea, 10, 166 C. harengus, 167, 231 Clupeidae, 167, 231, 234 Clupeoididae, 2 Cnesterodon, 84 Cobitidae, 229 Cobitis, 88 Codfish, 158, see also Gadus Atlantic cod, see Gadus morhua Colisa lalia, 228 Congiopodus, 151 Cormorants, 339 Coruina, 174, 179, 187 C . nigra, 22s. 230 Corydoras, 87 Cottidae, 228 Cottus, 180 C . bulbalis, 122 C. gobbio, 228 C. pnuo, 228 C . scorpius, 122, 165, 166, 228, 235 Crab, 104, 105

Crenilabrus C. griseus, 228 C . melops, 122, 228 Croacher, 152, 158, 160, 187 Croaching gourami, see Trichopsis uittatus Crustacea, 132 Ctenobrycon, 17, 18 Cyclopterus lumpus, 122 Cyclostoma, 2, 208, 223 Cymatogaster aggregatus, 228 C ynoscion C. nobilis, 158, 174 C . regalis, 218 Cyprinodont, 84 Cyprinus, 83, 86, 88, 94 Cyprodontidae, 228 C ypselurus heterurus, 2 Cyrpinidae, 11, 20-23, 51, 52, 84, 87, 208, 229, 231 D Dactylopteridae, 150 Dactylopterus, 149, 150 Dasyatis, 4, 553 Diplodus sargus, 112 Dogfish, see Acanthias, Mustelus, Scylliorhinus, Squalus, Scyllium smooth, see Mustelus canis spiny, see Squalus acanthias spotted, see Scyliorhinus caniculus spur, see Squalus acanthias Drumfish, 152, 158, 160, 187 fresh-water drumfish, see Aplodinotus

E Eel, 3, 34, 81, 95, 146 American, see Alosa rostrata Electric, see Electrophorus electricus European, see Alosa alosa Eigenmannia, 361, 382, 383, 409411, 415-417, 433, 450, 452, 479, 506508, 526, 533 Elasmobranchi, 2-4, 6, 8, 9, 12, 13, 21, 34, 39, 87, 89, 130, 143, 162, 165, 177, 181, 207, 208, 211, 215, 218, 219, 221-223, 225, 232, 234-236, 374, 451, 457, 494, 497, 500, 517, 550, 553, 555, 562, 563

SYSTEMATIC INDEX

Electrophoridae, 350, 382 Electrophorus electricus, 229, 349, 350, 360, 380-382, 388, 391, 392, 448, 451, 453, 457459, 471, 474476, 478, 480, 481, 483, 499, 515, 546, 550, 554, 555, 561, 562 Elephant nose fish, see Mormyridae Emboitocidae, 228 Entosphenus, 2, 94 Epinephelus striatus, 146, 147 E. guttatus, 151-153 Esocidae, 228 Esox, 82-84, 86, 90 E. estor, 96 E. Eucius, 215, 217 E. niger, 108

F Fitzroyia, 84 Flatfish, see Solea, Microstomus, Pleuronectes platessa, Platessa flesus Flying fish, see Cypselurus heterurus Flying gurnard, see Dactylopterus volitans Fundulus, 4 F. heteroclitus, 104, 228 G Gadid, 21 Gadidae, 174, 228 Gadus G. calkmias, 122, 187, 228 G. merlangus, 122 G. morhua, 176, 177 G. vivens, 122 Gaidropsarus, 174 Galeichthys, 159 G. felis, 112, 140, 144, 148, 188 Gasteropelecus, 342, 343, 482, 514, 545 Gasterosteus, 82-84 G. aculeatus, 93, 144 Gekko gekko, 46 Genomyrus, 437 G. donnyi, 437,439 Giguntura, 13 Ginglymostoma, 4 G. cirratum, 103, 105 Glandulocauda inequalis, 146 Gnathonemus, 228, 230, 437, 441443, 453, 456, 465, 477, 505, 521, 523,

589 529, 531, 537, 538, 542, 551, 554, 560 G. compressirostris, 441, 447 G. leopoldianus, 439 G. moorii, 447 G. numenius, 439 G. petersii, 439, 497, 498 Gnathostoma, 8, 208-211, 223-225 Gobies, 161, 187 Gobiidae, 106, 174, 228 Gobio, 82, 83 Gobius, 167, 174, 179 G. flavescens, 122 G. iozo, 219 G. niger, 228, 230 G. paganellus, 228 Goldfish, 11, 41, 42, 44, 60-70, 73-75, 90, 92, 97, 105, 267, 269, 273-275, 482, 545, see also Carassius Gourami, see Trichogaster Grouper, 150, 152, 174, 187 black, see Mycteroperca bonaci Nassau, see Epinephelus striatus Grunt, see Haemulon blue-striped, see H. sciurus white, see H. album Guitarfish, 553 Gymnarchidae, 350 Gymnarchus, 350, 355, 361, 393, 399, 415, 416, 419, 421, 432, 434, 435, 446, 450453, 456, 473, 494, 497, 512, 540, 556559, 562 G. niloticus, 350 Gymnocymbus ternetzii, 219, 226 Gymnorhamphichthys, 402, 408, 417, 478 G. hypostomus, 382 Gymnotidae, 229, 349, 351, 354, 355, 360, 361, 380-382, 384, 416, 432, 441, 451, 453, 456458, 467471, 474, 476, 494, 497-499, 503, 503, 506, 507, 509, 511, 512, 515, 516, 522, 524-526, 533, 534, 535, 541, 545, 547, 550, 553, 555-560, 562, 563 Gymnotus, 354, 390-397, 402, 417, 421, 451, 453455, 470, 471, 476, 479, 502, 509, 511, 533-535, 541, 545, 551, 555-558, 560 G. carapo, 351, 382

590

SYSTEMATIC INDEX

L

H Haddock, see Melanogrammus aeglefinus

Labridae, 228

Haemulon, 143, 145, 175, 186 H . album, 142 H . plumieri, 142 H . sciurus, 171 Hake, see Urophycis Hatchetfish, see Gasteropelecus Hemigrammus caudovittatus, 93, 229 Hepsitia stripes, 107 Herring, 160, see also Clupea Holocentrus, 158-160, 175, 553, 554 H . ascensionis, 151, 167, 171 H . rufus, 147, 151 Huso huso, 161 Hyborhynchus notatus, 92, 105 Hydrolagus afinis, 12 Hyperopisus, 437, 442 H . bebe, 439 Hyperprosopon, 168 Hyphessobrycon flammeus, 229 Hypopomus, 382, 383, 395402, 406, 409, 417, 428, 451, 453, 478, 479, 522, 525, 550, 560 H . artedi. 396

Lagodon rhomboides, 144 Lamna, 4 Lampetra, 86, 208 L . fluviatilis, 211 Lamprey, 3-5, 95, 224, see also Petromyzon Lebistes, 164, 174 L. reticulatus, 228 Lepomis, 22 L. gibbosus, 112, 122 Leptops, 554 Leuciscus L. dobula, 229 L. rutilus, 92, 101, 125-128, 223 Liparis montagui, 122 Lophius, 9 Lota, 21 L . lota, 82-84, 90, 97, 98, 242, 243, 249-251, 259, 260 L. vulgaris, 221, 222 Lucioperca sandra, ,177 Lungfish, see Protopterus sp. Lutjanidae, 48

I

M

Ictalurus, 9, 83, 90, 103, 109, 110, 166, 515 1. natalis, 102, 106, 107, 113, 122 1. nebdosus, 112, 113, 171, 174 ldus melanotus (Leucisctis i d u s ) , 229 Indian loach, see Botia hymenophysa Ipnops, 13 Isichthys, 437 I . henryi, 437, 439

Macro podus M . cupanus, 228 M . opercularis, 228, 230 Macrouridae, 149 Malapteruridae, 351 Makzpterurus, 441, 453, 454, 456 M. caballus, 439 M . rume, 441, 444446 Marcusenius M . isodori, 229, 230 M . plagiostoma, 439 Margate fish, see Haemulon album Melanogrammus aeglefinus, 158, 187 Menidia, 6 Mexican blind cave fish, see Anoptichthys jordani hlexican blind characin, see Astyanax mexicanus Af icrogadus tomcod, 103 Micropterus punctrilatus, 108 Microstomiis, 11 Midshipman, see Porichthys notatus

J Jack, see Caranx

K Kiaeraspis, 208 Killifish, see Fundulus sp. Knifefish, see Electrophortis electricus Kokanee, see Oncoihynchus nerka Kryptoperus bicirrhus, 500, 513, 515, 516, 559

SYSTEMATIC INDEX

Minnow, see Phoxinus bluntnose, see Hyborhynchus notatus freshwater, 187 Misgurnus, 94 Monomitpus, 148 Moray, see Muraenidae Mormyridae, 149, 350, 355, 360, 432, 436, 437, 439, 441, 446, 451, 453, 456, 465, 468, 472, 473, 475, 477479, 482, 494, 497, 498, 503, 505, 512, 515, 516, 521, 523, 524, 528, 529, 531, 532, 536-538, 540, 542544, 547, 548, 550, 551, 553-555, 557, 559, 560, 562, 563 Mormyrops, 437, 441, 442, 446, 473, 554 M . deliciosus, 439 Mugil, 2, 48, 174 Mugilidae, 48, 174 Mullet, see Mugil Mullidae, 174 Mullus, 174 Muraenidae, 82 Mustelus, 4, 12 M . cannis, 104, 218 M . mustelus, 112 Mycteroperca bonaci, 160 Mylinae, 108 Myliobatis, 4, 11 Myoinyrus macrodon, 439 Myxine, 208, 210, 211, 223, 225, 226 Myxinoididae, 210 Myxocephalus, 84, 145, 151

N Narcine, 369, 370, 373, 457, 495 Necturis maculosus, 46, 244, 247-250 Negaprion, 4, 12, 105 N. breuirostris, 178, 180, 181, 186, 232, 235 Nemacheilus barbatula, 229 Nerophis lumbriciformes, 122 Nolemigonus, 21 Notopterus ofer, 382 Nurse shark, see Ginglystoma cirratum

0 Oedemognathus exodon, 383 Omosidis, 13 Oncorhynchus, 10, 22, 109-111 0. kisutch, 92, 107, 109, 110

591 0. nerka, 22, 92, 105, 109 0. tshawytscha, 107, 109-111 Onos mustela, 122 Opisthoproctus, 13 Opsanus, 149, 150, 187 0. beta, 188 0. tau, 151, 157-160, 188 Orthosternarchus t a m n d u a , 383 Ostariophysi, 98, 108, 140, 152, 162, 164, 166, 170, 174, 179, 180, 227, 229232, 234, 235, 320, 321 Ostracodermi, 208

P Paralichthys, 8 Paramyomyrus, 437 P. aequipinnis, 437 Parasilurus, 88, 94 P. asotus, 102 Parrotfish, 144 Perca, 174 P. fluviatilis, 177, 229 Perch, see Perca common, see Perca fluviatilis Pike, see Lucioperca sandra stone, see Acerina cernua Percidae, 11, 174, 177, 228 Periophthalamus koelreuteri, 228 Petrocephalus, 437, 441, 442 P. sauvagei, 439 Petromyzon, 21 P. marinus, 108 Petromyzontidae, 210 Phoxinus, 63, 81-86, 88, 92, 93, 101, 106, 108, 164, 219, 234, 364 P. laevis, 107, 122, 123, 179, 209, 226 P. phoxinus, 81, 91, 93, 94, 214, 229, 230 Pigfish, see Congiopodus Pike, see Esox Pimephales notatus, 229 Pinfish, see Lagodon rhomboides Pipefish, see Syngnathus louisianae Platessa flesus, 226 Platichthys flesus, 122 Platyrhinoides triseriata, 554 Platyroctegen, 5 Platytrocetes, 5 Pleuronectes, 11 P. platessa, 122, 226

592

SYSTEMATIC INDEX

Plotosus, 550 P. anguillaris, 101, 102 Poeciliidae, 21, 174 Pollachius pollachius, 176 P. virens, 176 Pollack, see Pollachius Pomadasyidae, 142, 145, 174, 186 Porichthys, 137, 149, 150 P. notatus, 150, 157, 175, 188 Porotergus, 383 Priacanthidae, 143 Prionotus, 8, 87, 89, 149, 150 P. carolinus, 103 P. scitulus, 151 Protopterus, 271 Pseudopleuronectes, 8 P. americanus, 218 Puffer, 3, 82, 144 Pyrrhulina rachoviana, 229

R Rabdolichops longicaudatus, 382 Raja, 8, 130, 131, 225, 348, 350, 362, 472, 495, 500, 517, 547, 557-559 R. clavata, 216, 219, 220, 225, 233, 234 R. eghntaria, 375, 376 R. erinacea, 375-379 Rana, 22 R. esculenta, 106 R. palustris, 226 R. sylvatica, 226 R. temporaria, 106 Ray, 4, 81, 132, 143, 212, see abo M yoblatis, Dasyatis electric, see Torpedo marmorata thornback, see Platyrhinoides triseriata Rhamphichthys, 409 R. rostrattis, 382 Roach, see Leuciscus rutilus, Rutilus rutilus

5 Salamander, see Triturus, Salamandra, Necturus maculosus Salamandra, 106 Salmo, 11 S . fario, 553, 554 S. gairdneri, 110, 122 S . irrideus, 92, 109 S . salar, 101

Salmon, 10, 113, 189, 207 atlantic, see S. salar chinook, see Oncorhynchus tshwytscha coho, see 0. kiszctch pacific, see Oncorhynchus sockeye, see 0. nerka South American, see Hemigrammus caudovittatus Salmoniidae, 23, 95 Salvelinus, 11, 23, 123, 124 Sargus, 174, 179 S . annularis, 229, 230 Scardinus, 21, 22 S. erythrophthalamus, 107 Scaridae, 144 Scatophagus, 16 Schreckstoff, 185 Sciaenidae, 136, 137, 146, 151, 152, 158, 187, 229 Scombroid, 2 Scorpaenidae, 147 Sculpin, see Myoxocephalus, Cottus Scup, see Stenotomus chrysops Scyliorhinus, 8, 11 S. canicula, 178 S . stellaris, 112 Scyllium, 131, 132, 215 Sea bass, see Cynosion nobilis Sea horse, see Hippocampus Sea robin, 150, 151, 188, see also Prionotus, Trigla slender, see Prionotus scitulus Searsia, 5 Sebasticus, 147, 149 Selachians, 104 Semotilus, 181 S . atromaculatus, 122, 234 Serranidae, 48, 146, 174 Serrasalminae, 108 Shark, 21, 41, 81, 143, 178, 181, 186, 553, 555, 559, see also Ginglymostoma, Negaprion, Scyliorhinus, Sphyrna, Squatina, Mustelus bull, see Carcharhinus leucas lemon, see Negaprion brevirostris squaloid, see H ydrolagus affinis, Raja richardsonii Siluridae, 229, 231, 425 Siluroidea, 147, 163 Silversides, see Menidia Snapper, 174

593

SYSTEMATIC INDEX

Solea, 11 Sparidae, 144, 174, 229, 231 Sphenodon, 5 Sphyraena, 160 Sphyrna, 4, 105 Spinachia oulgaris, 122 Squalina, 4 Squalus, 12, 73 Squirrelfish, 140, 150, 152, 153, 187, 188, see also Holocentrus Stargazer, see Astroscopus Steatogenys, 383, 396, 402, 403, 405, 406, 413, 417, 451, 453, 454, 549, 551 S . elegam, 382, 404, 407-409 Stenesthes, 84, 86 Sternachidae, 349, 351, 361, 381-383, 416, 424, 433, 450452, 455, 479 Sternarchella, 383 Sternarchogiton, 383 Sternarchorhamphus, 383, 421, 423 S. oxyrhynchus, 383 Sternarchus, 354, 383, 416, 419423, 498 S . albifrons, 417 Sternopygidae, 351, 382, 451 Sternopygus, 354, 361, 382, 383, 409, 411-417, 419, 421, 433, 450, 452, 453, 479, 533 Stickleback, see Gasterosteus aculeatus Stingray, see Dasyatis Stomatorhinus, 437 S . corneti, 439 Sturgeon, see Acipenser fulvescens beluga, see Huso huso Surfperch, see Hyperprosopon Syngnathus louisianae, 145

Tilapia, 74 Tinca tinca, 94, 229, 276, 283, 303 Toadfish, 151-153, 187, see also Opsanrrs Tomcod, see Microgadus tomcod Torpedinidae, 350, 362, 369-371, 374, 451, 453, 456, 474, 483, 496, 562 Torpedo, 348, 350, 362, 371-374, 388, 449, 454, 460, 474, 480, 481, 548, 553, 561 T . marmorata, 161, 350, 371, 372 T . nobiliana, 371 Trichogaster, 89 T . leeri, 228 T . trichopterus, 228 Trichopsis uittatus, 137, 143 Triggerfish, 143, 145, 146, 187, see also Balistes, Rhinecanthus rectagulus Trigla, 136, 149, 150 Triglidae, 88, 150 Trout, 6, 63, 66, see also Saloelinuj, Salnio brook, 108 brown, 108 rainbow, 109, see Salmo irrideus, S . gairdneri Tuna, 105 yellowfin, sce Thunuzrs nlbacares Turtle, 49

U Umbra, 180, 230 U . limi, 228 U . pygmaea, 228 Unzbridus, 4 Uranoscopidae, 351, 361 Uranoscopus, 9 Urophycis, 87, 89

T Teleosti, 2-7, 9, 11, 13, 25, 34, 87-89, 97, 124-130, 132, 142, 143, 146, 152, 170, 227, 232, 243, 266, 267, 301, 451,457, 465, 468, 553 Tench, see Tinca tinca Tetraodontidae, 82 Tetraodontiformes, 144 Tetrapoda, 152 Therapon, 137, 147, 151, 157 Three-spined stickleback, see Gasterosteus Thunnus albacares, 175, 176 Tigerfish, see Therapon

W W'iiiteria, 13 X Xenoinystus, 382 Xenopus, 247, 258, 261 X. laeuis, 337

Z Zeus, 149 Zoarces uiuiparus, 122

SUBJECT INDEX A Accessory organs, 406, 408, 424, 425, 451, 457, 477 caudal filament, 381, 432 chin appendage, 437, 439 chin organ, 424, 425, 451, 456, 461 dorsal filament, 383, 416 postopercular organ, 406, 409 rostra1 organ, 402, 404, 405, 425, 457, 476, 550 submental organ, 405-409, 413 Acetylcholine, 460 Acetylcholinesterase, 459 Acousticolateralis system, 169, 21 1, 262, 500, 503, 567 Acoustics, see also Sounds, Auditory nerve, Auditory sac communication in fish, 183-189, 227 underwater, 135, 137-142, 155, 189191 Adenosine triphosphatase, 459 Alarm substances, 107-109 Ainacrine cells, 34, 47 Ampullae of Lorenzini, 131, 132, 164, 210, 211, 216, 219, 374, 494, 500, 501, 503, 517-520, 543, 544, 547, 550, 561 Aphakic space, 4, 5 Aqueous humor, 3 ATPase, see Adenosine triphosphatase Auditory nerve, 163, 164, 166, 170, 210, 213, 215, 225, 231, 266, 267, 269, 271, 273, 277, 278, 280, 315-320, 322, 324-329, 334, 338, 341 Auditory sac, 207, 500

B Barbels, 87, 88, 100, 103, 113, 114, 437 of catfish, 100, 102, 103 Basilar membrane, 180, 230, see also Ear Behavior, 101, 105, 123, 124, 137, 184, 186, 191, 480, 496, 555-561 aggressive, 185, 558 594

alarm, 142, 187, 188, 266, 278, 329, 332-337, 339-343 approach, 181 avoidance, 167, 171, 177, 338440, 441, 557, 558 courtship, 106, 143, 558 escape response, 123, 124, 181, 266, 335, 337, 339, 482, 556 feeding, 105, 107, 143, 185, 188 fighting, 106, 107 light avoidance, 124 parental, 79 reproductive, 102 schooling, 106, 107, 137, 143, 185, 188, 189, 191 searching, 104, 105, 122 social, 108 sonic, 157, 186-188, 191 spawning, 185, 187, 188 reproductive, 102 visual, 59-75 Brain, 95, 97, 98, 110, 112, 215, 231, 236, 266, 267, 269, 278-280, 285, 286, 311, 315, 321, 329, 331, 337, 338, 340, 341, 460, 553, 568 forebrain, 91, 105 morphology of, 89-91, 554, 555 Branchial muscles, 456, see also Gills Branchial nerves, 100 Bulbospinal relay system, 474, 475

C Canal organs, 242, 244-246, 248, 249, 251, 252, 258, 261, 499, see also Mechanoreceptor, Lateral line organs, Neuromast organ, Epidermal organs goblet cells, 246, 247 mantle cells, 246 mulberry cells, 248 supporting cells, 245, 249 Canalis transversus, 320, 321 Carr-Price reaction, 18

SUBJECT INDEX

595

Caudal filament, 381, 432 Caudal peduncle, 441, 497 Central nervous system, 92, 97-100, 105,

124, 127, 129, 131, 164, 182, 214, 217, 242, 329, 330, 343, 461, 471, 478, 481, 555, 560, 561, 567 Cerebellum, 110, 267, 269, 270, 554, 562 Cerebral cortex, 305 Chemical senses, 104 orientation by, 109-1 14 reproductive behavior and, 105, 106 Chemoreception, 79-114 biological aspects of, 104-114 chemical perception of foods, 104, 105 discrimination of body odors and schooling, 106, 107 chemical sense organs, anatomy of, 80-91 chemoreceptive functions, behavioral studies of, 91-94 alarm substances, 107-109 orientation by chemical senses, 109-

114 repellents, 109 Chemoreceptor responses, electrophysiological studies of, 94-100 Chemosensory system, 80 chemorensory organ, 109 Chin organ, 424, 425, 451, 456, 461 chin appendage, 437,439 Cholinesterase, 373, 430, 483 Cochlea, 180, 208, 227, 565, 566 Command system, 461, 467, 468, 471,

472, 474-476, 479-482 Corpus cerebelli, 553, 555 Corpus striatum, 305 Crista, 208-211, 217, 221 Crus commune, 208, 211 Cupula, 242, 244, 247-249, 251, 252,

258, 261, 499 Cutaneous structures, see also Skin nerves, 124, 128, 130 sense, 162, 167 sensory system, 163, 234

E Ear, 136, 140, 164,218,227 ampullae, 131, 132, 164 basilar membrane, 180, 230 canalis transversus, 321 cochlea, 180, 208, 227, 565, 566 crista, 208-210, 217, 221 crus commune, 211 ductus endolymphaticus, 207, 208 endolymph, 209, 211, 215, 218, 320 fenestra sacculi, 165 inner ear, 162-166, 170, 173, 174, 181-183, 208, 234, 252, 253, 258, 261 lagena, 163, 165, 166, 208, 209, 214, 223, 226, 2 3 4 2 3 6 macula, 165, 180, 210, 219, 221-226, 235 middle ear, 162, 182, 544 otolith, 164, 180, 208, 214, 223, 226, 234-236 sacculus, 163-165, 175, 208-210, 218. 219, 221, 223, 225, 226, 235, 274, 320, 321 sagitta, 164, 209 semicircular canals, 130, 164, 208, 210, 214-217, 219, 221 utriculus, 165, 208, 209, 218, 219, 221-223, 226, 321 Weberian ossicles, 145, 162-166, 174, 180, 182, 227, 231, 232, 234, 320, 321 Electric organs, 347484, 495, 496, 556, 560-563, 567 adaptations and convergent evolution of, 450-455 embryonic origin and development of,

455-457 evolution of, 561-564 of freshwater fish, 380-448 of marine fish, 362-380 membrane properties of, 4 5 7 4 6 2 neural control of, 4 6 0 4 8 3 Electrocytes, 352, 353, 355-460, 473,

475-481, 483

D Denticles, 142, 143, 145, 156 Dorsal filament, 383, 416 Ductus endolymphaticus, 207, 208

synchronization of activity of, 4 7 5 4 7 8 as experimental material, 457-460 Electrolocation, 495, 496, 543, 552, 556 Electromotor system, 477, 483

596

SUBJECT INDEX

Electroreceptor, 123, 257, 456, 484, 547, 552, 553, 558, 561-563, 566, 567 ampullae of Lorenzini, 131, 132, 164, 210, 211, 216, 219, 374, 494, 500, 501, 503, 517-520, 543, 544, 547, 550, 561 ampullary, 494, 496, 498, 500 in gymnotids and mormyrids tonic, 503515 characteristics in catfish of tonic, 515516 distribution of, 496-503 phasic, 494, 496, 497, 502, 520-541, 543, 548, 551, 559-561, 565, 567 pit-organs, 103, 549 tonic, 494, 496, 497, 500, 502-520, 524, 545, 548, 550, 556, 559, 561, 567 tuberous, 494, 496, 520, 540 Electrosensory system, 354, 385, 408, 424, 450, 452, 478, 483, 495-497, 552-558, 561-564, 567, 568 evolution of, 561-564 Eminentia granulosa, 555 Epidermal organs, 243, 244, 247, 248, see also Canal organs, Lateral line organs, Mechanoreceptors, Neuromast organs Eye, 1, 3, 4, 6-9, 11-14, 35, 36, 39, 69, 70, 73, 74, 82, 83, 104, 214, 215, 217, 219, 226, 253, 273, 278, 329, 341, 382, 437, 544, 553, 556, 563 choroid, 3, 4, 6, 12, 14 choroid gland, 3 ciliary body, 6 cornea, 2, 3, 5, 6, 8, 13, 14, 35-38 dilator, 9 irideal sphincter, 9 iris, 3-6, 8, 13, 14 lens, 3-8, 13, 14 oculomotor, 2 protractor, 3 structure of, 1-5 tubular, 8, 13, 14

F Facial lobes, 91 Facial nerve, 80, 87, 100-102, 164, 371, 500 Falciform process, 3

Fenestra sacculi, 165 Fins, 87, 89, 103, 124, 141, 153, 156, 186, 214, 215, 219, 278, 306, 343, 381, 382, 416, 437, 441, 497, 499, 515, 516 anal, 124, 381, 382, 385, 437, 556 caudal, 267 dorsal, 123, 382, 425, 432, 437, 556 fin spines, 137, 142, 144, 437 pectoral, 87, 145, 342, 343, 382, 437, 482 pelvic, 132 ventral, 437 First cranial nerve, see Olfactory nerve Foliaceous cell, 86

G Gas gland, 3, see also Swim bladder Genital papilla, 381 Gills, 82, 87, 276, 331, 335, 406, 409, see also Branchial muscles arches, 80, 87 rakers, 87, 100 slits, 161, 278 ventilation, 125 Glossopharyngeal nerve, 80, 87, 100, 164, 371 Gustatory organ, 87-89 receptors, 100-104 sense, 91-94, 103, 113 system, 105, 553 H Hair cell, 182, 211, 213, 214, 222-225, 241, 242, 247-253, 255-258, 261, 320, 566 kinocilia, 168, 169, 212, 213, 221, 222, 224, 245, 248, 253, 254 sensory excitation in, 248-255 sterocilia, 212, 225, 245, 248, 253-255 Hearing, 142, 163, 227-236, see also Acoustics bongo drum theory of, 180 capacities, 170-181 evolution of, 182 place theory, 180, 190 pitch discrimination, 229-234 volley theory, 180 Heart, 461, 482, 559 Hemoglobin, 15

597

SUBJECT INDEX

Herring’s theory, 52 Hodgkin-Huxley equation, 479-536 Hunter’s organ, 385, 457, 474, see also Canal organs, Epidermal organs, Lateral line organs, Mechanoreceptors Hypoglossal nerve, 152 Hypophysis, 98 Hypothalamus, 90, 105

I Inner ear, 162-166, 170, 173, 174, 181

183, 208, 234, 252, 253, 258, 261 Interocular transfer, 69-71 Intragemmal plexus, 88 Iris, 3-6, 8, 13, 14

J Jamming avoidance response, 417, 432,

478,557, 559, 560 Jaws, 82, 223, 273, 278, 341, 382, 437

K Kinocilia, see Hair cell Klinokinesis, 112

L Labyrinth, 163, 164, 207-236, 321 structure of the, 207-214 Lagena, 163-166, 208, 210, 219-221,

223, 225, 226, 320, 321 Lateral line, 124, 132, 142, 162, 167,

176, 177, 183, 184, 207, 227, 235, 501, 558, see also Canal organs, Epidermal organs, Mechanoreceptors, Neuromast organs hearing and, 167-170 mechanoreceptors, 241-262, 562 nerves, 123, 124, 340, 497, 498, 500,

530, 554, 558 organs, 101, 123, 124, 131, 132, 170,

181, 182, 234, 241-248, 258, 259, 261, 263, 567 system, 163, 164, 169, 173, 177, 182, 257, 261, 340, 563 Lens, 3, 5-8, 13, 14 pad, 14 Lips, 383

Lissmann’s hypothesis, 562 Lithocyst, 218

M Macrosomatic, 80, 82 Microsomatic, 80, 82 Macula, 165, 180, 210, 219, 221-226,

235 Mauthner cell, 163, 166, 181, 266-344,

481, 482, 563 A, unit, 286-288, 290, 292-294, 296, 297, 301, 302, 304, 306, 308-312, 314, 335, 342 function of, 310-312 A? unit, 286, 287, 288, 290, 293, 294, 309, 313 system, 266, 267 activation of Mauthner neuron, 271277 anatomy of Mauthner neuron, 267-271 excitation of, 315-331 function of, 331-344 glia cells, 319, 325 group A, 286, 287, 289, 292, 302, 337 group B, 286, 287, 289, 292, 302, 337 Mauthner reflex, 278-284, 286, 287, 293, 295, 306, 308, 315, 330, 331, 338343 minimum discrimination time, 282284, 288, 294-297, 310-315, 338 Mechanoreceptor, 123, 125, 128, 182, 241-262, 558, 562, see also Canal organs, Epidermal organs, Neuromasts, Pacinian corpuscle Medulla oblongata, 91, 167, 231, 261,

266-269, 270, 273, 277, 281, 371, 411, 461, 465, 469, 471474, 476 Melanophore, 12, see also Pigment Memory, 92, 112 Mesencephalon, 91, 461 Midline nucleus, 472, 474, 481 Mitral cells, 90, 97 Mouth, 80, 87, 267, 382, 383, 406, 409, 437 Mucosal potentials, 94, 95 Muller cell, 34, 47

N Nares, 382, 383, 437 Nernst equation, 389

598

SUBJECT INDEX

Neuromast organ, 168, 182, 207, 247250, 259, 502, see also Canal organs, Lateral line organs, Mechanoreceptors Nictitating membrane, 2 Ninth cranial nerve, see Glossopharyngeal nerve Nose, 81, 83, 92, 96, 99, 104-106, 110, 111 Nucleus princeps trigemini, 274

0 Occipital nerve, 152 Oculoniotor muscles, 2 Oculomotor nerve, 9, 462, 553, 554 Ociilomotor neurons, 471, 472 Odors attractant, 112 predator, 108 repellent, 109 Olfaction, olfactory bulb, 80, 84, 89, 90, 96, 97, 99, 108111, 554, 567 center, 89 epithelial system, 84, 86 epithelium, 82-84, 86, 87, 93-95, 98, 99, 106, 107 knob, 83, 86 lobe, 90, 93, 106 nerve, 80, 84, 89, 90, 94-96, 108 activity, 95, 96 organ, 81-87, 104, 253 pits, 81, 82, 84 receptor, 82, 84-86 rosette, 81, 82 sac, 94, 96, 97, 105, 106 sense, 91-93, 104, 106-108 system, 81, 94-100, 105 tract, 80, 90, 91, 94, 97-99, 104 electrical activity and central regulatory system, 97-100 Operculum, 145, 273, 278, 280, 341, 406 Optic lobes, 110 Optic nerve, 13, 14, 33-35, 38 Optic sense, 104 Optic tectum, 39, 61, 68, 70, 72, 74, 553, 554 Orthokinesis, 112 Otolith organs, 164, 180, 208, 214, 223, 226, 234-236

function of, 218-226 structure of, 209-211 Otocyst, 218

P Pacemaker neurons, 461, 4 6 9 4 7 6 , 478, 479, 482 Pacinian corpuscle, 253, 255 Palatal organ, 87, 100 of carp, 100, 101 Palatine nerve, 87 Pars inferior, 164, 219, 234 Pars superior, 164, 219, 234 Pectoral girdle, 145 Perilyniphatic fluid, 167, 174, 320 Pharynx, 80, 87, 142, 143 Photomechanical movements, 9-12 Photopigments, see Visual pigments Photoreceptor, 31, 40, 44, 46, 47, 49-51, see also Retina, Vision early and late receptor potential, 43, 44 response of, 4 3 4 7 Pigments, 9, 12, 14, 26, 43, 44, 52, 53, 87, see also Retina, Vision black, 13 epithelium, 4, 9-12, 39 falciforni process, 3 melanophore, 12 opsin, 14, 15, 18, 23, 24 photophores, 13 porphyropsin, see Visual pigments retinal melanin, 9, 11 rhodopsin, see Visual pigments Pit organs, 103, 549 Placode, 207 Pneumatic duct, 146 Postopercuhr organ, 406, 409 Protractor muscle, 3 Pseudobranch, 3 Pupil, 3, 4, 8, 9, 11, 12

R Receptor potential, 248-250 origin of, 253-255 properties of, 251-253 Receptors auditory (acoustic), 565 bimodal, 128 cutaneous, 127

599

SUBJECT INDEX

electroreceptors, 123, 257, 347, 456, 484, 517, 552, 558, 561-563, 566, 567 exteroceptor, 565 mechanoreceptors, 128, 495, 562, 563, 565, 567 multimodal, 129 stretch, 132 thermoreceptors, 121-132 touch-temperature, 127 visual or optic, see Vision Repellents, 109 Rete mirabile, 3 Retina, 3, 5, 6, 8-14, 19, 26, 33-39, 41, 44, 48-50, 60, 70, 74, 75, 556, 557 accessory retina, 13, 14 electrophysiology of, 33-53 C response, 48-50 electroretinogram, 3 5 4 0 component analysis of, 37-39 L response, 48-50 localization of components of, 3 9 4 0 luminosity type, 48, 49 as mass response, 35-37 S cell, 49, 50 S response, 48-50 fovea, 4-6 ganglion cells, 3335, 39, 47, 50, 52, 53, 63, 75 receptive field, 4 1 4 3 response of single, 4 0 4 3 horizontal cells, 34, 47-50 inner nuclear layer, 34, 39, 40, 50, 51 responses in, 47-50 S cells, 49, 50 S potential, 40, 47-52 Retinomotor movements, 9, see also Photomechanical movements Retractor lentis muscle, 6 Retractor muscle, 3 Retroorbital muscle, 279 Rheotaxis, 112, 113, 142 Rostra1 accessory organ, see Accessory organs

5 Sacculus, 163-165, 175, 208-210, 218, 219, 221, 223, 225, 226, 235, 274, 320, 321 sagitta, 164, 209

Sach's organ, 385, 457, 474 Schooling, 106, 107, 137, 160, 161, 185, 188, 189, 191 Sclera, 2, 14 Segmental cutaneous system, 123-125 Semicircular canals, 130, 164, 208, 210, 219, 221 function of, 2 1 4 2 3 6 structure, 209 Sherrington concept, 471 Sinus impar, 320, 321 Skin, 243, 382, 501-505, 513, 515, 524, 529, 532, 540, 541, 544548, 552, 559, 563, 564, 567, see also Cutaneous structures Skull, 145, 147, 148, 166, 167, 177, 215, 222, 2-33, 234, 277 Snout, 82, 382, 383, 397, 409, 563 Sonic drumming muscle, 145 Sonic mechanisms, 142-154, 161, 482, 563 Sonic muscles, 146-151 Sonic organ, 164-170 Sonic species, 137-139, 158 Sounds, 154-162 detection mechanisms of, 136, 162182, see also Hearing hydrodynamic and swimming, 142, 153, 154, 186 production, 135-162 stridulatory, 142, 156, 157, 186 swim bladder, 157-160, 184 Spinal system, 315, 330, 331, 381, 412, 417, 424, 441, 465, 472474, 476, 479 anatomy of, 297-310 circuitry, 266, 281, 293-295, 297, 471, 476 cord, 124, 266, 271-273, 277-279, 281, 282, 284-286, 288-290, 292, 293, 295, 297, 298, 300, 303-305, 307, 311, 317, 327, 331, 33&338, 340343, 411, 412, 416, 417, 419, 425, 461, 468, 470, 471, 473, 475, 479 nerves, 80, 87, 89, 103, 152, 381, 385, 406 responses, 28C294 Spindle cells, 88 Statocyst, 218

SUBJECT INDEX

600 Steller ganglion cell, 34 Stretch receptors, 132 Stridulatory mechanisms, 142-145 Stridulatory sounds, 142, 156, 157, 186 Stridulatory teeth, 137, 142, 186 Submental filament, see Accessory organs Swim bladder, 3, 136, 142, 143, 162, 163,

177, 181-183, 186, 227, 231, 232, 234, 261, 312, 320, 329, 338, 342 elastic spring, 148, 150, 152 hearing and, 166, 167 red glands, 150 sound mechanisms, 145-153 sounds, 157-160, 184 Synaptic noise, 310, 311, 330

T Tail, 272, 273, 277, 278, 280, 281, 283,

286, 335, 376, 412, 455,

306, 321, 322, 329-331, 334, 337, 338, 340, 341, 354, 374, 382, 396, 397, 404, 409, 411, 415, 423, 425, 427, 432, 442, 456, 477, 482, 549, 556, 557 Tapetum lucidum, 3, 4, 9, 11-13 choroidal, 11, 12 retinal, 11 Taste buds, 87-89, 100, 102, 103 Taste receptors, 89, 100, 427 Teeth, 382, 383, 437, 439 Telencephalon, 97 Temperature receptors, 121-132 electrophysiology of, 125-132 in Elasmobranchs, 130-131 in Teleosts, 125-130 Tenth cranial nerve, see Vagus nerve Thermal sensitivity of fishes, 121-123 acclimation temperature, 124 Thyroxine, 22 Trigeniinal nerve, 86, 87 Trunk, 273, 277, 278, 281, 282, 285, 286, 322, 329, 331,332

U Utriculus, 165, 208, 209, 218-220, 222,

223, 226, 321 lapillus, 209

V Vagal lobes, 80, 87, 91, 105, 553, 554 Vagus nerve, 80, 89, 164, 371, 500 Vertebral column, 167, 276 Vestibular system, 275, 277, 320-322,

324, 329, 338, 339, 342 Vision, 8, 183, 503, 556, 563 accommodation, 5-8, 13 achromatic, 48 adaptation, light and dark, 8-12, 35-

39 binocular, 4, 5, 7, 13, 73 C cell, 50 C response, see chromaticity type chromaticity type, 48-50, 52 color, 25-26, 41, 43 retinal mechanisms of, 51-53 dichromatic, 48 emmetropic, 6-8 monocular, 70 myopic, 6, 7 opponent color theory, 61 retinoscopic, 7 Visual behavior configurational properties of shapes,

64, 67 experimental analysis of, 59-75 perceptual equivalence and change in spatial position, 68-72 relative discrimination weaknesses, 61-

64 selective attention, 72, 74 toward unified outlook on, 74-75 Visual pigments, 14-26, see also Pigments color, 25-26 methods of study of, 15-18 multiplicity of opsins, 23-25 photochemistry of, 14, 15 photopigments, 13 porphyropsin, 15, 18-23 retinene, 14-18 rhodopsin, 9, 15, 18-24 Vitreous humor, 3

W Weberian ossicles, 145, 162-166,

174, 180, 182, 227, 231, 232, 234, 320, 321