INTERNATIONAL REVIEW NEUROBIOLOGY V 6, Volume 6 (v. 6)

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sF INTERNATIONAL REVIEW O

Neurobiology VOLUME 6

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INTERNATIONAL REVIEW OF

Neurobiology Edited by CARL C. PFEIFFER New Jersey Psychiafric lnsfitute Princeton, New Jersey

JOHN R. SMYTHIES Department of Psychological Medicine University of Edinburgh, Edinburgh, Scotland

Associate Editors V. Amassian

E. V.

J. A. Bain

H. J. Eysenck

D. Bovet Lord Brain Sir John Eccles

G. W. Harris R. G. Heath

VOLUME

F.

Evarts

Georgi

C. Hebb K. Killam S. Mirtens

6 1964

ACADEMIC PRESS

0

New York and London

COPYRICHTO 1964,

BY

ACADEMICPRESS INC.

ALL RIGHTS RESERVED NO PART OF THIS BOOK MAY BE REPRODUCED lN ANY FORM, BY PHOTOSTAT, MICROFILM, OR ANY OTHER MEANS, WITHOUT WRI'ITEN PERMISSION FROM T H E PUBLISHERS.

ACADEMIC PRESS INC. 111 Fifth Avenue, New York, New York 10003

United Kingdono Editwn published by ACADEMIC PRESS INC. (LONDON) LTD. Berkeley Square House, London W . l

IABHAHI- OF CONGRESS CATALOG CARDNUMBER: 59-13822

PRINTED IN THE UNITED STATES OF AMERICA

CONTRIBUTORS P. 0. BISHOP,Department of Physiology, University of Sydney, Sydney, Australia

QUENTINBONE, The Marine Biological Association, Citadel Hill, Plymouth, England CARMINE D. CLEMENTE, Department of A n a t m y , School of Medicine and the Brain Research Institute, University of California, Los Angeles, California and the Veterans Administratiort Hospital, Sepulveda, California

Josh M. R. DELGADO, Yale University School of Medicine, New Hauen, Connecticut

EDWARD F. DOMINO, Department of P h u m o l o g y , University of Michigan, Ann Arbor, Michigan Department of Bwchernisty, the College of Physicians and Surgeons, Columbia University, and New York State Research Institute for Neurochmisty and Drug Addidion,

ABEL LA-,

Ward's Island, N e w York, New York THOMAS H . MEIKLE,JR., Departwnt of Anatmtj, Cornell University Medical College, New York, New York

M . SPRAGUE, lnstitute of Neurological Sciences, University of Pennsylmnia, Philadelphia, Pennsylvania

JAMES

V

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PREFACE Progress in neurobiological research must maintain a delicate balance between the fascination of basic explanation of clinical and physiological phenomena by means of chemical and physical concepts on the one hand and the pressing needs for the development of new and effective treatments of disease on the other. Advances in basic biochemistry and biophysics often give rise to developments in the clinical field, but mature judgment is required to select from the vast detail of biochemistry and biophysics, those parts which are likely to apply to human disease. The aim of this review is to enable active workers in such fields as neurobiology, psychopharmacology, and psychology, as well as those in biological psychiatry and neurology to give a full account of recent progress in their fields. The review covers the whole field of neurobiology and includes work within a particular science as well as in neurology and psychiatry. Particular emphasis has been laid on the recent development of ideas that are of fundamental importance and general interest and also those that are likely to further our understanding of nervous and mental disease. In the past the basic neurobiological sciences have played no little part in progress toward these ends. They are most active at present and hold great promise for the future. These reviews and summaries ordinarily are by invitation, with an annual deadline for receipt of manuscripts by October 1. The editors, however, will be happy to review unsolicited manuscripts if submitted in outline form.

Decembw 1963

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CONTENTS CONTRIBUTORS .

PREFACE

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V

vii

Protein Metabolism of the Nervous System ABEL LAJTHA I. I1. I11. IV. V. VI . VII. VIII .

Introduction . . . The Pool of Free Amino Acids Cerebral Proteins . . Protein Turnover . . The Formation of Proteins Protein Catabolism . . Protein Metabolism in Nerve Conclusions . . . References . . . .

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2 3 19 28 45 60 72 82 83

Patterns of Muscular Innervation in the lower Chordates QUENTIN

BONE

I . Introduction . . . . . . . . . . . I1. Motor and Sensory Innervation of Myotomal Muscles of Lower . . . . . . . . . . Chordate Groups I11. Discussion . . . . . . . . . . . . References . . . . . . . . . . . .

99 108 133 141

The Neural Organization of the Visual Pathways in the Cat THOMAS H . MEIKLE.JR., AND JAMES M. SPRAGUE I. I1. I11. IV. V. VI . VII .

Introduction . . . Historical Preview . . Primary Optic Terminations Mesencephalic Projections Thalamic Projections . . Neocortical Projections . Recapitulation . . . References . . . .

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150 150 152 165 173 177 182 184

X

CONTENTS

Properties of Afferent Synapses and Sensory Neurons in the lateral Geniculate Nucleus P. 0. BISHOP I . Introduction . . . . . . . . . . . 192 I1. The Lateral Geniculate Nucleus . . . . . . . 194 . . . . . 196 I11. Waveforms of Geniculate Responses . IV . Spontaneous Activity . . . . . . . . . 209 v. Repetitive Firing . . . . . . . . . . 212 . . . . . 223 VI . Fractionation of Unit-Waveforms . . . . . . . 236 VII. Refractory Period of Geniculate Neurons VIII . Recovery of Responsiveness . . . . . . . . 241 . . . . . 242 IX . Phamiacology of Geniculate Iieurons . . . . . . . . . . 248 X. Concluding Remarks . References

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251

Regeneration in the Vertebrate Central Nervous System CARMINED. CLEMENTE Introduction . . . . . . . . . Developmental Considerations . . . . . . Theories of Nerve Growth and Orientation . . . Regeneration in the Central Nervous System of Primitive . . . . . . . . . Vertebrates . L-. Regeneration in the Central Nervous System of Fishes VI . Regeneration in the Amphibian Central Nervous System VII . Regeneration in the Reptilian Central Nervous System . 17111 . Regeneration in the Central Nervous System of Birds . IX . Regeneration in the Mammalian Central Nervous System . . . . . . . . . References .

I. I1. 111. IV .

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258 258 261 264 264 268 276 277 281 293

Neurobiology of Phencyclidine (Sernyl). a Drug with an Unusual Spectrum of Pharmacological Activity EDWARDF. DOMINO I. I1. I11. IV . V. VI . VII .

Introduction . . . . . . . . . . . Chemical Structure of Phencyclidine and a Related Derivative . Neuro-Psychopharmacological Actions . . . . . . Cardiovascular and Respiratory Actions . . . . . . Metabolic Actions . . . . . . . . . . Discussion . . . . . . . . . . . . Summary . . . . . . . . . . . . References . . . . . . . . . . . .

303 304 305 333 338 342 346 346

xi

CONTENTS

Free Behavior and Brain Stimulation Josh M . R. DELGADO

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I. Introduction

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I1. Conditioned Reflexes. Instrumental Responses. and

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I11 IV. V. VI . VII.

. . . . . Free Behavior . Study of Free Behavior . . . . Study of Evoked Behavior . . . Types of Behavior Evoked in Free Situations Fragmental Organization of Behavior . Summary . . . . . . . References . . . . . . . .

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SUBJECT INDEX .

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AUTHORINDEX

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351 353 374 385 430 436 438

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451 473

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PROTEIN METABOLISM OF THE NERVOUS SYSTEM' By Abel Lajtha

Department of Biochemistry. The College of Physicians and Surgeons. Columbia University. and New York State Reseorch Institute for Neurochemistry and Drug Addiction. Ward's Island. New York. New York

I. Introduction .

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11 The Pool of Free Amino Acids .

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. A . The Penetration of Amino Acids . B . Amino Acid Flux and Transport .

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C . Amino Acid Metabolism . . . . D. Compartmentation . . . . . . E. Changes in Cerebral Amino Acid Levels F. Conclusions . . . . . . . . I11. Cerebral Proteins . . . . . . . A . Purified Protein Fractions . . . . B . Protein Composition of Cerebral Areas C . Amino Acid Content of Proteins . . IV. Protein Turnover . . . . . . . A . The Dynamic State . . . . . . B . Half-Life of Cerebral Proteins . . . C . Protein MetaboIism during Development D. Regional Metabolism . . . . . E . Heterogeneity of Turnover Rates . . F. Localization of Protein Metabolism . V. The Formation of Proteins . . . . . A. General Problems . . . . . . B . The Precursors . . . . . . . C . Amino Acid Incorporating Systems . D. The Mechanism of Protein Synthesis .

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' The work in which the author participated was supported by grants from the following: National Institutes of Health. United States Public Health Service (grants B226. B557. and 3226); Scottish Rite Masons of the Northern Jurisdiction. United States; and the United States Office of Naval Research. 1

2

ABEL L A P H A

VI. Protein Catabolism . . . . . . . A. Cerebral Proteinases . . . . . B. The Mechanism of Breakdown . . . C. Relation of Breakdown and Formation VII. Protein Metabolism in Nerve . . . . A. Proteins of Peripheral Nerve . . . B. Incorporation in Nerve . . . . . C. Axoplasmic Flow . . . . . . D. Wallerian Degeneration . . . . E. Nerve Growth Factor . . . . . VIII. Conclusions . . . . . . . . . References . . . . . . . . .

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60 61 66 69 72 72 73 74 78 80 82 83

I. Introduction

Protein metabolism has in recent years become of prime interest to a great many investigators, not only in biochemistry, physiology, and neurochemistry, but also in such fields as microbiology, pathology, and genetics. In few fields of biological research have important advances been made so rapidly during the past few years as in the chemistry, structure, and metabolism of proteins. Many areas, such as the mechanism of protein synthesis and the mechanism of genetic determination of the specificity of this synthesis, areas formerly thought inaccessible to investigation, have been recently elucidated. Furthermore, the great rate of advance of knowledge in this field makes it likely that other questions that can be answered only partially today will be clarified in the near future. The various aspects of protein metabolism touch upon too many areas and problems to deal with adequately in a limited space. Therefore, only those aspects have been selected that are most pertinent to a discussion of cerebral function and malfunction. Areas recently discussed in other reviews-such as the work on cerebral protein metabolism in which the author participated (reviewed by Waelsch and Lajtha, 1961)-will be mentioned more briefly. Several areas of the general problems of protein metabolism in other organs and organisms were the subject of numerous recent reviews [among others, for example: Loft6eld, 1957; Askonas et al., 1957; Chantrenne, 1958; Campbell, 1958; Hoagland, 1960; Zamecnik, 1960; Berg, 1961; Sirlin, 1962; Simpson, 1962); some of the recent reviews discussing areas of importance in cerebral protein metabolism are those of

PROTEIN METABOLISM OF THE NERVOUS SYSTEM

3

Waelsch ( 1957, 1958), Richter ( 1959, 1961), Hyden ( 1959), Waelsch (1960) and Waelsch and Lajtha (196l)l. Even though it seems unlikely that the basic mechanisms of protein synthesis and breakdown in the nervous system are signscantly different from those in the rest of the organism, very interesting features justify a separate discussion. In the brain, more than in any other organ, the functional metabolism seems to involve changes in the proteins of the organ. The controlling factors of cerebral protein metabolism are of interest, particularly because there seems to be very little regeneration in the central nervous system. Some diseases of the nervous system are apparently caused by the lack of some enzyme proteins normally present; in other diseased states, the presence of abnormal proteins, and structural changes in normal proteins, have been found. Also of interest are the reported effects of a number of drugs on cerebral protein metabolism. II. The Pool of Free Amino Acids

Any measurement of cerebral protein metabolism will have to take the free amino acid pool or pools in the organ into account. Measurements of incorporation into or release from proteins of labeled amino acids are influenced by the rate of passage of the free amino acid into and out of the brain, brain cells, and brain cell compartments. There is considerable evidence that at least in the quantitative sense the passage of amino acids to the brain is different from that to other organs in that changes in blood levels do not result in changes of comparable size in the brain. The passage of the amino acids into and out of the brain seems to be governed not by diffusion but by a complex system constituting one aspect of the brain barrier system (Lajtha, 1962a). It is also likely that the brain barrier system by influencing the level and distribution of the amino acids available for protein synthesis exerts an important influence on in v i m protein metabolism. Therefore, in the following the cerebral passage of amino acids will be briefly discussed.

A. THEPENETRATION OF AMINOACIDS After the elevation of the plasma level of most substances their uptake by the brain as compared to other organs is limited. Among

4

ABEL LAJTHA

the amino acids the cerebral uptake of glutamic acid was strongly restricted; that of glutamine was not as strongly restricted ( Schwerin et al., 1950). Lysine uptake (Lajtha, 1958a), proline uptake (Dingman and Sporn, 1959), and leucine and phenylalanine uptake ( Lajtha and Toth, 1961) is also restricted, that of tyrosine (Chirigos et al., 1950) somewhat less. This restriction is not usually absolute, since cerebral glutamine, methionine, histidine, lysine, and arginine (but not glutamic or aspartic acid) increased during continuous infusion (Kamin and Handler, 1951), and in most cases, if blood levels were sufficiently elevated, there was some increase in the brain too. The role of the brain barrier system in influencing measurements of protein metabolism was recognized when it was discovered ( Friedberg et aZ.,1948) that more radiomethionine was incorporated into brain proteins after intracisternal administration than when it was injected intravenously. As compared with the intravenous administration, the intracisternal dose resulted in higher radioactivity in the free amino acid pool and consequently in the proteins. The influence of the levels of amino acids on protein metabolism in vivo is not known. It is important in this respect that in order to initiate protein synthesis at a rate consistent with cellular survival and growth in cultiired hiunan cells, the intracellular amino acid concentrations have to exceed a critical threshold level. Increasing the minimum effective amino acid concentrations 2 to 4 times increased protein synthesis from 20% of maximal rate to 80% of maximum, i.e., about fourfold (Eagle et al., 1961). It is possible that the concentration of five amino acids in the pool that is available for protein metabolism is one of the controlling factors of the rate of protein synthesis and breakdown. The permeability to amino acids decreases during development. h greater uptake in the young as opposed to the adult has been shown for glutamic acid (Himwich d al., 1957), lysine (Lajtha, 1958a), and leucine (Lajtlia and Toth, 1961). A t present, there are not enough data to call the greater permeability of the young brain a general rule, since greater permeability during development was found for phosphate (Fries and Chaikoff, 1941; Bakay, 1953), thiocyanate, and chloride (Waelsch, 1955; Lajtha, 1957), but not for Gypan blue (Crontoft, 1954; Grazer and Clemente, 1957). There was some restriction of amino acid uptake even in young brains in

5

PROTEIN METABOLISM OF THE NERVOUS SYSTEM

the experiments quoted above (with glutamic acid, lysine, and leucine); y-aminobutyric acid was taken up in immature brains only in cases of some damage (Purpura and Carmichael, 1960). Whether the changes of permeability influence protein metabolism during development or are only the consequences of the greater need for substrates in the growing animal has not yet been established.

B. AMINOA m FLUXAND TRANSPORT Although net uptake of the amino acids is restricted if the plasma levels are elevated, a rapid exchange takes place between plasma and brain amino acids ( Waelsch and Lajtha, 1961).From the rate of appearance of radioactivity in the brain fonowing intravenous administration of tracer doses of an amino acid (which does not disturb physiological plasma levels), the flux of the amino acid can be estimated and, from it, the half-life of the compound in the brain. Such estimates are only approximate, as the changes of the specific activities in plasma are very rapid. (A few minutes after intravenous administration more than 90% of the injected radioactivity has disappeared from the circulating plasma.) The flux rate and half-lives of cerebral free amino acids have been estimated (Table I). The TABLE I HALF-LIFETIMEOF CEREBRAL FREEAMINOACIDS~ ~~

Half-life time in minutes Lysineb Brain, young Brain, adult Liver Muscle

27 20 6

-

LeLcinec

Glutamic acidd Methioninee

-

-

34 15 14

600

5

-

-

-

-

-

a Because of very rapid changes in plasma following the administration of labeled amino acids the above values of the rates of exchange between the free amino acids of plasma and those of other organs are only approximate. The flux of glutamic acid is comparable to that of the other amino acids but because of low plasma and high cerebral glutamic acid levels the half-life time is greater. The table clearly shows the rapid exchange of cerebral amino acids, which is considerably greater than their incorporation into cerebral proteins. b From Lajtha et al., 1957. In mice. c From Lajtha, 1959.In mice. d From Lajtha et al., 1959.In rats. From Appel et al., 1960. In rats. 6

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ABEL LAJTHA

flux of glutamic acid was comparable to that of the other amino acids, but because of the high levels of this compound in the brain such flux yields larger half-lives. The rate of exchange of tyrosine (Chirigos ct al., 1960) was also high. The term "exchange" means that a flow of equal size occurs simultaneously in both directions, that is, that influx equals efflux. The rate of exchange is not necessarily equal and usually is greater than the rate of unidirectional net flow. This latter, which most likely depends on experimental circumstances, such as the concentration gradient between plasma and brain, was measured as efflux of amino acids after their level was increased in the brain by subaraclmoidal administration (Table 11). TABLE I1 RELATIVEEFFLUXRATESOF CEREBRAL AMINOACIDS Relative efflux rate"

Form rd-hnino acid u--\niiiio acid

-

Leucine* Pheriylnlanineb Lysineb hfethioninec I4 28

27 3 '7

1-10 200

34

-

-,-Aminobut,yric acidd 21

' l'imr i n niinutes required for thr ccrehral levels of administered substrates t o tlecrcnsci dOCL.In referenccs c and d, efflux RBS not measured but here it is c~rtim:ittdfrom siinilnr data. From I ~ j t h mid a Toth, 196'7. In rats. Fi-oni f hitonde and Richter, 1956. In rats. l'rom R i d w t s ef a!., 1938. In mice.

The rapid exchange between plasma and brain makes the measuremcnt of small continuous uptake difficult, but it seems likely that the needs of the brain can be replenished by the circulating plasma. Obviousl~,since adult brain is not able to synthesize at all, or at a sufficient rate, a number of the amino acids, the net uptake from plasma has to replace any portion that is metabolized. The first evidence for active amino acid uptake by brain was found in citro. Brain slices can accumulate from the incubating medium, against a concentration gradient, most of the amino acids that have been examined, such as L-glutamic acid (Stern et al., 1949), D-glutamic acid ( Takagaki et al., 1959), aspartic acid ( Korey and Mitchell, 1951), L- and D-tyrosine (Guroff et al., 1961), y-

PROTEIN METABOLISM OF THE NERVOUS SYSTEM

7

aminobutyric acid (Elliott and Van Gelder, 1958; Tsukada et al., 1960), histidine, proline, lysine, ornithine, methionine, and arginine (Neame, 1962a), and 5-hydroxytryptophan (Schanberg and Giarman, 1960). When the utilization of available energy was inhibited, no concentrative uptake could be shown with L-glutamate (Stern et d.,1949), aspartate (Korey and Mitchell, 1951), y-aminobutyrate (Elliott and Van Gelder, 1958), tyrosine (Guroff et al., 1961), histidine (Neame, 1961a), and 5-hydroxytryptophan (Schanberg and Giarman, 1960). The active uptake is fairly specific for brain slices with y-aminobutyrate (Elliott and Van Gelder, 1958) and tyrosine ( Guroff et al., 1961), although glutamate uptake could be shown with other tissues as well (Stern et al., 1949). Histidine, proline, tyrosine, and ornithine (Neame, 1962a, b) were taken up by some of the other tissues tried, against a concentration gradient ( tissue: medium ratio was larger than one). With a few exceptions (such as Sarcoma DR 3, or, with some amino acids, the intestinal rnucosa), the value of this ratio was the greatest for brain. It is of interest that, although in the living animal the brain is the organ that takes up the smallest amounts of amino acids from the blood if blood levels are elevated, with tissues slices, brain is quantitatively the most active organ in this respect. The reasons for this difference are not obvious. Neame (1962) considered three possibilities: ( a ) it is merely incidental to other metabolic processes peculiar to brain; ( b ) it may be a compensating response to the blood barrier which prevents the access of amino acids; or ( c ) it may have a function of removing from the intercellular spaces amino acids, such as glutamic and y-aminobutyric acids, and Ei-hydroxytryptophan, which might interfere with normal brain function. The absence of circulation in slices and the different route of entry (and therefore the lack of influence of vascular permeability) in slices suspended in a medium obviously are significant differences between the in vitro experiments and those in the living animal. It is not very likely, however, that the processes studied in brain slices are completely insignificant in the living brain. Another at least partial explanation for the opposite behavior of slices and living brain in this respect would be that the mechanisms in slices and in the living brain are similar but the controling factors are different or missing, and the direction of the metabolic pump gets reversed from

Y

ABEL LAJTHA

stronger efflux in vico (keeping substances out) to stronger influx in slices (resulting in net uptake). If we consider this last explanation, then the permeability behavior of tissue slices reflects at least some of the properties of the tissue under living conditions, and the experiments discussed above again show clearly the quantitative differences between the brain and other organs and point to active processes that require energy and that are capable of transport against a concentration gradient. At present they do not definitely show qualitative differences, i.e., the existence of mechanisms in the brain that are specifk for this organ alone. Indications that active, or carrier-mediated, processes are operative also in the living brain have come from several experiments: a. By the above discussed rapid exchange in spite of a barrier to net uptake, amino acids pass rapidly in and out of the brain, although a net increase is inhibited ( Waelsch and Lajtha, 1961). This behavior cannot be explained by a non-facilitated passive diftusion through a membrane. b. There is some inhibition of uptake into the brain of one amino acid by another in vioo (Chirigos et al., 1960, Schanberg et al., 1961), and also in cifro (Neame, 1961b). This phenomenon, which has been observed in other tissues previously (Christensen et al., 1952), is best explained by assuming that there is competition for the site on a common carrier, and that increasing the concentration of one amino acid decreases another amino acid's attachment to the carrier and thereby its transport. c. There is transport from the brain against a concentration gradient (Lajtha and Toth, 196l), i.e., an elevated brain amino acid level decreases to normal in uiuo, even if the blood level is kept several times above that of the brain throughout the experiment (Fig. 1). Again, passage against a diffusion gradient (in this instance, out of the brain) cannot be explained by passive diffusion alone. d. The exchange rate of an amino acid is increased if the cerebral levels of this amino acid are increased (Lajtha and Mela, 1961), a phenomenon observed in other systems and explained by the mechanism of exchange diffusion rather than passive diffusion ( Ussing, 1949; Heinz, 1954; Heinz and Walsh, 1958). e. PLctive transport of phenolsulfonphthalein and diodrast from

PROTEIN METABOLISM OF THE NERVOUS SYSTEM

9

the cerebrospinal fluid to blood has also been shown (Pappenheimer et al., 1961).

800

Adult Plasma

7501

Newborn

700

20

Minutes

FIG. 1. Leucine (in cg/gm) transport from the brain. In the beginning of the experiment ( 5 minutes after the administration of leucine), brain levels of adult and newborn animals are about the same. During the experiment (from 5 to 20 minutes) the average plasma levels are also about the same in the two ages, adult plasma levels actually being slightly higher. During the experiment, adult brain leucine levels decrease against the elevated plasma levels (leucine is pumped out of the brain against a concentration gradient). No such transport from newborn brains can be demonstrated; newborn brain leucine levels increase with time but do not reach plasma levels. (From Lajtha and Toth, 1961; Lajtha, 1962a.)

Most likely more pathways of transport than one are involved. Tyrosine uptake (Chirigos et al., 1960; Guroff et al., 1961) and 5hydroxytryptophan uptake (Schanberg et al., 1961) are inhibited by some and not influenced by other amino acids, i.e., there is competition for the same carrier by only a few amino acids, and other amino acids are transported by a different carrier. There are indications that exchange diffusion and active transport involve the same carrier (Heinz and Walsh, 1958; Johnstone and Quastel, 1961); further support for several specific carriers is the finding that the effect of compounds on increasing exchange also shows specificity (Lajtha and Mela, 1961) in that one amino acid increases its own exchange rate while that of another is affected slightly or not at all. Exchange rates are not uniform throughout the brain but seem to depend on the level of the compound in a particular area (Lajtha, 1961a). In general, the distribution and supply of the amino acid cannot be considered uniform throughout the brain (Lajtha, 196213). The picture that emerges is that the central nervous system is by no means completely impermeable to amino acids. There is rapid

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ABEL LAJTHA

exchange between plasma and brain, and a small increase of influx over efflux can supply the amino acids needed. In addition to passive diffusion, mediated or facilitated transport plays an important, perhaps the major, role; these transport mechanisms can be metabolite- and area-specific in that the passage of one amino acid can be altered without affecting many others, and that passage in one area can be altered without affecting other areas. The brain barrier system for amino acids in this sense becomes a homeostatic control mechanism capable of reacting to altered situations. It is possible that the barrier system can transport needed metabolites, among them amino acids, when and where necessary. Such a system not only can replace in a specific way substrates that have been used up but, by generally controlling the level of substrates and their metabolic products, it will have a profound influence on the rates of cerebral metabolism. IVhether or not the brain barrier system has such an influence over cerebral protein metabolism as well, is not known at present. (For recent discussions of the brain barrier system see Lajtha, 1962a, b.)

c. A M I N O ACID METABOLISM The source of cerebral amino acids is not only the plasma; some amino acids can be synthesized by the brain itself. The metabolite used for synthesis that has been most often studied is glucose. The very interesting observation that newborn brain tissue was able to incorporate C1*from glucose into some essential amino acids, although adult brain only incorporated it into the nonessential amino acids: (Rafelson et al., 1951; Winzler ct al., 1952; Sky-Peck et al., 1956), indicates a unique synthetic ability of the young brain. This transitory ability and its functional significance deserve further careful study. The authors point out that the appearance of labeled carbon from glucose in amino acids in uiuo does not necessarily indicate the net synthesis of these compounds, since it is possible that products of amino acid metabolism are in reversible equilibrium with intermcdiates derived from glucose. This means that the label could appear via isotopic exchange rather than net unidirectional movement along a metabolic pathway. The metabolic relationship of glucose to amino acids has been studied also in the adult brain. In most experiments, the greatest amount of label from glucose was found in glutamic acid. The rela-

PROTEIN METABOLISM OF THE NERVOUS SYSTEM

11

tionship in brain of glutamic acid and glucose was shown in experiments in which the glutamic acid level of brain slices was restored when glucose was added to the medium, or no decrease of glutamic acid was shown in the presence of glucose ( Waelsch, 1949). Nine per cent of the label from glucose metabolized by brain slices was found in glutamic acid (Beloff-Chain et al., 1955) and a significant portion of the label in cat brain following perfusion with glucose was found in glutamic and aspartic acids (Barkulis et al., 1960) and consequently in protein (Geiger et al., 1960). In one study it was estimated that glucose supplies 10 times as much carbon to the glutamic acid and glutamine moiety of brain as does plasma glutamic acid (Flexner et al., 1958); in another study, at 30 minutes after administration, less than 2% of the label remained in glucose and about 92%was in amino acids, mainly aspartic and glutamic acids ( Gaitonde et al., 1962). Again it has to be emphasized that the appearance of label from glucose in a compound might occur through equilibration rather than net synthesis. As an example, let us suppose that glutamic acid is in isotopic equilibrium with ketoglutarate, i.e., by transamination unlabeled glutamate becomes unlabeled ketoglutarate and an equal amount of ketoglutarate labeled from glucose becomes labeled glutamate; there is no net change in the concentration of either compound, but glutamate becomes labeled. This reaction, shown below, is extremely rapid in brain, the equilibrium being reached almost instantaneously (Rossi et al., 1962). In this case, there is no net 0

II

HOOC-C-CHZ-CHz-COOH a-Ketoglutarate

NHB NHc 4- H

S +O

I

HOOC-CH-CHz-CH~-COOH Glutamate

formation of glutamate from glucose, and the labeled glucose can be quantitatively oxidized via the Krebs cycle, therefore going through ketoglutarate rather than glutamic acid, but a considerable portion of the glutamate becomes labeled. In vitro experiments can shed more light on the possible metabolic pathways of glucose in the brain but again cannot decide the quantitative contribution in vim of these reactions, Therefore, although it is an intriguing thought that in brain some part of glucose is not utilized directly via the well-known pathways of glucose metabolism, such as the Krebs

.%nmLAJTHA

12

cycle, but is converted in the brain h s t into other compounds such as amino acids that are more suitable for cerebral metabolism, no present finding can be accepted as convincing proof for this hypothesis. Although amino acid metabolism has not been investigated in detail in the brain, there is at present no reason to believe that reactions present in other tissues are absent in the brain. It has become recognized recently that the earlier belief that brain tissue is capable of oxidizing only glutamic acid was due to the fact that this amino acid is metabolized at a higher rate than any other. With experiments designed to measure lower metabolic rates, the metabolism of other amino acids, such as proline (Sporn et al., 1959), was also established. Homogenates of immature brain oxidized all of the 13 amino acids investigated, the per cent of the added amino acid recovered as C 0 2 after 1 hour incubation varying from 8%with glutamic acid to 0.0048 with lysine ( Schepartz, 1961) . Cerebral amino acids give rise to other compounds such as amines, and the liberation of amino acids by breakdown of proteins might result in the production of amines or other active products of amino acid metabolism, thereby connecting protein metabolism with other reactions.

D.

COM PA~.HTMESThTIOS

An important pliasc in our understanding of brain protein metabolism is the knowledge of the qualitative and quantitative contribution of the metabolic pools. The morphological heterogeneity of the brain, brain areas, and cells is well known and requires no discussion here. More and more evidence points to the metabolic heterogeneity which parallels the morphological one. Knowledge of the metabolic pools of protein precursors is important for understanding the detailed mechanism of synthesis in the cell. The question has to be answered whether the breakdown products of newly degraded protein molecules are used preferentially or the newly synthesized protein molecules are derived chiefly from amino acids taken up by the cell for this purpose. Another important question is whether the free amino acids are in a homogeneous pool within the cell or in several distinct pools, only some of which supply the amino acids for protein synthesis. In liver, in spite of the administration of relatively large amounts of amino acid into the circulation, the intra-

PROTEIN METABOLISM OF THE NERVOUS SYSTEM

13

cellular pool still supplied more than half of the amino acids for the newly synthesized protein (Loftfield and Harris, 1956), showing that the pool utilized for protein synthesis may not be in rapid equilibrium with the amino acids in the circulating blood. These results have been confirmed in a number of systems (most recently, see Walter et al., 1962). Several recent studies showed that substrates in the cell may be localized in one or the other particulate fraction, that various pools of a metabolite, physically or chemically separate, can coexist in the cell. Such findings were made with a number of compounds, such as citrate (Schneider et al., 1956) and phosphoglycerate (Moses et al., 1959), but only the compartmentation of amino acids in the cell will be discussed here. Evidence that the free amino acids may not all be in a homogeneous pool came from several laboratories studying pools in bacterial cells. These studies showed that a part of the acidsoluble amino acids in these cells is in a loosely bound form (Britten et aZ., 1955) and may consist of several kinds of pools such as metabolic, synthetic, and reservoir, or expandable and internal pools (Cowie and Walton, 1956; Cowie and McClure, 1959; Halvorson and Cowie, 1961).Some of these pools exchange with the external milieu rapidly and others, where the amino acids in the internal pool are the ones used for protein synthesis, only slowly. Thus, in an experiment to measure protein turnover in which the incorporation of an administered labeled amino acid is estimated, the intracellular amino acid used for protein synthesis may have higher or lower specific activity than the administered acid present in the plasma even after prolonged infusion, and may also differ from the average specific activity of the brain pool of free amino acid. If, for calculations of protein turnover, the incorporation of the amino acid with this average specific activity is assumed, a false high or low value may be obtained. Similar considerations apply to the measurements of the release of labeled amino acids from proteins, even in the presence of large amounts of unlabeled amino acids in the plasma. Evidence was found that gIutamic acid, glutamine, glutathione, and glycine pools are heterogeneous in vivo in mammalian organisms (Lajtha et d.,1959; Waelsch, 1960), and it seems likely that when other amino acids are investigated, many will show similar heterogeneity. Glutamine was shown to exist in heterogeneous pools in carrot roots (Steward et al., 1956).

14

ABEL L A F A

It was found in our laboratory that upon injecting C14-glutamic acid the specific activity of glutamine was the highest in the plasma of all tissues investigated (liver, kidney, lung, muscle, brain, spleen, red cells), even if the liver and a large part of the body were excluded from the circulation. A newly formed glutamine from a pool of higher than average specific activity in the cells thus entered the circulation without reaching prior equilibrium with the other glutamine pools within the cells (Lajtha ct ul., 1959, 1960). Similarly, if labeled glutamic acid was injected intracerebrally (Berl et al., 1961), the specific activity of glutaniine in the brain in less than 1 minute became higher than that of its precursor glutamic acid, showing that the administered glutamic acid entered into an active pool and formed glutamine and glutathione without equilibration with the less active glutamic pool in the rest of the brain. The tentative explanation of the above finding ( Waelsch, 1960) was that the glutamic acid from the circulation is in more rapid equilibrium with the pool of the endoplasmic reticulum; it forms glutamine in situ, and the freshly formed glutamine subsequently enters the circulation without prior equilibrium with the rest of the intracellular glutamine. The endoplasmic reticulum thus will have glutamic acid and glutamine of a specific activity which is greater than that of these amino acids in the rest of the cell. The changes in the specific activity of glutamic acid and glutamine were too rapid for even an approximate estimation of the size of the active glutamic acid pool in brain that was utilized for the rapid glutamine formation, but it is likely that it is not greater than 20%of the total and probably considerably less (Berl et ul., 1961). These results have been further extended with the use of N*5-labeled ammonia ( Waelsch, 1961b; Berl et al., 1962), where the pools in liver and brain could be studied in greater detail and, in the glutamine, the metabolic rates of the amide group could be compared with the amino group. While any localization of the intracellular amino acid pools is speculative at the moment, heterogeneity in amino acid pools will greatly influence not only our measurements of the rates of protein metabolism but also this metabolism itself. Another as yet tentative proof for heterogeneity of amino acid pools is the finding in our laboratory (Garfinkel and Lajtha, 1963) that the glycine moiety of newly formed hippuric acid has a higher specific activity than the average glycine pool. In this case, the cell

PROTEIN METABOLISM OF THE NERVOUS SYSTEM

15

particulates that have access to the “active” glycine pool seem to be the mitochondria. Hippurate seemed to be synthesized preferentially from a glycine pool which was more nearly in equilibrium with plasma glycine than was the average intracellular glycine. It is obvious that compartments exist on many separate levels such as brain areas, brain cells, and cell particulates; the size, turnover and function of these various pools can only be speculated upon. Pools might be defined histologically (one closer to surface, one bound in a particulate fraction, etc.), functionally (one that is lased mainly for storage, one depleted first, one needed for certain metabolic events, etc. ), metabolically (higher or lower turnover rates), physically (one in closer diffusion equilibrium with the outside, etc.), and in many other ways. There is evidence that the level of the amino acids is not uniform throughout the brain (Tallan, 1957; Furst et ul., 1958; Berl and Waelsch, 1958; Okumura et al., 1959; Fukai, 1959; Price and West, 1960; Lajtha and Mela, 1961), but varics according to brain area. Since exchange and uptake measured in the various areas show similar inhomogeneity, i.e., exchange or uptake of added amino acids is greater in those areas that have higher amino acid levels under normal conditions (Lajtha, 1961a), it is likely that regional amino acid distribution in normal conditions is regulated by a homeostatic control mechanism. The computation of the rates of metabolism and rates of exchange of the multiple pool of one compound in a cell poses formidable problems at the present time. Pools of digerent size and different rates of turnover and equilibration also may be different in their quantitative or qualitative contribution to the metabolic events. A further understanding of all these factors will be necessary before a final accurate evaluation of the metabolic rates of proteins can be made. In our thinking, we must thus be aware of the heterogeneity not only as far as the proteins and their metabolism is concerned but also of the precursors of the proteins, i.e., of the amino acid pools.

E. CHANGES IN CEREBRAL Aximo ACIDLEVELS Cerebral amino acids may take part in a host of reactions. They can be metabolized, and they are also the precursors of highly active substances such as amines. Changes in amino acids or their metabolic derivatives may signify or accompany pathological changes or

16

ABEr.4 LAJTHA

prevent such changes from occurring. A short discussion of the extensive investigation of changes in the free amino acids is included here, since such changes may secondarily influence protein metabolism or may be the result of an alteration in protein metabolism. Especially those changes caused by insulin hypoglycemia were investigated, often to see if the therapeutic effect of this treatment is due to metabolic changes in the central nervous system. A decrease in glutamate, glutamine, and y-aminobutyrate with an increase in asparate was observed (Dawson, 1950, 19%; Cravioto et al., 1951); in such cases systemically administered glutamate, and also glucose, restored cerebral glutamate levels to normal (Himwich and Petersen, 1958). Such changes (rise in aspartate and ethandamine, fall in glutamate, glutamine, yaminobutyrate, alanine, and glycine) were found with insulin but not oral hypoglycemic agents, most likely because the latter did not reduce blood sugar levels enough ( DeRopp and Snedeker, 1961a). It seems likely that in the absence of a sufficient amount of glucose a number of amino acids are metabolized in place of glucose, causing their level to fall; the total amino N in the brain does not change greatly, however, as the concomitant degradation of phosphatides results in increase in such compounds as serine, ethanolamine, and choline (Knauff and Bock, 1961). The reason for the accumulation of aspartate is probably that, because a smaller amount of glucose is available, there is less acetyl coenzyme A to combine with oxalacetate to form citrate, and so the oxaloacetate forms aspartate via transamination (Roberts and Eidelberg, 1960). The decrease of glutamic acid does not occur through decarboxylation ( through the y-aminobutyric acid pathway), since insulin also lowers glutamic acid levels in deoxypyridoxine treated animals (where glutamic decarboxylase is inhibited) (Massieu et al., 1962). It seems that the changes in amino acids are the results rather than the causes of convulsions. In an investigation of the effect of a number of convulsive agents, no common amino acid pattern emerged which would have reflected either convulsive activity or protection from convulsive activity ( Kamrin and Kamrin, 1961) , although it was reported that rat brains susceptible to acoustic epileptiform seizures contained larger amounts of free amide N (Kolousek et al., 1959) and human epileptic brain showed some differences (Yamamoto et al., 1961). Epileptogenic foci in cat cortex,

PROTEIN METABOLISM OF THE NERVOUS SYSTEM

17

produced by freezing, contained less glutamic acid, glutamine, and glutathione, while the 7-aminobutyric acid levels were kept constant (Berl et at?., 1959); the low glutamic acid levels were localized at the sites of strong epileptogenic discharge, whereas contralateral active mirror foci did not show such changes (Aelony et al., 1962). Strychnine convulsions also resulted in decreased glutamic acid levels (Haber and Saidel, 1948). Some convulsants increased free alanine (DeRopp and Snedeker, 1961b), but glutamine was not changed (Richter and Dawson, 1948). Electric shock caused small increases in basic and in aromatic amino acids (Hemmer, 1958). In anesthesia glutamic acid falls (Dawson, 1951, 1953), and therefore the total free amino N (Ansell and Richter, 1954a) falls too; histidine and arginine are not affected (Clouet et al., 1957), valine and leucine slightly ( Soep and Janssen, 1961). A number of other effects on cerebral amino acid levels were studied. Ethanol ( Hakkinen and Kulonen, 1961) increased glutamic, aspartic, and y-aminobutyric acids and decreased glutamine. Deoxycorticosterone increased glutamic acid, decreased glutamine and asparagine (Woodbury et d.,1957). Hypophysectomy decreased glutamic, aspartic, and y-aminobutyric acids, serine, alanine, and glycine, and did not affect some others (Nishioka, 1959). Hepatectomy greatly increased free amino acid levels, especially that of glutamine (Flock et al., 1953); infusion of ammonia greatly increased cerebral glutamine, while the levels of glutamic acid and y-aminobutyric acid were not changed ( Waelsch, 1961b; Berl et al., 1962). Tranquilizers in uitro influenced amino acid levels ( Ernsting et az.,1960). It must be realized that the cerebral levels of amino acids are determined by a number of processes such as cerebral amino acid and protein metabolism, as well as amino acid transport from the plasma. In some cases it has been found that with slight decreases in cerebral amino acid levels there is a greater release of amino acids from the liver resulting in increased plasma levels tending to compensate for the changes in brain (Hemmer, 1958). F. CONCLUSIONS The proteins of the nervous system derive mainly, and probably exclusively, from the free amino acid pool of the organ. How far changes in this pool affect protein metabolism is not clear at present.

18

ABEL LAJTH.4

The local concentrations of cerebral amino acids at the sites of protein formation, and any concentration changes at these sites, have not yet been studied, but it seems likely that especially large vaiiations in free amino acid levels might alter some aspects of protein metabolism such as turnover rate or the type of protein formed or broken down. The nervous system occupies a special position regarding its composition of free amino acids. In a qualitative sense, compounds found rarely, or not at all, in other organs are y-aminobutyric acid (Roberts and Frankel, 1950), acetylaspartic acid (Tallan et d., 1956), cystathionine (Tallan et al., 1958), homocarnosine (Pisano d al., 1961 ), and perhaps some others. Quantitatively most pronounced are the high concentrations of glutamic acid and its metabolic derivatives, which comprise more than half of the free amino N in the brain ( Waelsch, 1949, 1951; Tallan et al., 1954). Another special property of the cerebral amino acid pool is the small uptake of some amino acids by the brain following considerable increases of concentrations in the plasma. Although only small net increase occurs under such circumstances, there is a very rapid exchange of amino acids between plasma and brain. It seems that at least as far as the amino acids are concerned the brain barrier system is not a protective mechanism barring any entry of metabolites, but a homeostatic control mechanism consisting mostly of carrier mediated transport processes. These processes are present not only at the surfaces of the organ or at the capillary walls but are most likely components of all membranes present in the nervous system, including cell membranes and the membranes within the cells such as nuclear and mitochondria1 membranes. This barrier system that is active in amino acid influx and efflux can supply the brain with any specific amino acid at the time and at the place it is needed. This, the regulation can be metabolite- and area-specific (Lajtha, 1962a,b ) . Although the barrier system regulates cerebral amino acid levels, changes in these levels occur as a result of a number of conditions, such as the administration of insulin or drugs, and convulsions. In addition, protein synthesis involves the reutilization of amino acids liberated as a result of protein breakdown, and therefore protein metabolism can proceed at a considerable rate even with a limited external supply of amino acids.

PROTEIN METABOLISM O F THE NERVOUS SYSTEM

19

The intracellular amino acid pool that is utilized for protein synthesis is not necessarily in rapid equilibrium with the total amino acid pool of the brain. Within the brain, the free amino acids are in several pools or compartments which can be distinguished kinetically and some of which might have metabolic roles different from others. This compartmentation has to be considered in any experimental calculation of incorporation rate of amino acids into proteins (Waelsch and Lajtha, 1961) or the estimation of any effects the changes in amino acid levels may have on protein metabolism. To establish the properties of the amino acid pool that is utilized for protein synthesis remains one of the important tasks in this field. Ill. Cerebral Proteins

It is obvious that for a better understanding of the function of the proteins in the brain, the role played by individual protein species has to be known. One prerequisite for solving this problem is the availability of methods for isolating proteins in purified form. Although methods suitable for purifying proteins with known function are available, our present methods for quantitative fractionation of organ extracts are not adequate for meaningful studies. The metabolic activity of and changes in a number of protein fractions are of great interest, and a number of studies are in progress on this subject in various laboratories. In using protein fractionation procedures on brain tissue extracts, each group of investigators has obtained several fractions from brain, each of which probably contains a great number of individual protein species. Because of the divergent methods used, it is difficult at the present time to compare these results; the composition of each fraction will depend on the method used. Among other variables, any alteration in the way the brain extract is obtained may qualitatively or quantitatively influence the fractions obtained-the previous handling of the organ, such as freezing; the type of buffer used and concentration of the extracting solution; the duration, temperature, and pH of the extraction, etc. The effect of pH and salt concentration was the subject of a study by LeBaron and Folch (1959). At pH 6-9 and ionic strength of 3, the maximal yield of proteins was obtained, which was from white matter about 20%of the total organ proteins, and from gray matter considerably more. The solutions could be freed of lipids by

20

ABEL LAJTHA

centrifugation. Albumins were lipid-free, but the globulin fraction contained 25%lipids. After a recent survey of the extraction of soluble brain proteins, the authors think that, although in some of the studies 3050%extraction was achieved, most previous extraction methods used (including those of Kaps, 1954; Hofman and Schinko, 1956; Robertson, 1957; Dingman et al., 1959; Karcher et al., 1959; Kiyota, 1959b; LeBaron and Folch, 1959; etc.) usually extract less than 20!%of the proteins of the brain (Bauer et al., 1962). It has to be kept in mind, therefore, that the present methods probably leave the major portion of cerebral proteins unextracted. Treatment with lysolecithin resulted in extraction of about 70%of the total proteins from gray matter and about 40% from white matter without an inhibition of those enzymatic activities that were tested (Bauer et al., 1962). It is to be hoped that as more refined methods become available, future studies will utilize better defined fractions that will permit comparison between those obtained by the various investigators.

A. PURIFIED PROTEIN FRACTIONS The method most often used to obtain purified protein fractions was the electrophoretic separation of the soluble proteins into several fractions, and characterization of them by their migration pattern, especially in comparison with the migration pattern of the serum of the same animal under similar conditions. This, of course, does not mean that the cerebral fractions are identical to the serum fractions. There are differences between the proteins of spinal fluid and serum; protein fractions in the cerebrospinal fluid that are not present in the serum have been shown immuno-electrophoretically ( Clausen, 1961) . From brain, at first only a few fractions were isolated electrophoretically (Demling et a?.,1954), giving the following pattern: 15%albumin, 48%a-,284: p-, and 9!%7-globulins. Later more fractions (up to 9) were isolated and the above figures were considerably modified (Keup, 1955a; Hofman and Schinko, 1956; Caravaglios and Chiaverini, 1956; Robertson, 1957; etc.; for more references see LeBaron, 1959). Also, previous fractions were further purified (Sheng and Tsao, 1957). With the use of electrophoresis in starch gel, 14 fractions were separated and estimated (Bailey and Heald, 1961a, b). Electrophoretic separations have the advantage of minimal alter-

PROTEIN METABOLISM OF THE NERVOUS SYSTEM

21

ation of the proteins during the procedure, but the proteins in solution react with components of the buffer, and therefore the type of buffer will have an important influence on the composition of the fractions. This is one of the explanations of the great divergence of reported values in the literature. The fractions gained by this method exclude the organ proteins that are not dissolved in the buffers used, which, as already mentioned, amounts to at least half of the total proteins and usually considerably more. The fractions also vary from one study to another and each is composed of a great number of proteins. Nevertheless, electrophoresis is a valuable tool for comparative studies where the variables are reduced to a minimumfor example, the same procedure used on the same species of animal before and after insulin shock-to compare the fractions from various sources or under various conditions. Further progress will come from the combination of this technique with a number of other methods of separation, such as differential centrifugation or column chromatography, which will result in better defined fractions. With electrophoresis in agar gel, differences in protein composition of the brain of different species were found (Bailey and Heald, 1961a). Similar species differences have been found with starch gel electrophoresis (Bernsohn et al., 1961); differences were found with age (Kiyota, 1957, 1959a; Palladin, 1957). Electrophoresis can be used for enzyme preparation; with this method, nine esterases have been shown to occur in brain extracts (Barron et al., 1961) . Other methods of fractionation were also employed to obtain separate fractions, some of which were obtained in highly purified form. Mucoproteins showed changes in development with histochemical methods ( Sulkin, 1955). Three different protein fractions that contained copper (Porter and Folch, 1957a) were separated from brain. One of these fractions was about 85%homogeneous in the ultracentrifuge-cerebrocuprein I (Porter and Folch, 1957b). A cerebrocuprein I which is similar to the bovine fraction in containing 0.3%copper and having a molecular weight between 30,000 and 40,000, but different in electrophoretic mobility, has been isolated from human brain (Porter and Ainsworth, 1959). This protein fraction contains only a small portion of the cerebral copper content. One point of interest in copper-proteins is their role in hepatolenticular degeneration. A fraction investigated in greater detail is the protein-lipid com-

22

ABEL LAJTHA

plexes. Proteolipids, isolated by Folch and Lees (1951), are not present in fetal brain but appear at the time of myelination. This and their distribution suggest that they are, in most part, the protein component of myelin. They are in highest concentration in white matter, and only 1/20 to 1/30 of this amount occurs in peripheral nerve (Folch et al., 1958). They consist of about one-third lipid and two-thirds protein. The lipids are mostly phosphatidylserine, phosphatidylethanolamine, and phosphoinositides. The turnover of the phosphoinositide portion is rapid, that of the protein fraction very slow (Folch et al., 1957; Furst et al., 1958; and Gaitonde, 1961a); the incorporation of P32varies in different electrophoretic lipoprotein fractions (Robertson, 1960b). Phosphatidopeptides have also been isolated from brain ( Folcli and LeBaron, 1953; LeBaron and Rothleder, 1958). These are composed mostly of phosphoinositides combined with shorter peptides. Lipophylic peptides and proteins were studied by Uzman (1958) and U n a n and Rosen (1958), and a membrane protein fraction, neurosclerin, was isolated, whose relationship to myelin proteins is not clear at present. Neurokeratin was demonstrated to be a component of myelin and was the protein network remaining after treatment with lipid solvents and proteinases. The earlier methods of preparation, however, would result, according to LeBaron and Folch (1956), in splitting phosphatidopeptides and proteolipids and leaving proteolipid protein (which is resistant to proteolytic enzymes, Folch and Lees, 1951) with the neurokeratin fraction, showing that this fraction contains at least partly a mixture of degradation products probably originating from lipoproteins. Neurokeratin, as opposed to other keratins, is not solubilized by thioglycolate and does not lose its trypsin resistance by this treatment; this shows that the solubility properties and its resistance to proteolytic enzymes are not mainly due to S-S linkages but possibly to linkages to lipids because of its high leucine and phenylalanine content (Stary and Arat, 1957). A number of enzymes, phosphatases, cytochrome oxidases, and succinic dehydrogenase were found to be localized in the neurokeratin network ( Tewari and Bourne, 1961) . There have been several studies of phosphoproteins in the brain (for summary see Heald, 1960). The phosphorus was found to be attached to serine (Heald, 1958; Vladimirov et aJ., 1956; Heald,

PROTEIN METABOLISM OF

“€E NERVOUS SYSTEM

23

1961a). One interesting aspect of this class of proteins is the metabolic activity of their phosphate moiety. Administered P32is rapidly incorporated into cerebral phosphoproteins and its specific activity is comparable to or higher than other P32 fractions in the tissue (Marshak and Calvert, 1949; Schmidt and Davidson, 1950; Strickland, 1952; Johnson and Albert, 1953). This incorporation in vitro is dependent on the presence of oxidative metabolism (Vladimirov, 1953; Findlay et al., 1954; and Heald, 1958). ATP was a more immediate precursor of the incorporated P32into the proteins than was inorganic phosphate, and GTP was a more immediate precursor than ATP ( Heald and Stancer, 1962). The incorporation increases under stimulation in vivo (Vladimirov, 1953), and in slices it doubles with electrical stimulation (Heald, 1956, 1959). One theory to explain such changes upon stimulation proposed that phosphoproteins are associated with structures involved in maintenance and restoration of ionic gradients (Heald, 1961b) and the reversible phosphorylations are part of this mechanism. Cerebral phospholipoproteins contain sequences of adjacent phosphorylserine groups and thus could be acting as a polyelectrolyte ion exchange resin. The folding and unfolding of these proteins may be involved in the active transport of ions (Heald, 1962). A partial purification ( 1 5 2 5 fold) of cerebral phosphoproteins has recently been accomplished ( Rose, 1962a). A collagen-like protein has been isolated from spinal cord (Roboz et al., 1958) and its activity in producing allergic encephalomyelitis has been investigated (Kies et al., 1958).

B. PROTEIN COMPOSITION OF CEREBRAL AREAS A number of studies have shown that the morphologic, metabolic, and functional heterogeneity of the nervous system is reflected in the heterogeneity of its protein composition. Determining the protein concentration of different areas of the brain, Clouet and Gaitonde (1956) found variations from one area to another, with the sequence changing during development, The protein content of the phylogenetically older parts were the first to increase; these reached their maximum value at an earlier age (Kelley, 1956). The variation in composition among brain parts and in development was shown in changes in the ratio of the protein bound amino acids arginine to histidine (Clouet and Gaitonde, 1956), and in the increase of in-

24

ABEL L A P A

soluble and decrease of soluble proteins in development ( Kiyota, 1957). In adults the soluble protein fraction varied in different areas, and there were si@cant differences in the various brain areas in the relative amounts of the electrophoretic fractions of the soluble proteins. The a-globulin, apparently an important component in determining the hydrophilic property of brain tissue, was highest in the cerebral cortex ( Kiyota, 1959b). Differences in the quantitative distribution of the electrophoretic fractions have also been shown by Karcher d al. (1959), who found more 7-globulins at the level of brainstem and medulla than in the higher regions of the brain. Robertson (1960a) found that the cerebellum was much richer in soluble proteins than the other regions. White matter contained more of one fraction than another, while in gray matter the ratio was reversed. The albumin: globulin ratio varied between 0.33 in the pons to 0.86 in the medulla. Booij { 1960) found quantitative electrophoretic differences between frontal and occipital cortex, also that the latter could be separated into 8 fractions while under the same circumstances the former yielded 7 fractions. Bailey and Heald ( 1961a) used starch gel electrophoresis with greater resolving power than the previous methods and recognized 15 components: human cortex yielded 10 components and the cerebellum 8, the cortex containing 3 components not present in the cerebellum. There were qualitative differences in the whole brain proteins between the various species and between the dBerent strains of mice, although no such differences were detected in some inbred strains of rats. Other protein fractions, the distribution of which was investigated, included phosphoproteins. This fraction was investigated in different brain areas, especially its distribution in particdate and soluble fractions ( Heald, 1959; Rose, 196%). h recent study of the distribution of proteolipids in about 30 different areas ( Amaducci, 1962) confirmed previous findings ( Folch et al., 1958) that this fraction is at its highest level in central white matter, and is only 1/5-1/10 of that in gray matter and 1/20-1/30 of that in peripheral nerve. There were differences in proteolipid content, not only between various gray matter areas but also between white matter from various areas, and between various parts of the spinal cord. In central nervous white matter proteolipids were 2% of wet weight, but in spinal roots they were only 0.1-0.18% (Wolfgram and Rose, 1961). The distribution did not support the

PROTEIN METABOLISM OF THE NERVOUS SYSTEM

25

thesis that this fraction derives from myelin alone, unless the composition of the myelin from each area was assumed to be different ( Amaducci, 1962). Numerous studies established the difference in protein composition between the central and peripheral nervous system, among them those of Caravaglios and Chiaverini (1956). Palladin and his co-workers (Poliakova, 1956; Palladin, 1957; Palladin et al., 1957a) found that nerve was rich in an “albumin” fraction which was low in the brain and in spinal cord, and in turn nerve was lower than the central nervous system in other fractions. A higher content of neurosclerin in brain than in nerve (Uzman and Rumley, 1960) and differences in trypsin-resistant protein between the central and the peripheral nervous system (Tuqan and Adams, 1961) have been reported. Electrophoretic fractions from peripheral nerves have been studied by, among others, Deuticke et al. (1952), Keil (1954), and Palladin ( 1957). The distribution of several enzymes, especially cholinesterase, has been measured in numerous studies. In a recent paper (Rose, 1962b), the distribution of phosphoprotein phosphatase in various brain areas was measured and compared to the distribution of other enzymes, in the same brain areas as determined by other workerssuch as the distribution of choline acetylase (Feldberg and Vogt, 1948), cholinesterase (Burgen and Chipman, 1951) and acid, plus alkaline, plus pyrophosphatases ( Gordon, 1953) . Certain similarities could be found, such as the relatively low activities in the corpus callosum and medulIa and high activities in cerebral cortex. Methods for localization of enzymes in very small amounts of tissue samples have been worked out by Lowry and by Robins, and their associates. These ingenious methods (e.g., Lowry et al., 1954) permit determinations in a very small area, down to enzyme measurements in single cells. Examples of this work are: the determinations of 7 enzymes of glucose metabolism in 10 brain regions (Buell et al., 1958) or in 12 layers of the retina (Lowry et al., 1961), of 12 enzymes in peripheral nerve ( McCaman and Robins, 1959), of P-glycosidases in the layers of the cerebellar cortex (Robins, 1961). Pope and his co-workers (Pope, 1952, 1955) measured a number of enzymes, among them peptidases in various cortical layers. Proteinases were also determined in various brain areas and brain particulate fractions (Lajtha, 1961~).

26

ABEL LAJTHA

The distribution of cerebral enzymes among the particular fractions of homogenates have been measured in a number of instances. For example, Palladin ( 1961) compared the distribution of 7 enzymes. A more detailed discussion of the numerous important studies of enzymatic distribution among the various elements of the nervous system is outside the smpe of this review; the examples mentioned will sufficientlyshow the heterogeneity of the distribution of the enzymes in the nervous system. The above findings show clearly the expected heterogeneity of protein distribution as far as the different elements of the nervous system are concerned. Not only is one protein in relatively higher concentration in one element than in another but some of the protein fractions present in one element may be absent in another. These qualitative and quantitative differences exist between the central and the peripheral nervous system, between the various areas of the brain, between the various cells, between particulate fractions of cells, between myelin and other structural elements, between various layers of the same structure, etc. There are also variations of protein content during development. Further refinements in the methods of separation and isolation are necessary to facilitate the chemical analysis, as well as measurements of distribution and metabolism of well-defined proteins in the nervous system. It is likely that considerably greater variations than known at present will be found between the various elements of the nervous system with future methods. The linowledge of quantitative and qualitative distribution of the proteins is essential for the understanding of the role they play in the metabolism of the organ.

C. Axriwo ACIDCONTENT OF PROTEIXS The inability to obtain well-defined purified protein fractions from brain makes the interpretation of the results of amino acid analysis from proteins difficult. The total composition of the brain is comparable to other organs (Block and Bolling, 1951). Recently Knauff et al. (1961) determined the amino acid content of whole brain proteins and compared the levels of free and protein-bound amino acids. The ratio (protein-bound: free form) varied greatly; it was the lowest with glutamic acid ( 8 . 5 ) , higher for glycine and aspartic acid (about 30), and over 100 for most others, the highest for the basic amino acids being over 600, i.e., there is about 100

PROTEIN METABOLISM OF THE NERVOUS SYSTEM

27

times as much protein-bound amino acid as in the free form. Protein-bound amino acid determination from the whole organ can be useful for purposes of comparison; such a study is the measurement of changes in arginine and histidine during development (Clouet and Gaitonde, 1956), showing differences between the chemical composition of proteins in the young and in adults. Changes during development in protein-bound amino acids of cerebral white matter have also been shown ( Wender and Waligora, 196l), in fetal white matter containing more of some and less of other amino acids than after birth. There were changes during development also in the amino acid composition of proteins of gray matter, but such changes were different from the changes in the amino acid composition of white matter proteins ( Wender and Waligora, 1962); also the developmental changes in guinea pig (Wender and Waligora, 1962) differed from those in rat (Clouet and Gaitonde, 1956). The composition of the myelin sheath proteins also differed from other structural components of cerebral white matter. The amino acid content of proteolipids has been measured by Folch and Lees (1951) and Folch (1959); the composition is dependent on the procedure used to obtain this fraction (Gaitonde, 1961b). Amino acid analysis has also been performed on proteolipid proteins from white matter and from spinal roots, and on neurosclerin from white matter (Wolfgram and Rose, 1961). In another study (Uzman, 1958) the lipophilic peptides showed changes in composition at different ages, but the neurosclerin protein fraction had a constant amino acid content throughout development, although its concentration increased in the older animals. Neurosclerin obtained from mice was distinctive in containing a large proportion (close to 50%of total) of alanine, glycine, isoleucine, and leucine residues ( Uzman and Rosen, 1958). Somewhat less (below 40% of total) of these amino acid residues was found in neurosclerin from bovine white matter in an extensive study of myelin protein components (Wolfgram and Rose, 1961). This study showed a relative constancy of the amino acid pattern of the proteolipids regardless of human or bovine, or of central or peripheral, origin. The demonstration of heterogeneity of protein composition of brain areas and the variation of amino acid content in the individual proteins, although not unexpected, is a step toward identifying individual proteins and establishing their distribution. Only after con-

28

ABEL LAJTHA

siderable further study can we attempt to investigate such problems as the nature of changes in development and in function. The question to be answered is whether proteins are modified in a chemical or physical fashion to assume new or additional roles, or whether completely new proteins are formed during development. The effect of drugs, dietary deficiencies, or other diseases on the composition and distribution of proteins is another important area, Some of these topics will be discussed along with the influence on protein metabolism, further on. IV. Protein Turnover

A. THEDYm;Zfxc STATE

The question of whether or not neurons are formed in the adult nervous system has not been answered. There seems to be some regeneration (Windle, 1956) and there are suggestions of proliferation (Altman, 1962). However, there is little doubt that cell renewal in the adult brain is very much below that of other organs. For this reason, though general problems of protein metabolism are outside the scope of this review, a short discussion of the dynamic state of proteins is of interest. It has not been established definitely that all proteins in the living organism are in a dynamic state, i.e., undergo continuous degradation and resynthesis, but the available experimental evidence at present is in favor of such a state for most proteins. The reasons for such a continuous turnover are not clear, and it seems possible that some proteins, once laid down, are not metabolized during the lifetime of the organism. If such proteins exist, however, they probably form only a small fraction of the total protein of any organ. The doubts about the dynamic state of proteins have come from the work with induced enzymes in bacteria (Hogness et al., 1955), where the newly formed enzyme appeared to be stable. A number of other studies established the predominantly dynamic state in microorganisms (Mandelstam, 1957; Borek et al., 1958). Some of the findings of apparent metabolic stability may be due to differences in breakdown rate between nongrowing and rapidly growing organisms. During rapid growth, breakdown is considerably decreased in bacteria (Mandelstam, 1958; Urba, 1959) and in yeast ( Halvorson, 1958b). This finding, which demonstrates that growth

PROTEIN METABOLISM OF THE NERVOUS SYSTEM

29

can be regulated either by increasing synthesis or by decreasing breakdown, will be discussed later. Administered label that was incorporated previously will be released during the rapid growth phase of microorganisms at a much lower rate than any new label is incorporated and could go on, therefore, undetected under the experimental conditions. Studies in resting yeast ( Halvorson, 1958a) in nonmultiplying animal cells (Harris and Watts, 1958), and in nongrowing mammalian cell cultures (Eagle d al., 1959) showed active turnover of the constituent proteins. Indications for the metabolic stability of cerebral proteins came from experiments in which, following the administration of labeled glycine into pregnant animals, the label remained constant in the brain of the fetus, whereas in adult brain it decreased in time. This was taken as evidence for permanent deposition of some protein fractions during growth ( Still, 1957). The alternate explanations, namely, increased reutilization of radioglycine or the deposition of label from glycine in the relatively stable myelin lipids have not been excluded by these experiments. The conclusion can be drawn that lack of incorporation in the early experiments was due more to the experimental circumstances (such as poor penetration of the amino acid used for the test into the brain) than to the predominant metabolic inertness of the organ. It has to be emphasized again that although predominant metabolic stability can be dismissed, it has never been shown that all proteins are replaced during the lifetime of the organ; it might well be that some proteins are not degraded after their formation, and that other proteins are replaced so slowly that only part of the total is degraded during the lifetime of the organism. One protein fraction in brain with a low rate of metabolism is the proteolipids, some of which might have a half-life greater than the life-span of the animal (Furst et al., 1958; Davison, 1961 and Gaitonde, 1961a).

B. HALF-LIFEOF CEREBRAL PROTEINS Protein metabolism in vivo has usually been estimated by the measurement of incorporation of administered labeled amino acids. When such experiments were performed with brain, rapid incorporation of all the measured amino acids was found, including methionine, lysine, leucine, phenylalanine, glutamic acid, etc. After intracisternal injection of radiomethionine, more label was incorporated

30

ABEL LAJTHA

into brain proteins than after intravenous injection of the amino acid ( Friedberg et al., 1948). The difference was probably due to greater penetration with intracisternal administration. The methionine that entered the acid-soluble phase of brain was quickly incorporated into the proteins. The rate of uptake varied in rats of two different genetic strains but was constant in those of uniform strain (Gaitonde and Richter, 1955). In our laboratory the specific activity of the amino acid, in the pool of free amino acid and also protein-bound, was measured, and from the changes in specific activity in time the turnover rate of the proteins was estimated. Such experiments were performed with lysine (Lajtha et al., 195%) and with leucine (Lajtha, 1959). To estimate turnover rates (or half-lives) of cerebral proteins in experiments of this type, certain assumptions must be made: For instance, the free amino acid in the total pool of the organ, with its measured specific activity, is the true precursor of protein, and incorporation occurs by de novo synthesis rather than by exchange. (These will be discussed later.) At this time, it must be emphasized that, because certain conditions are only assumed, not proved, and because there are a number of technical difficulties in separations and measurements, protein half-lives are very rough approximations-the averages of widely divergent metabolic rates. They should be used only at present for comparative purposes. The true rate of metabolic activity of any single cerebral protein has yet to be determined. From the values, presented in Table 111, two main conclusions were drawn: One, that the turnover rate of cerebral protein is in the same order of magnitude as that of other tissues, whole brain turnover rate being lower than that of liver but higher than muscle, and two, that the value of turnover rate is not constant but depends on the experimental time. This second finding, which will be discussed in greater detail later (Section IV. E ) was interpreted as showing the heterogeneity of metabolic rates. Similar values to those shown (Table 111) have been reported from other laboratories. Gaitonde and Richter (1956) estimated 13.7 days for the average half-life times of cerebral proteins in rats, and Maurer and co-workers (Maurer, 1957; NiMas et al., 1958) estimated 6.2 days for rabbit brain and 8.9 days for rat brain proteins. There was similar approximate agreement for the average half-life of liver and muscle proteins from the different laboratories.

31

PROTEIN METABOLISM OF THE NERVOUS SYSTEM

TABLE I11 TURNOVER RATES OF ORGANPROTEINS IN ADULTMICE* Half-life time in days Experimental Brain time (minutes) Lysineb Leucinec 2

3 5 10 30 60

3

-

-

4

4 6 7 15

8 11 20 27

Liver Lysineb 0.9

Muscle

Leucinec

-

Lysineb Leucinec 4

-

0.4

-

1.3 2.6

0.9 1.3 3.9 -

8 8 13

-

-

24

5

7 14 28 34

a The half-life times increase with increasing experimental time. This is interpreted ag the organs being composed of proteins with widely divergent metabolic rates, the above values representing averages. Active turnover in brain can be seen; the metabolic rate of cerebral proteins is Iower than that of liver proteins but is higher than that of muscle proteins. From Lajtha et al., 1957a. c From Lajtha et al., 1959.

What the above experiments show is that at least some, probably a major portion, of cerebral proteins turn over at an appreciable rate, similar to proteins in other organs. This turnover occurs in adult as well as growing animals. The reasons for and the significance of this turnover have now to be established.

C. PROTEINMETABOLISM DURING DEVELOPMENT A study of protein metabolism in the growing organism is of great importance, not only for investigation of the metabolism and mechanism of cellular growth, of myelination, but also because it might shed light on factors regulating growth, as well as on the mechanism of protein metabolism in general. Perhaps functional metabolism can be studied since there should be parallel changes in protein metabolism as the functional requirements change with development. During development a great number of changes occur in brain. With the onset of myelination both the lipid content and lipid composition change. There are changes in the extracellular space, water, and protein content and in the level of a number of other

32

ABEL L A W

substances, many of which participate in protein metabolism, such as energy rich phosphates and amino acids (for a discussion of changes in the penetration of amino acids during development see Lajtha, 1962a). With all these alterations, parallel changes in protein metabolism during development can be expected. The increase in total proteins, and certain enzymes, occurs disproportionately; some enzymes even decrease. Many enzymes have characteristic patterns of changes during growth, and there are developmental stages in which sudden changes occur in the enzyme composition of the organ (Potter et al., 1945; Flexner, 1952; Rudnick and Waelsch, 1955; Cohn and Richter, 1956). The soluble protein content decreases while insoluble proteins increase during development; there are differences in electrophoretic fractions, heat coagulability, etc. ( Kiyota, 1959a, b ) . The enzyme analyses mentioned confirm the results of the analysis of protein bound amino acids during development, which showed differences in the amino acid composition of proteins between young and adult animals (Clouet and Gaitonde, 1956; and Wender and Waligora, 1961, 1962) with characteristic changes differing from one brain area to another. Only a few well-characterized proteins have been investigated up to the present, but it is likely that the protein distribution and composition are altered quite markedly during development. Changes during development affect the amino acid pool as well. The ability of newborn brain to incorporate label from glucose into essential amino acids-a property which is lost in the adult organ ( Winzler et al., 1952)-has been already mentioned. The measurements in our laboratory of flux rates of free amino acids from plasma to brain showed a rapid exchange of lysine (Lajtha et al., 1957b), glutamic acid (Waelsch, 1958; Lajtha et el., 1959) and leucine (Lajtha, 1959), but were not sufficiently accurate to enable us to detect flux rate changes between young and adult animals. In another study (Flexner et al., 1958; Roberts et al., 1959) the flux from plasma to brain of phenylalanine, leucine, and isoleucine mixture was about double, and that of glutamic acid about ten times as much in adult as in newborn. Any interpretation of differences in the metabolic activity between young and adult brain proteins has to take these above discussed factors into consideration. In the first comparison of incorporation rates (Greenberg et al.,

33

PROTEIN METABOLISM OF THE NERVOUS SYSTEM

1948) in chick brain homogenates in witro, 2%times faster incorporation of C14-glycinewas found in embryos than in newly hatched chicks. The incorporation into brain was twice as fast as in liver. In uivo, the uptake of S35-methioninewas higher in younger cerebral proteins than in older ones, but no such variations were found with liver (Gaitonde and Richter, 1956). A decreasing rate of cerebral methionine incorporation with development was also found by Palladin et al. ( 1957b), and Pantchenko ( 1958). In our laboratory the incorporation of labeled amino acids from the cerebral free amino acid pool into brain proteins was measured in newborn and young animals, with lysine (Lajtha et al., 1957a), and with leucine ( Lajtha, 1959) (Table IV) , If the values of turnTABLE IV TURNOVER RATESOF ORGANPROTEINS IN YOUNGMICE" Half-life time in days Experimental Brain time (minutes) Lysineb Leucine0 3 5 10 30 60

-

3 2

3 -

0.7 1.2 2 3 4

Liver

Muscle

Lysineb

Leucine0

Lysineb

Leucinec

-

0.2 0.4 0.7 2 5

-

0.9 1.5

1.7 1.1

3

-

2 2 2

-

2 3

5

A comparison of the data of this table with those of Table I11 shows smaller differences between the various organs in young animals. The metabolic rate of liver proteins is also higher here than that in the other organs tested. There is an increase in half-life times with increasing experimental times with leucine; this is not apparent with lysine. b From Lajtha et al., 1957a. In l0day-old animals. c From Lajtha, 1959. I n newborn animals.

over rates in newborn and young (Table IV) are compared with the values found for adults (Table 111) a decrease in turnover during development can be seen. The increased turnover rate in newborn and young as compared to adult is due to several reasons. Among them, the following may be most influential: ( a ) that there are fractions with slow and fractions with fast turnover (see below discussion of heterogeneity) at both ages, but the relative concentra-

34

ABEL LAJTHA

tion of the fast fraction is larger in the young; ( b ) that in young there are protein fractions that turn over faster than any corresponding fraction in the adult; ( c ) that the same fraction that turns over slower in adult turns over faster during growth; and ( d ) that there is deposition of proteins during the development of the organ, i.e., net increase due to growth. How much of the increased turnover in newborn or young is due to the rapid turnover of fractions not present in adults (point b ) , and how much to the greater abundance or faster turnover of these fractions that are present in adults (points a and c ) cannot be ascertained at the present time, but it was estimated that the deposition of new proteins during growth (point d ) could account for only a small portion of the increased rate of incorporation in young. Roberts et al. (1959) found indications that the differences in the in oico measured metabolic rates of proteins between young and adult depended on the amino acid used for the measurements. The apparent rates of incorporation of the amino acids investigated differed from the ratio of the concentration of these amino acids in the proteins. In newborn liver and brain cortex the essential amino acids tried were incorporated at a higher rate than in adult tissues. The non-essential amino acids tried: aspartic acid, glutamic acid, and glutamine-were incorporated at an equal rate in the young and adult cortex, while young liver incorporated aspartic acid faster and glutamic acid and glutamine slower than adult. The authors think that their results cannot be fully explained by the difference in protein composition between young and adult, but suggest that some incorporation occurs by an exchange mechanism rather than a de rim0 synthesis of the whole protein molecule. In view of the differences between the protein fractions of young and adult brain, and also the lack of certain data, such as differences in the rate of breakdown of proteins during development, incorporation data do not give us a clear picture for explaining events of growth and development in terms of mechanism of protein metabolism. The comparison of the turnover rates of identical (and pure) cerebral protein fractions from young and adult might give further information on this subject. It is of interest in this respect that while serum albumin turnover decreases as the animal matures, the yglobulin turnover increases (Jeffay, 1960). There seems to be good evidence at present as shown by the studies quoted above that the

PROTEIN METABOLISM OF THE NERVOUS SYSTEM

35

relative concentration of more active components is higher in younger animals, although some fractions may be more active in adult than in young. It is not known whether some or many proteins are formed or broken down without a change in their metabolic rate throughout development, nor do we know enough at the present time to describe protein fractions characteristic and specific for the developing brain from either analytical or metabolic points of view. At any rate, the turnover of proteins is rapid in young brain, and turnover proceeds at a much higher rate than the rate at which net protein formation, that is, growth, occurs. Because of this a proportionately small increase in synthetic rates or a small decrease in catabolism could account for the deposition of proteins during development. In such respects also, cerebral protein metabolism is not unique. For example, so much protein is involved in daily serum albumin turnover that a net change of 6 to 12%in synthetic or breakdown rate (or perhaps in both) could account for albumin accumulation during growth (Jeffay, 1960).

D. REGIONALMETABOLISM Further light may be shed on the role the metabolism of protein plays in the over-all metabolism and function of the brain by investigation of the relationships and differences existing in protein metabolism between the functionally different areas and cells of the nervous system. Up to the present time, the incorporation of labeled amino acids into the proteins of the various areas has been measured mainly either by counting the purified protein fraction or by autoradiography of sections from various areas. Each method has its advantages. The determination from a whole area rather than autoradiography of a section has the advantage of using a sample that represents the average of the whole area, also makes the determination of radioactivity of the free amino acids of the area possible, and thus permits expression of results in terms of specific activities. While this type of determination does not solve all the methodological problems connected with the estimation of protein turnover rates, it is able to decrease interference of such factors as amino acid transport and amino acid metabolism-factors about which the autoradiographic methods yield very little information. On the other hand, autoradiographic methods have resolution of detail that is

36

ABEZ LAJTHA

several orders of magnitude greater and that is able to add information on the level of cellular and subcellular elements. It must also be emphasized here (and it will be discussed in greater detail in the section on protein breakdown) that any definite rate of incorporation does not necessarily mean that the breakdown occurs at the same rate, or that the locus of formation and breakdown have to be necessarily the same. Half-life times of proteins in monkey brain areas are presented in Table V (Furst et uZ., 1958). Although the absolute value of the ThRLE V HALF-LIFETIMES OF PROTEINS IN VARIOCSCEREBRAL AREASOF MONKEYSO Half-life in daysh

~

_

-

White matter Cerebral cortex Cerebellum Medulla-pons Thalsmus-h ypothalamus Spinal cord

5 minutes

10 minutes

4

4

6 5

-

8

ti 11

8

I

-

t

10

45 minutes

Relative half-life

9 13 14 14 17 19

43 65 68 70 77 100

From Furst el al., 1958. T h e half-life time increases with increasing experimental time, showing the hetcrogeneous composition of each area measured. There are differences between the protein composition and turnover rates of the various areas. Similar results were obtained if the brain was subdivided into several more areas. For a proposed explanation of the high turnover in white matter see text.

half life depends on the duration of the experiment, the sequence of values in decreasing order is white, cortex, cerebellum, medullapons, thalamus-hypothalamus, spinal cord. In these experiments the change with time of the specific activity of free and of protein bound lysine in an area was measured, and for the calculations of half-lives it was assumed that the lysine with average specific activity in the free amino acid pool is the presursor of the protein bound form. Thus, although the radioactivity in white matter was relatively low, the specific activity of free lysine was relatively even lower in this area, making the calculated turnover rate high. It was proposed in this study (Furst et al., 1958; Waelsch and Lajtha, 1961) that the unexpectedly high turnover rate of proteins in white matter, and

PROTEIN METABOLISM OF THE NERVOUS SYSTEM

37

perhaps parts of the medulla, is most likely only an apparent one and may be explained by assuming that the average free lysine is not the true precursor and therefore not the proper reference for the calculations of turnover rates of this area. Obviously, if the specific activity of the lysine that was incorporated in the proteins was twice as much as the measured average specific activity in the free amino acid pool, then the true turnover rate is only half as much as the one calculated from the average rather than the true higher specific activity. In other laboratories the incorporation of methionine S35 was measured without the determination of the specific activity of the methionine in the free amino acid pool. Palladin et al. (1957b) found that the rate of incorporation decreased in the following order: gray matter, white matter, nerve. Pantchenko (1958) in a similar study found the following decreasing order: in adults, cerebellum, cerebral cortex, optic region, hypothalamus, medulla-pons, spinal cord, white; in young, cerebellum, medulla-pons, optic region, spinal cord, cerebral cortex, hypothalamus, white. This work shows that the pattern of heterogeneity of protein metabolism changes during development, the relative order of activities of the various areas being different in young as compared with adult. Differences during development that were found previously were that incorporation was diffuse in one day old brain, and that in adult the areas with cellular layers containing a high proportion of nerve cell bodies were more active than areas rich in white matter (Cohn et al., 1954). Incorporation seemed to be the highest in the cerebellar cortex and lowest in white matter, with other areas of the brain intermediate between these two values (Richter et al., 1960). Another autoradiographic study of P5-methionine incorporation showed the following distribution: highest, cerebellum (medial parts the highest), then cortex, pons, cord, peripheral nerve. In general, white matter was lower than grey, and the incorporation was not uniform throughout the gray matter (Fischer et al., 1956). Maurer and his collaborators in an extensive set of publications (see for example Maurer, 1957; Schultze et al., 1959; Goslar and Schultze, 1958) measured with radiomethionine the turnover of the different cell types in different brain areas and compared this with the metabolic activity of cell types from other organs. According to activity the cells could be divided into four groups with a more than

38

ABEL LAJTHA

200-fold difference between the most active and the least active cell types, the ganglion cells and the epithelial cells of the choroid plexuses belonging to the most active group with such other cells as the protein secretory cells in the pancreas and stomach or cells of the reticuloendothelial system. Ganglion cells were estimated to have a half-life of eight hours versus glia with seventeen days. In a somewhat Werent approach to the same problem, radioautography following the intrathecal administration of labeled methionine or glycine showed that nerve cells lost most of their initial radioactivity in a few hours while other cell types retained their radioactivity for considerable periods of time. Some cells such as astrocytes and microglia incorporated very little label (Koenig, 1959). The above discussed studies, only a sample out of many, extend our knowledge of the morphological heterogeneity of the brain, and the observations of the uneven distribution of the cerebral protein fractions in the various brain areas (see Section 111. B ) in that they show that this heterogeneity exists from the metabolic point of view as well. It is clear that the average turnover rates of the various brain areas and cell types show great divergences. Such a finding was to be expected after the finding of heterogeneous distribution of protein fractions and of enzymes mentioned in Section 111. B, since probably the greatest contributing factor to metabolic heterogeneity is the heterogeneity in the distribution of proteins. This is further discussed in the next section.

E. HETEROGEATITY OF TURNOVER RATES I'ariations in the metabolic rates of proteins exist not only from one brain area to another but also within the area, indeed, within a single cell. In general, it is likely that each protein has its own characteristic turnover rate when the organism is in equilibrium, although the rates may be varied under some circumstances. Indications for variations in the turnover rates of single protein species came first from a study of the turnover of muscle proteins; aldolase was found to turn over almost twice as fast as dehydrogenase (Simpson and Velick, 1954), and a study of seven purified proteins from muscle showed half lives varying from 20 days for light meromyosin to 100 days for dehydrogenase ( Velick, 1956). For brain proteins, the indication for metabolic heterogeneity

PROTEIN' METABOLISM O F THE NERVOUS SYSTEM

39

came from the finding that the half-life time value calculated depended on the duration of the experiment both with lysine (Lajtha et al., 1957b; Waelsch, 1958) and with leucine (Lajtha, 1959). When less time elapsed between the administration of the labeled amino acid and the killing of the animal, the half-life calculated was shorter than it was in experiments of longer duration (Table 111). This finding was interpreted as a result of the fact that in short term experiments mainly the protein fractions with high rates of turnover influence the incorporation, whereas in long term experiments the rapid fractions turn over several times and even lose some of their initial label, and fractions with low rates of turnover have greater influence on the calculated results. This also shows that for the estimation of the rapid fractions short term experiments are necessary, which in turn are not suitable or sensitive enough for the measurement of less active fractions. Thus metabolic heterogeneity of the various brain areas is a reflection of their protein composition, areas with a relatively high proportion of rapidly turning over proteins showing the higher metabolic rates, areas richer in cells with rapid turnover also being more active. Experiments with S35-methionineincorporation showed different activity in cerebral protein fractions obtained by extraction with various solvents. Saline extracted the fraction with the highest activity, and the fraction remaining after NaOH extraction showed the lowest activity (Kravchinski and Silitch, 1957); by employing somewhat similar extraction methods heterogeneity could be shown within the microsomal fraction, which could thus be further divided into subfractions of various activity ( Clouet and Richter, 1959). Protein fractions with considerably lower than average metabolic activity are the proteolipids. In our experiments ( Furst et al., 1958), the rate of incorporation of lysine into proteolipids was ten to forty times lower than into the rest of the proteins, corresponding to halflives between 90 and 600 days in mice, depending on the area measured, if calculated from a 45 minute experiment; it was even longer if calculated from an experiment of longer duration. The experimental difficulties prevent the precise assignment of a half-life time to this fraction, because falsely long half-life can result from reutilization of the breakdown products. The penetration of a labeled amino acid marker to the myelin sheath, and its passage from it, might be considerably slower than in other areas, also tending to

40

ABEL LAJTHA

make the measured half-lives longer. A half-time of 600 days or longer, however, would mean that at least a part of the fraction does not turn over during the life time of the animal. These experiments have been recently confirmed and extended (Davison, 1961; Gaitonde, 1961a), and the low activity of this protein fraction as compared to the average proteins seems to be well established. The suggestion has been advanced that this above finding may indicate the metabolic stability of the myelin proteins, but this cannot be accepted as fact. The finding that in adult animals incorporation occurs into this fraction speaks against its absolute metabolic stability; also it has not been established whether the proteolipids make up the total of the myelin sheath proteins, or if, in this sheath, other proteins, possibly with higher metabolic activity, are present. There is at present no reason to suppose that all brain proteins are turning over rapidly or that all those that are metabolically stable are restricted to a single class of proteins. For an investigation of the proteins of lower activity experiments of long duration are necessary. The few such experiments that have been performed showed that in some organs a considerable portion has rather low turnover rate. Thompson and Ballou (1956) exposed rats to a constant level of tritiated water from conception to 6 months of age (this method labels all compounds containing hydrogen) and they then studied the rats for periods up to one year. It was found that each tissue contained components with a long half-life; collagen was found especially stable, confirming a number of previous studies on the relative metabolic stability of some tissue proteins, among them collagen. In this study, the long lived component comprised about 54%of the brain with a half-life of 150 days. While this method did not necessarily label the total organic matter in the brain, the halflife of whole brain (including lipids and carbohydrates as well as proteins) is given; the portion of the relatively or absolutely stable protein elements has not been established. More recently (Buchanan, 1961), in continuous feeding experiments with adult rats and mice, it was found that some portions of muscle, brain, bone, cartilage, and skin may not be replaced during the life span of the animal. In brain and muscle about 50% replacement occurred in 21-25 days, 70%replacement after 58 days. Again, average turnover time could be calculated, which is the average of divergent values; in brain 38%had a mean turnover time

PROTEIN METABOLISM OF THE NERVOUS SYSTEM

41

of 9.8 days, 30%in 74 days and 32%in 100 or more days. These values (similar to those of Thompson and Ballou, 1956) refer to the total carbon of the tissue including lipids and carbohydrates and do not establish the quantitative relationships between protein fractions, but they do establish the dynamic state of a large portion of the cerebral constituents. Since the relative metabolic stability of other cerebral constituents, some lipid components of myelin, for example ( Waelsch et al., 1940), has been established, part of the more stable fraction seems most likely to be composed of compounds other than proteins. The studies discussed above clearly show the metabolic heterogeneity of brain components, including proteins, in every level investigated, from the whole organ to small parts of cells. Our present methods of isolation of pure proteins and of measuring turnover rates are too crude to assign any definite turnover rate to a single protein fraction at the present time. Problems of penetration of the label will have to be solved to be able to measure the most rapidly metabolizing fraction, and problems such as the reutilization of amino acids gained from protein breakdown in situ for the resynthesis-a turnover undetected by the present methods-will have to be solved to establish the quantitative parameters of the more stable fractions. This metabolic inhomogeneity is not restricted to brain. Experiments on purified muscle proteins have been mentioned already (Simpson and Velick, 1954; Velick, 1956), and the dependence of half-lives on the duration of the experiments in our studies was also found for the proteins of muscle and liver, the other organs investigated (Lajtha et al., 1957a; Lajtha, 1959). The finding for the liver was confirmed in a study in which the half-life of liver proteins changed from 1.8 to 3.8 days depending on the length of time the liver was exposed to the label. This also was interpreted as showing the heterogeneity of the metabolic activity of liver proteins. In this study the reutilization of lysine originating from the degradation of liver proteins was estimated to be about 50% ( Swick, 1958). It is obvious that at present we face a great number of technical difficulties in attempting to assess such factors as the role played by local permeability barriers, or the heterogeneity of the precursor amino acid pool, or the extent of reutilization of the amino acids, to name only a few. The values of turnover rates of proteins can be

42

ABEL L A W

utilized, therefore, only with these limitations in mind, even for comparing the metabolic rates of different tissues or different parts of the same tissues. The establishment of this metabolic heterogeneity, however, is of great significance, and presents a great challenge to establish the mechanism and the controlling factors of turnover and of the variations in turnover rates. In general, it is believed that proteins connected with structure are more stable than the ones concerned with possible functional role-but the validity of this idea is uncertain. It is also important to establish whether the wide spectrum of turnover rates is an intrinsic property of the proteins themselves, or whether the heterogeneous distribution of the systems connected with protein metabolism plays a considerable role. It is likely that we will have to consider not only the individual proteins such as collagen, but also their relationship to amino acid activating enzymes, proteinases, collagenase, etc. in the living organism. In other words, it is possible that a protein molecule would not be metabolized at the same rate at two different sites if, for example, the enzyme responsible for its breakdown is more active at one site than at the other.

F. LOCALIZATION OF PROTEIN METABOLISM Important problems which follow the recognition of the metabolic heterogeneity of cerebral proteins are the localization of the metabolically active fractions and the establishment of the site where protein formation or incorporation occurs. When, following in vitro incorporation of a labeled amino acid, the brain was fractionated by centrifugation, the fraction with the highest rate of incorporation was the microsornes (Furst et al., 1958; Clouet and Richter, 1957, 1959; Palladin et al., 1959). The specific activity of protein bound lysine was highest in the microsomes for all thc brain areas that were investigated (Furst ct al., 19%> (Table VI ) . In this respect, brain is not unique since in other organs the highest incorporation rate among the particulate fractions was also found in the microsomes (Borsook et al., 1950; Hultin, 1950; Keller et al., 1954). The microsomal fraction, which in the brain is considerably more heterogeneous than in other tissues (Petrushka and Giuditta, 1959), could be further fractionated by salt and alkali extraction; the alkali extractable liponucleoproteins had the highest specific activity with an estimated half-life of about

43

PROTEIN METABOLISM OF THE NERVOUS SYSTEM

TABLE VI DISTRIBUTION OF RADIOACTIVITY IN CEREBRAL PARTICULATE FRACTIONS FROM MONKEYBRAIN^

Whole homogenate, absolute specific activityb Whole homogenate, relative specific activity" Nuclear fraction Mitochondria1 fraction Microsomal fraction Supernatant fraction

Cortex

Pons-medulla

0.26 100 97 82 390 150

0.13 100 80 100 180 I20

0 I n each area the highest specific activity following the administration of a labeled amino acid (in this case lysine) is initially in the microsomal fraction. Such findings focused attention on the microsomal fraction as the site of protein formation. From Furst et al., 1958. b Counts/minute/pg lysine. 0 Activity of whole homogenate taken as 100.

one hour (Clouet and Richter, 1959). The above experiments could be extended in vitro, where isolated microsomes from brain, when fortified with the necessary systems, could be shown to incorporate labeled amino acids (Satake et al., 1960; Lajtha, 1961b) (Table VII), and in uitro incorporation could also be shown on further fractionation of the microsomes into ribosomes (Acs et al., 1961). While with microsomal preparations liver incorporates more than brain, with ribosomes brain is the more active tissue: in liver microsomes and ribosomes incorporate amino acids at about the same rate, but in brain incorporation into ribosomes is considerably higher than into microsomes (Acs et al., 1961). It is of interest in this respect that brain slices incorporated amino acids at about the same rate as did liver slices ( Mase et al., 1962). Histological, especially autoradiographic, evidence showed active incorporation around the nucleolus and nuclear membrane, some of the active fractions being the lipoproteins rich in SH groups; initial distribution of the label also paralleled the distributions of the Nissl bodies (Vraa-Jensen, 1956; Niklas and Oehlert, 1956; Picard et al., 1957; Droz and Verne, 1959). In ganglion cells the incorporation occurred in the chromatin associated with the nucleolus itself, in the nuclear membrane and its immediate surrounding, and in the cytoplasm in the Nissl bodies (Schultze et al., 1959).

4 4

ABEL

WJTHA

TABLE V I I PROTEINSYNTHESIS I N In Vitro SYSTEMS Experimental system Brain Complete system S o energy source S o activating enzyme Xuclei instead of microsomes XIitochondria instead of microsomes Ribosomes instead of microsomes Ribosomes, no energy source Liver microsomes ribosomes

Remar&

Specific activity

hfererice

55 4 12 19

18 470 4

A

E

480 300

d d

-4= contains microsomes, pH 5.2 supernatant (activating enzymes), creatine phosphate (CP), adenosine triphosphate (ATP), guanosine triphosphate (OTP), C1-l-I,-Ieucine and creatine phosphoferrwe (CI’ enzyme). B = CY, ATP, GTP and CP enzyme omitted. (’ = pH 5.3 supernatant omitted. I ) = the activity in these fractions might be due to microsomal contamination. E = ribosomes are the pellets obtained by centrifugation of deoxycholate treated microsomes. From Lajths, 1961b. From Kaelsch and Lajtha, 1961. From Acs et al., 1961. The data are recalculated to be proportioned t o the rest of the table. Highest activity is obtained with microsomes and ribosomes.

The large cells in spinal ganglia have been found to undergo a series of metabolic cycles shown by variations in the distribution of enzymes. The perinuclear region was the site of considerable metabolic activity from which the waves of enzymatic activity proceeded to the peripheral cytoplasm. It was proposed that such metabolic cycles may be related to the synthesis of protein and enzymes ( Tewari and Bourne, 1962). Radioautographic investigation of the in zjiua incorporated H:’ labeled leucine, methionine, or glycine in nuclei showed a high uptake within the nuclear chromatin, of cerebral pyramidal cells, cerebellar Purkinje cells, and the liver cells, but not by their nucleoli; this points to an active synthesis of chromosomal proteins in the nucleus (Carneiro and Leblond, 1959). The finding of incorporation

PROTEIN METABOLISM OF THE NERVOUS SYSTEM

45

in vitro, mostly in microsomal or ribosomal fractions, in the same fractions that incorporate at the highest rate in the living animal also, opened up the possibility of studying the mechanism of protein synthesis in isolated systems. This will be discussed in the next section. V. The Formation of Proteins

A. GENERALPROBLEMS Obviously the difficulties in interpreting measurements of protein metabolism in other organs apply also to experiments with brain, but because of space limitations only a few of these questions will be discussed, and for a more detailed discussion the reader is referred to the previously mentioned articles summarizing the present concepts of protein metabolism in other organs. Some questions such as the dynamic state of cerebral proteins have already been touched upon. One of the questions is whether in a cell-free system the amino acid incorporation represents the net synthesis of new protein molecules. This has not been studied in the brain. The chance of artifacts, i.e., that an amino acid may become attached to a protein not through a peptide bond (protein formation), is greater than is generally realized, Sulfur amino acids may also be bound through disulfide bonds, and glycine after conversion to serine can appear (as phosphatidylserine) as protein-bound lipid; part of the proteinbound leucine and valine can be removed by treatment with agents such as performic acid, thioglycolic acid, or sodium hydroxide that do not remove these amino acids from peptide bonds. (For a discussion in detail see Tarver, 1954 and Loftfield, 1957.) These artifacts show the necessity of careful procedures. When purified proteins are isolated and identified, incorporation may measure protein synthesis. Such evidence comes from studies of a cell-free system containing ribosomes from rabbit reticulocytes in which indications for new formation of hemoglobin could be shown (Borsook et al., 1957; Schweet et al., 1958; Allen and Schweet, 1962). Though the available evidence is rather slight, it seems that net formation can be achieved in cell-free systems, where the mechanism can be studied more closely than in the living animal. Another problem of such studies is that incorporation of amino

46

ABEL L A W

acids might occur by an exchange mechanism rather than by the breakdown and resynthesis, that is, turnover of the whole protein molecule. Incorporation thus would measure the turnover of only a part of the protein molecule, or in the extreme case the exchange of a single protein bound amino acid residue with the labeled one from the free amino acid pool. Because of technical difficulties this question cannot be finally settled; while a few experiments (e.g. Rabinovitz et al., 1954) are compatible with the existence of exchange, most studies do not support it (e.g. Askonas et al., 1955; Penn et nl., 1957), and one may conclude that most of the available evidence is against such an exchange mechanism playing a major role (see Loftiield, 1957, 1962). For the brain Roberts et al. (1959) found some evidence, especially in newborn cortex, that the rate of incorporation of several amino acids did not correspond to the average concentration of these acids in the proteins and suggested that exchange may be an important process in incorporation. As the authors state, such a finding does not provide proof of exchange. There are possible explanations other than exchange for this non-proportional labeling. One is that the protein fraction that incorporates the label most rapidly differs in amino acid composition from the average. If this active fraction contains twice as many phenylalanine residiies and half as many glutamic acid residues than is the average for the cortex proteins, the labeling of phenylalanine will be more and that of glutamic acid less than expected from average concentrations. Another explanation could be that the labeled administered phenylalanine or glutamic acid is not uniformly mixed with the pool and the specific activity of the acid which is incorporated into protein differs from the measured average specific activity in the free amino acid pool. Inhomogeneity (compartmentation) in the free amino acid pool has been discussed (see Section 11. D). Because of these uncertainties the role of exchange in the brain cannot be definitely evaluated at present but must await further study. If the parallelism with other organs holds in this respect also, then it is not likely that it plays a major role. A problem of special interest for the nervous system is the control of the rate of protein metabolism-a problem not studied extensively up to the present time. The roles of such control mechanisms in growth, in regeneration, axon formation, etc., are of particular importance. Some aspects of factors that can stimulate or inhibit nerve

PROTEIN' METABOLISM OF THE NERVOUS SYSTEM

47

growth ( Levi-Montalcini, 1958; Levi-Montalcini and Booker, 1960a) will be discussed in Section VII.

B. THEPRECURSORS There is little doubt that proteins are formed ultimately from free amino acids; the identity of the precursors just preceding the final protein molecule is not as well established. The pathway which is today regarded as the main one from amino acids to proteins will be discussed further below. In addition to this pathway other precursors have been proposed: (1) peptides, in which case protein formation would be a stepwise process by which first the amino acids would form peptides and subsequently the peptide chains would be assembled into proteins, and ( 2 ) proteins, in which case a protein molecule would be modified to become another protein or the protein precursor would dongte peptides for the assembly of the new proteins. The finding (Roberts et al., 1959) which might be interpreted as exchange of amino acids or as formation of proteins through peptides in the brain has been discussed already, and difficulties in unequivocal interpretation of the data have been pointed out. In the following, a few examples in favor of free amino acids as the precursors of proteins and examples for peptide utilization for protein formation will be briefly discussed. Evidence that free amino acids are the immediate precursor comes from a number of experiments some of which are discussed here. a ) One system is induced enzyme synthesis in bacteria, in which the enzyme protein being induced is more rapidly formed as compared to other proteins in the organism. When the bacterial proteins were already labeled, before induction, but the free amino acid pool was not, the newly formed enzyme did not contain label. When the bacterial proteins were not labeled, but the free amino acid pool was, the newly formed enzyme also incorporated label (Monod et al., 1952; Halvorson and Spiegelman, 1952; Rotman and Spiegelman, 1954). Thus the induced enzyme was formed from free amino acids and not from the proteins which were already present. b ) In muscle, with aldolase and glyceraldehyde-3-phosphatedehydrogenase (Simpson and Velick, 1954; Simpson, 1955), the study of incorporation of several amino acids produced evidence that the proteins are derived from free amino acids in that a given amino acid was equally labeled regardless of its position in the protein

48

ABEL LAJIH-4

molecule; the specific activity ratios of 8 amino acids tried in the two proteins were equal. c ) The analysis of a number of peptides from partial hydrolysates of casein and p-lactoglobulin from milk after the injection of labeled amino acids showed that the radioactivity was distributed uniformly within the protein, i.e., the synthesis occurred by a net formation from free amino acids ( Askonas rt al., 1955). d ) Ferritin in liver was also found to be formed from free amino acids after various intervals following the infusion of three labeled amino acids. The ferritin isolated had the same radioactivity in these amino acids as was present in the free amino acids in the liver ( Loftfield and Harris, 1956). There are many other experiments supporting the idea that amino acids are the immediate precursors of proteins, such as studies of antibody formation (Taliaferro and Talmage, 1955), which cannot be discussed in detail here. Evidence from several laboratories investigating various protein fractions has been interpreted as showing that not only free amino acid intermediates participate in protein synthesis. Mainly two different kinds of evidence could be shown, one, the non-uniform distribution of label within a protein molecule, and two, the incorporation of label from one protein molecule to another without going through the free amino acid stage. Nonuniform incorporation in different parts of ovalbumin (Steinberg and Anfinsen, 1952), insulin and ribonuclease (Vaughan and hnfinsen, 1954), and silk fibroin (Shimura et al., 1956) have been found; the specific activity of an amino acid could differ as much as 10-fold depending on the part of the protein from which it was isolated (for discussion see Steinberg et al., 1956; and Loftfield, 1962). These findings can be interpreted as synthesis from peptide intermediates, one peptide precursor being labeled differently from another; or as the exchange of some but not all of the amino acid residues of a protein with the free amino acid pool; but also as synthesis which takes long enough for the specific activity of the free amino acid to change markedly during the assembly of the protein molecule. In the last instance synthesis would occur from free amino acids. Evidence for the utilization of labeled proteins without their complete breakdown to amino acids for new protein synthesis was shown with embryonic tissue cultures ( Francis and Winnick, 1953),

PROTEIN METABOLISM OF THE NERVOUS SYSTEM

49

Walker carcinoma tumor (Babson and Winnick, 1954) , chick embryonic protein (Walter and Mahler, 1958), and Ehrlich ascites cells (Chin et al., 1959), among others. The interpretation of the evidence either for or against amino acids as the immediate precursor of proteins is not simple. Because of technical difficulties, evidence for the participation of free amino acids is easier to make persuasive than the evidence to the contrary. Isolation of peptide fragments of proteins and showing that the label is uniform throughout the molecule is fairly convincing evidence that free amino acids are the precursors. Although it can be assumed in experiments of this kind that any particular type of peptide residue does not originate in only one part of the molecule, and thus the values measured represent averages of divergent specific activities, it is not too likely that all the different peptides isolated would show the same average specific activities if the radioactivities were unevenly distributed throughout the molecule. Non-uniform labeling, a finding that could be interpreted as being against protein formation from free amino acids, might on the other hand be the result of slower synthesis, where the specific activity of the precursor free amino acid is changed during the assembly of the protein molecule. It is even more difficult to interpret the preferential utilization of proteins. The amino acid pools of organs are not homogeneous (see Section 11. D ) and, furthermore, these organ pools are not in rapid equilibrium with the plasma pool. Loftfield and Harris (1956) found, for example, that the bulk of the free amino acids in liver is derived from endogenous protein even after infusion of considerable amounts of amino acids into the circulation. The precursor amino acid pool might be preferentially labeled from amino acids derived from the administered labeled proteins without necessarily being in equilibrium with the plasma amino acids. Therefore, apparent preferential labeling of the cellular proteins from an administered labeled protein might only mean that the breakdown products (which might well be amino acids) of the administered protein reach the site of new protein synthesis faster than some other amino acids. There is also a possibility that in some areas the entire administered labeled protein has been selectively absorbed by a specific organ. For the reason discussed above, and because no peptide inter-

mediates have been found up to the present time, the available evidence favors the free amino acids as immediate precursors in protein synthesis. The possibility of further modification of some protein molecules cannot be excluded and will be discussed with protein breakdown. C. AMISO ACIDINCORPORATING SYSTEMS In few fields of biochemistry has there been such a rapid advance as in the study of the mechanism of protein biosynthesis, and as a result our knowledge has greatly increased in this area. Since a number of reviews have surveyed the field very well, in spite of the great importance of these results only a brief summary is needed here. At present there is no reason to believe that the mechanism of protein formation in brain differs significantly from that in other organs. Although almost all of the work which will be discussed was first done in other tissues, similar results that have been recently obtained with nervous tissues will be discussed at the appropriate places. Soon after labeled amino acids became available, it was found that there is a rapid flow between the free amino acid pool and proteins in all living systems studied (Schoenheimer et al., 1939; for summary see Schoenheimer, 1942; Rittenberg, 1950; Zamecnik, 1960). When radioactive amino acids were added to a great variety of living systems the radioactivity rapidly appeared in proteins (e.g. Melchior and Tarver, 1947; Zamecnik et al., 1948). Interferences with energy utilization inhibited incorporation ( Winnick et al., 1947). When, following the in viuo incorporation of the labeled amino acid, liver was homogenized and separated by centrifugation into nuclear, mitochondrial, and microsomal fractions, most of the radioactivity was found initially in the microsomal fraction (Borsook et al., 1950; Hultin, 1950; Keller et al., 1954). This was the fraction in which most of the cytoplasmic RNA was found, A close connection between RNA and protein synthesis was implied (Brachet, 1950; Caspersson, 1950), and therefore it seemed possible that these particles are the primary site for protein synthesis. By further fractionation lipoproteins could be extracted from microsomes, and the remaining ribonucleoproteins seemed to be most active in incorporation ( Littlefield et al., 1955). Following in uiuo incorporation, brain could be separated into particulate fractions; the one with the

PROTEIN METABOLISM OF THE NERVOUS SYSTEM

51

highest initial specific activity was the microsomal fraction ( Waelsch, 1957; Clouet and Richter, 1957; Furst et al., 1958) (Table VI), which could be further fractionated into more active and less active fractions (Clouet and Richter, 1959). These findings in the living animal made a closer study of the processes in isolated system possible. With fractionation and further purification the requirements of the incorporating system were further studied and the requirement for ATP (Zamecnik and Keller, 1954), for GTF', and soluble pH 5 enzymes (Keller and Zamecnik, 1956; Littlefield and Keller, 1957) could be established. Brain slices in vitro incorporated labeled amino acids and later the presence of the cell free incorporation system with similar requirements to that found in other tissues could be shown in brain (Satake et al., 1960; Lajtha, 1961b) (Table VII). Further studies of particulate systems other than microsomes revealed that nuclei ( Allfrey et al., 1955; Logan et al., 1959) and mitochondria ( McLean et al., 1958; Roodyn et al., 1961,1962; Truman and Korner, 1962) in isolated systems when fortified with the necessary requirements are also capable of incorporating labeled amino acids into proteins. Although in detailed requirements or perhaps in some detail of the biosynthetic pathway these particulates seemed to differ somewhat from the microsomes, it could be shown that these differences were not all in the primary reactions involved in protein synthesis. The requirement for Na in nuclear protein synthesis was due, for example, to a Na-dependent amino acid transport into the nucleus (Allfrey et al., 1961). On closer investigation many similarities of mitochondrial (Kalf et al., 1959) and nuclear (Frenster et al., 1961) systems to the microsomal incorporation could be established. Incorporation in isolated mitochondrial or nuclear fractions when fortified with the necessary requirements was also found in brain ( Lajtha, unpublished). The nucleoprotein particles possibly present in each of these particulate systems are the ribosomes. Ribonucleoprotein particles with the properties of ribosomes have been isolated in brain from microsomes and have also been isolated from various bacteria, bean and pea seedlings, yeast, ascites cells, liver, pancreas, reticulocytes, appendix, and also from mitochondria and nuclei. Incorporation of amino acids into ribosomal proteins (prepared from microsomes ) has been shown in brain preparations ( Acs et al., 1961).

52

ABEL LAJTHA

Brain microsomes incorporate at a considerably lower rate than do microsomes from liver, but ribosomal preparations from these tissues incorporate at the same rate because brain ribosomes are more active than brain microsomes (Acs et al., 1961). One explanation could be the inhibition by the lipid components of the cerebral microsomes. When free fatty acids were added to cerebral ribosomes the incorporation was inhibited. Since brain microsomes were found to contain free fatty acids which increased upon incubation, it was pointed out that such fatty acid production may be involved in the regulation of protein formation ( Acs et al., 1962).

D. THE MECHANISM OF PROTEIN SYNTHESIS From the works briefly mentioned and from numerous other studies, a picture of protein synthesis starting from amino acids emerged which seems to be the major pathway in those systems that have been studied up to the present time, It is generally considered to consist of several steps, and although not all of the steps can be separated from each other, the mechanism will be artificially subdivided into five parts for the sake of easier visualization, It has to be emphasized that in reality there might be more or there might be fewer steps necessary. Other reactions indirectly necessary, such as the supply of energy, will not be considered here. Starting from amino acids, the polypeptide chain is formed utilizing the energy derived from A", and nucleic acids determine the sequence of amino acids in the peptide chain. First step: amino acid actication (Hoagland et al., 1956; Berg, 1956; Davie et al., 1956; DeMoss et al., 1956; Cole et a,?.,1957; Schweet and Allen, 1958; Preiss et al., 1959; Moldave et al., 1959; Lipman et al., 1959; Allen d al., 1960; Berg, 1961; Bergman et at., 1961; Webster and Lingrel, 1961). The energy required for amino acid activation is supplied by ATF'. The reaction is catalyzed by amino acid activating enzymes that are usually present in the supernatant portion of the cell homogenates, although they have been found in other parts such as the nucleus or mitochondria as well. The enzymes are amino acid specific, i.e., a separate enzyme is involved in the activation of each amino acid. In the reaction the carboxyl group of the amino acid displaces the terminal pyrophosphate of ATP and aminoacyladenylate is formed. As shown in ( l ) ,the resulting amino acid adenylate

PROTEIN' METABOLISM OF THE NERVOUS SYSTEM

AA + A M P - P P + Amino ATP acid

E

$AMP-Ah-E+

Activating enzyme

Amino acid adenylate (enzyme bound)

PP

53 (1 1

pyrophosphate

remains attached to the amino-acid-activating enzyme. The aminoacid-activating enzymes also catalyzes the second step of the synthetic reaction. Amino-acid-activating enzymes have been found in the brain (Lipmann, 1957). More recently, ( Wender and Heerowski, 1962) activation in white matter of 7 of the 14 amino acids studied was established. The activity of the cerebral amino-acid-activating enzymes changed during development, but this did not seem to be the factor responsible for the changes in amino acid composition during development in the proteins of the cerebral white matter (Wender and Waligora, 1961). Second step: transfer t o sRNA (Hoagland et al., 1957; Ogata and Nohara, 1957; Zachau et al., 1958; Hecht et al., 1959; Holley et al., 1960; Allen and Schweet, 1960; Singer and Cantoni, 1960; Dunn et al., 1960; Herbert and Canellakis, 1960; Wong and Moldave, 1960; Lipmann, 1961; Berg et al., 1961; Benzer and Weisblum, 1961; Rendi and Ochoa, 1961). In the second step, the amino acid is transferred from the adenosine monophosphate to a terminal adenosine of a low molecular-weight ribonucleic acid ( sRNA or transfer-RNA) . AA-AMP-E

+ sRNA

A*i- sRNA

+ AMP + E

(2)

In the amino acid adenylate, the bond is an anhydride, joining the carboxyl group of the amino acid and the phosphate group of adenosine monophosphate; in the amino acyl-sRNA the bond is between the carboxyl group of the amino acid and one of the free ribose hydroxyl groups of the terminal sRNA nucleotide. The active amino acid now is an amino acid-RNA ester, and in this step the amino-acid-activating enzyme again becomes free. The sRNA is also amino-acid-specific; a separate sRNA seems to exist for each amino acid, though the separation of separate sRNA's is a difficult task. There is aIso some species-specificity of sRNA. For example, the threonine activating enzyme from calf liver reacts with sRNA from animal sources but not with sRNA from yeast or bacteria. The sRNA is spec& in this way for the particular amino acid adenylateenzyme complex. The end of the sRNA to which the amino acid is

54

ABEL LAJTHA

attached has the same structure in each sRNA: -cytosine-cytosine -adenine, with the amino acid attached to the terminal adenylate. The terminal end-group on the opposite end of each sRNA seems to be guanosine. Since the terminal end to which the amino acid is attached is the same in each sRNA, the specificity, i.e., the part that determines which amino acid will be attached, is dependent on the inner structure of the molecule. The next step of the reaction is the further transfer of the amino acid from the sRNA. The fate of the sRNA is not entirely clear. One possibility is that after entering the ribosome particle it remains attached to it, or at least a part of the sRNA remains attached. If only a part of the sRNA remains attached, then it would be used up in the reaction and would have to be resynthesized. Another possibility is that after this step the sRNA is again liberated in an unchanged form and can react with an amino acid again-thereby the role of sRNA is one of specific catalytic agent. The available evidence favors this latter possibility. The second step confers further specificity of the pathway. Although the first step is fairly specific, it was found that a few aminoacid-activating enzymes activate not only their specific amino acid, but also, although to a much smaller extent, another amino acid. In the second step, however, only the specific amino acid gets transferred. In the second step the amino acid is transferred from one adenine to another, but this time to an RNA of a specific structure. The role of this sRNA is to further transfer the amino acid (hence the better name, transfer-RNA ); because of its specific structure, it can guide the amino acid to its position on the template. The next step, therefore, is the transfer of the amino acid to the template-RNA. Third step: transfer of amino acid-sRNA to the ribosome (Littlefield et al., 1955; Keller and Zamecnik, 1956; Palade and Siekevitz, 1956; Hoagland, 1958; Hoagland et al., 1958; Tissieres, 1959; Hultin and von der Decken, 1959; Nathans and Lipmann, 1960; Takanami and Okamoto, 1960; Kurland, 1960; von der Decken and Hultin, 1960; Huxley and Zubay, 1960; Aboul-Nour and Webster, 1960; Tissieres et al., 1960; Bishop and Schweet, 1961;Dintzis, 1961; Goldstein and Brown, 1961; Nirenberg et al., 1962; Wood and Berg, 1962). The activated amino acid attached to sRNA in this step is assembled into the proper sequence on the ribosomal ribonucleoprotein, probably in a set of reactions in which the exact fate of

PROTEIN METABOLISM OF THE NERVOUS SYSTEM

55

sRNA is not clear. According to the most likely scheme the amino acid would not be transferred from a smaller sRNA to a template RNA, but the peptide bonds would be formed before the full amino acid complement is assembled on the ribosome, and several aminoacyl-sRNA molecules could be present on the template at the same time. In this sense, steps 3 and 4 would be in reality a single step; for further clarity they will be separately discussed here. It is not clear at which stage the amino acid gets detached from the sRNA. It seems that the peptide chain is assembled stepwise by single additions of amino acids starting at the amino end and growing to the carboxyl end. In short-term experiments only the carboxyl end of the newly formed proteins is labeled; with increasing time, the label spreads from this end to the rest of the molecule. An alternate possibility would be that the peptide bond formation starts only after most or all of the amino acid-sRNA residues are attached to the ribosomes. In this case step 3 would be the assembly of the various amino acid-sRNA's on the ribosome, and the linking of the amino acids into the protein would be an additional step. Soluble ribonucleic acid (sRNA) can be obtained free of proteins; its molecular weight is fairly similar for all sRNA's varying from 20,000 to 30,000. It is of interest that it has considerable amounts of unusual bases-including a relatively high content of pseudouridine, which might have something to do with the function of the molecule. The RNA in the ribosome is of considerably higher molecular weight, 106-2 x loGor about 50-100 times sRNA. In mammalian cells the ribosomes are attached to a lipoprotein membrane, which complex constitutes endoplasmic reticulum (or sometimes ergastoplasm). When the tissue is homogenized, the membrane is fragmented, but most of the ribosomes are still attached to it. This broken membrane-ribosome fraction sediments at high centrifugal forces (usually 100,000 g for an hour) and is called the microsomal fraction. The lipoprotein membrane component can be separated from the ribosomal particles in various ways, including extraction with salt, lipid solvents, or detergents. The ribosomes from all sources have a roughly spherical shape with a diameter of 200300 A and are approximately 50%protein and 50%RNA in content. They may contain very small amounts of other substances, such as lipids or amines, that may, however, play an important role. Ribosomes

56

ABEL L A F A

seem to require the presence of low concentrations of magnesium to maintain their structure. By increasing the magnesium levels, two ribosomes can form a dimer particle, and in very low magnesium concentrations each ribosome is reversibly dissociated into two particles, one being two-thirds, and the other one-third, of the original particle. These dissociated particles are not able to incorporate amino acids. The detailed requirements and the components of the system responsible for step 3 are not conipletely clear at the present time. SH components generally are stimulatory, and glutathione may be involved, but these factors may be needed only for the next step. Guanosine triphosphate is a component of the system although the role it plays is not known. There is also evidence that one or more protein fractions (transfer enzymes) may be required for the transfer of aminoacyl-sRNA to the ribosome. Auxiliary reactions. Not in the direct pathway, but necessary to protein synthesis, is the production of all molecules that take part in the reaction. It has been mentioned already that ATP is used up in the activation step; therefore, a fresh supply of ATP is required for the activation of further amino acids. There i s little direct evidence concerning the site of formation of the ribosomal RNA, but indirect evidence points to the nucleus as the primary site. Much of the evidence (e.g., lack of reconstitution of cytoplasmic RNA without the nucleus) is compatible with the alternate explanation, that ribosomal RNA precursors are formed in the nucleus that induce the formation of the proper ribonucleoprotein, with the final assembly of the ribosome or ribosomal RNA occurring in the cytoplasm. Because the genetic information is deposited in the DNA i n the nucleus, this molecule must in some way be the controlling influence on amino acid sequence. The most attractive proposition is that the nuclear DNA participates in the formation of an RXL4which in turn participates in the formation of the protein. Most likely the RNA strand that is the template for the protein is complementary to the D N A strand in its base composition. This RNA then would migrate to the ribosomal particle, and as a messenger of the genetic information would determine the sequence of amino acids in the protein. This newly formed RNA would be ( a ) incorporated into the ribosomal structure to form the protein coding unit, which would

PROTEIN METABOLISM OF THE NERVOUS SYSTEM

57

be relatively stable and capable of forming many protein molecules; or ( b ) loosely bound to a preformed ribosome and would catalyze the formation of one or at the most a few protein molecules before its degradation and replacement. This latter scheme ( b ) would correspond to the “messenger” concept of Jacob and Monod (1961). Further experimental work would be necessary to determine whether the concept of a rapidly turning over messenger-RNA is applicable not only to microbial systems but also to mammalian cells in general and to brain in particular. Fourth step: peptide chain synthesis on the ribosome ( Brachet, 1955; Bonner, 1958; Chantrenne, 1958; Hoagland, 1960; Bishop et al., 1960; Riley et al., 1960; Hoagland and Comly, 1960; Berg, 1961; Hayashi and Spiegelman, 1961; Matthaei and Nirenberg, 1961; Nirenberg and Matthaei, 1961; Dintzis, 1961; Lengyel et al., 1961; Tissieres and Hopkins, 1961; Chamberlin and Berg, 1962; Weisberger, 1962; Martin et al., 1962; Speyer et al., 1962; Ofengand and Haselkom, 196.2). The condensation of the sRNA-amino acids to yield the finished protein, is the least known part of the reaction sequence. If the genetic information originally present in the DNA molecule is transferred to the ribosome in the form of an RNA coding unit, then the base sequences of the template-RNA must determine primary amino acid sequences in proteins. According to present theory (Berg, 1961) , “The process of sequence specification occurs by base-pairing through hydrogen bonding, between the amino-acyl RNA and a complementary segment of RNA, contained within the nucleoprotein particle. Peptide bond formation b e t y e g these specifically oriented amino acids occurs at the expense of the free energy change which accompanies the cleavage of the amino acyl RNA linkage. Folding of the polypeptide chain occurs spontaneously or by an as yet unspecified mechanism.” The problem of determining which nucleotide sequences on the template RNA (and the complementary sequences on the transfer RNA) correspond to a given amino acid constitutes the problem of the determination of the genetic code. If the template is the ribosomal RNA exclusively, as is most likely, then the role of the sRNA would not be to determine the sequence but only to locate and facilitate access to the active site. The strongest influence is probably the primary chemical structure

58

ABET-. LAJTHA

of the messenger-RNA, that is, the base sequence and composition. The base sequence. of the template-RNA, which was determined by the DNA, in turn determines the amino acid sequence of the protein. The most widely accepted theory is that three bases determine the position of a single amino acid, i.e., that the genetic code is a triplet code. If we substitute the first letters as abbreviations for the bases (A, G, U, and C for adenine, guanine, uridine, and cytosine), then the triplet base sequences on the RNA might be such combinations as UUU, UAU, AGU, CCC, AGA, and so on, and each one, or more than one, would correspond to a single amino acid. If UUU corresponds to phenylalanine then template-RNA consisting of U’s only (polyuridylic acid) should catalyze the formation of a peptide chain consisting of phenylalanine only ( polyphenylalanine ) , and if the RNA consists of U’s and A’s then the peptide chain should contain the amino acids corresponding to UAU, UUA, AUU, etc., in the same proportion as the statistical occurrence of each triplet in the RNA. With such methods the coding has recently been established for the amino acids. It is most likely that the growth of the peptide chain occurs through a single addition of the amino acids, i.e., stepwise, and at any moment during the assembly of the protein molecule there will be a partially assembled peptide chain on the ribosome, sRNA-amino acids on their way to this chain, and free or activated amino acids on their way to sRNA. If a labeled amino acid is added after the assembly started (in assembly the first step of amino acid activation or transfer to sRNA must be included too), it is to be expected that the resulting protein will be unequally labeled, since the parts of the molecule that are already assembled will not become labeled. If a labeled amino acid is added when the chain is almost compIeted, it will be incorporated near the carboxyl end, since the assembly starts at the amino end. Uniform distribution of the label is to be expected only if the amino acid w7as added before the assembly of the molecule started and if the amino acid equilibrated with all the pools taking part in the synthesis of the particular protein molecule (which might inchide some sRNA-amino acid complexes). It is possible that part of the already formed protein becomes detached from the ribosome (even though the other part, not completely assembled, remains attached to it) and the assembly of a new protein starts

PROTEIN METABOLISM OF THE NERVOUS SYSTEM

59

at the space liberated; this means that on a single ribosomal RNA molecule two or more protein molecules might be assembled simultaneously. The mechanism of the condensation of the amino acids into the peptide chain is little known. Probably one or more enzymes are involved. Fifth step: release of the newly formed protein from the ribosome (Hultin and Beskow, 1956; Simkin, 1958; Magasnik et al., 1959; Yarmolinsky and de la Haba, 1959; von der Decken and Hultin, 1960; Webster, 1961; Lamfrom, 1961; Hultin et al., 1961; Morris and Schweet, 1961; von Ehrenstein and Lipmann, 1961; Lamborg, 1962). Relatively little is known about the release of the newly synthesized proteins from the site of formation. One of the reasons for the interest in this step is that the regulation of the rate of formation of the individual proteins might occur through inhibition or activation of this step. Obviously, the assembly of the next protein molecule cannot be finished if the previously formed protein is not released from the site of formation, and therefore release might be one of the control mechanisms of the rate of protein metabolism. Release seems to depend on the ionic composition of the medium, and can be influenced by corticoids. It also seems to require an energy-dependent reaction; it is enzyme catalyzed, requires Mg, and ATP seems to be the specific source of energy. The rate of release is also to a certain extent dependent on the conditions of the cells; there is an increased release in liver systems following hepatectomy. Nothing is known at present about the regulating factors of this important step or the physical structure of the protein being released. Summary of steps one to fioe. There seems to be little doubt that, utilizing energy derived from ATP, peptide chain formation can occur through amino acid activation, formation of amino acid adenylates, transfer of the activated amino acids onto a smallermolecular-weight sRNA, assembly of the aminoacyl-sRNA's with the help of the template into the proper amino acid sequence, formation of the peptide chain, and release of the completed molecule from the template. While the transfer-RNA (sRNA) is specific for the amino acid, it is the template-RNA that determines the amino acid chain composition of the protein through its base sequence. This base sequence

60

ABEL L A P A

on the (messenger)RNA is determined, in turn, by the nuclear DNA. In this way aspects of energy metabolism and nucleic acid metabolism are intimately connected with protein metabolism. Difficulties in assessing the in vioo pathways of protein biosynthesis have not all been eliminated. The effect of the homogenization of the tissue with the resulting disorganization of the inbacellular relationships needs to be better understood. This is especially true when the localization of the steps in vivo is discussed, because enzymes attached to membranes might well be solubilized during the preparation of the in uitro test systems. Also the effect of other tissue components such as lipids that are diluted or eliminated during preparations will have to be studied. A number of other difficulties in the interpretation of the results have been discussed already; for example, it has not been demonstrated that in most cases incorporation shows the net synthesis of a well-defined protein. Although evidence for a similar mechanism of protein formation has been reported in each study of the various living organisms, it is not certain if the above is the only pathwav through which protein synthesis occurs. Indications for pathways of protein synthesis which would not go through the amino acid activation step or would not utilize transfer RNA (sRNA) have been reported from several laboratories (Beljanski and Ochoa, 1958; Cohn, 1959; Rendi and Hultin, 1960; Stone and Joshi, 1962). With a11 the above difficulties in mind, many more significant contributions can be expected before all the details of protein biosynthesis are worked out, but is seems that a major pathway has been clarified, in this problem which not so long ago was considered almost unsolvable. VI. Protein Catabolism

While a great deal is known about the mechanism of protein synthesis, few observations have been made on the mechanism of breakdown. The dynamic state of cerebral proteins requires that active swthesis and breakdown be operative in the brain. Processes of protein breakdown might play a role not only in the turnover of proteins, but also in tissue damage and repair, growth and regeneration, pathological states, excitation and exhaustion, regulation of amino acid supply, to name a few. It is obvious that the regulation of protein metabolism might occur partly through the catabolic steps. Growth in a dynamic sys-

PROTEIN METABOLISM OF THE NERVOUS SYSTEM

61

tem can be induced equally well by increasing synthesis or by decreasing breakdown.

A. CEREBRAL PROTEINASES 1. Cathepsins ( Proteinuses Active in the Acidic p H Range) The presence of proteinases in the brain has been known for some time. More recently Kies and Schwimmer (1942) found a high level of catheptic activity in brain which was several times that in muscle. Cathepsins were found in isolated nuclei from cortex ( Richter and Hultin, 1951). Cerebral cathepsin tested with hemoglobin was found to be more active in the gray matter than in white; it had an optimal pH of 3.5 to 3.8 and was not affected by cysteine, glutathione, cyanide, or iodacetate (Ansell and Richter, 1954b). Highest catheptic activity, as tested with autolysis at pH 3.8, was found in the mitochondrial fraction (Lajtha, 1961c) (Table VIII). TABLE VIII PROTEINASE ACTIVITYIN SUBCELLULAR FRACTION OF RATBRAIN'

Whole homogenate Nuclear fraction Mitochondrial fraction Microsomal fraction Supernatant fraction Mitochondrial subfractions I-myelin fragments 11-vesicles 111-mitochondria Nuclear subfractions I-nuclei 11-residue

pH 3 . 8

pH 7 . 8

218 190 350 (1200) 230 (450) 60

125 160 125 (350) 47 (250) 95

130 (250) 260 (600) 620 (4500)

165 (530) 150 (620) 21 (540)

110 130

143 0

Ratio pH 3.8/7.8

0 . 8 (0.5) 1.7 (1) 30 (8)

Protein breakdown is expressed in terms of pmoles amino acid (as glutamic acid) released/gm particulate proteinfiour. Values in parenthesis refer to breakdown in presence of hemoglobin as substrate. Numerals refer to selected subfractions prepared by sucrose gradient centrifugation and are given together with their approximate composition. The ratio of acid to neutral proteinase serves to illustrate the association of pH 3.8 enzyme with mitochondrial subfraction 111. Highest proteinme activity is in the mitochondrial fraction while protein synthesis is most active in the microsomal fraction, the two activities are thus separated spatially. Upon subfractionation of the mitochondrial fraction the neutral proteinases can be separated from the cathepsins (acidic proteinases). From Marks and Lajtha, 1963.

@2

ABEL LAJTHA

The mitochondrial fraction was further separated into subfractions, and the distribution of acid proteinase activity was studied (Marks and Lajtha, 1962). At least some of the catheptic activity in liver was found in particles that under usual circumstances sediment with mitochondria but can be separated from them (DeDuve et al., 1955). Although these particles, called lysosomes, have not yet been isolated from brain (Beaufay et aE., 1957; DeDuve, 1959), histological evidence indicates that they are present in the brain and sediment with the mitochondrial fraction (DeRobertis et d.,1962). About 10 acid hydrolases are concentrated in these particles, and they may have a hydrolytic function. It is clear that enzymes capable of breaking down proteins are not all localized exclusively in one kind of particle. The distribution of a number of proteolytic enzymes was studied in liver and kidney homogenates, among them cathepsin A, cathepsin B, catheptic carboxypeptidase, aminopeptidase, and dipeptide esterase; and it was found that all the enzymes were not contained in the same particulate fractions (cathepsin A was mainly in light mitochondria, carboxypeptidase mainly in heavy mitochondria), The relative distribution in kidney was different from that in liver (Rademaker and Soons, 1957; Hanson et al., 1959). The enzyme content of such particles can be measured only after their disruption, presumably so that the enzymes should be available for the substrates; detergents, by solubilizing the membrane lipoproteins, have such disruptive action on the particles, resulting in maximum enzyme activity (Wattiaux and DeDuve, 1956). Catheptic activity does not show homogeneous distribution in different brain areas, although in each area the “mitochondrial” fraction is the most active. At optimal p H (about 3.8) the activity is high, the initial rate of autolytic activity being 4 9 %proteins solubilized per hour in mitochondria; in an 8 hour incubation as much as 20%of the total brain proteins could be solubilized (Lajtha, 1961~). This very high activity shows that catheptic splitting can be significant even if the pH of the cell is far from 3.8, since a small fraction of the maximal catheptic activity still could be contributing significantly to the rate of breakdown that is required for turnover, or tissue degradation or degeneration. For an average half-life of 10 days, about 0.2%of the brain proteins turn over per hour, which is less than one-tenth of the catheptic activity at p H 3.8. For measuring full activity the particle bound enzyme has to be solubilized, and

PROTEIN METABOLISM OF THE NERVOUS SYSTEM

63

the higher activity under anaerobic conditions or in the presence of SH compounds suggests that the enzymes are sensitive to oxidation (Lajtha, 1 9 6 1 ~ ). A detailed study of catheptic activity in mitochondrial and nuclear subfractions prepared by sucrose gradient centrifugation techniques showed that enzymes were present in most fractions but were associated more with the heavier particles. The highest level of activity was found in the fraction containing true mitochondria and lysosome type structures and lowest activity in the lighest particles consisting of myelin and glial cell fragments; other elements of crude mitochondrial fractions consisting of vesicle and nerve ending structures were intermediate in activity. In nuclear subfractions the residue material sedimenting in 1.0 M sucrose had the highest activity (Marks and Lajtha, 1963). Comparison of acid proteinases in different rat tissue homogenates showed that the level in brain was threefold that in muscle but only half that in liver while kidney and spleen were, respectively, 3 and 5 times that of brain ( Marks and Lajtha, 1963). 2. Peptidases The presence of various peptidases in the brain has been established; the distribution of these enzymes in different brain areas (Pope and Adinsen, 1948) was related to nerve cell bodies. The detailed study of the distribution of dipeptidase activity in the various layers of the cortex (Pope, 1952, 1955,1959) on animal and human material showed that the activity was localized in the neuronal and neuroglial cell bodies. The presence of a polypeptidase which split peptone was found (Ansell and Richter, 1954b); its enzymatic activity ceased after one hour at pH 7.4 under anaerobic conditions, apparently because of the instability of the enzyme. Peptidases show uneven distribution in the brain particulate fractions (Hanson and Tendis, 1954). The substrate specificity of brain peptidase was studied with a number of dipeptides and some higher peptides (Uzman et al., 1961) ; hydrophilic side chains decreased activity, the side chains at a distance from the peptide bond also having influence. The effect of metal ions on peptidase activity has been studied; a number of dipeptidases were activated by Zn, and a Co-activated glycylglycinase was found, while no metal requirement could be shown for tripeptidases (van der Noort and Uzman, 1961;

Uzman et al., 1962), making it likely that cerebral peptidases are similar to peptidases isolated from other sources. The metal activation shows the similarities in behavior between cerebral peptidases and peptidases from other organs. Aminopeptidases, on the other hand, from the nervous system were different in their activationinactivation reactions from such enzymes from other tissues ( Adams and Glenner, 1962). 3. Neutral Proteinases

Protein breakdown in the brain at neutral pH was first observed by Ansell and Richter ( 1 9 5 4 ~ )Examined . under anaerobic conditions activity was unstable and ceased 1-1.5 hours after death; it was not increased b y cysteine or cyanide and was inhibited by iodoacetate and coppkr sulfate. There was 75%more activity in white matter than in gray matter. The initial activity was approximately 0.2%protein split per hour, which is in the range required by the turnover rate of the tissue. A system obtained from brain and liver mitochondria that split added serum albumin was described. Activity was dependent on energy yielding processes and was increased by ATP and coenzyme A and inhibited by microsomes. When albumin labeled with C14phenylalanine was used the major portion of the labeled breakdown product was free phenylalanine, but a small fraction of the label was in a combined form with a nucleotide-type spectrum (Penn, 1960). Coenzyme A also increased the breakdown of endogenous proteins by liver mitochondria. The initial rate of breakdown was about twice as high as would be needed for the in vivo rate of protein turnover in this organ (Penn, 1961). The behavior of breakdown of brain proteins near neutral pH in brain homogenates was found to be similar to such breakdown described in other systems in that it was more active in oxygen atmosphere and was inhibited by anaerobiosis, cyanide, and dinitrophenol, indicating a requirement for energy (Lajtha, 1 9 6 1 ~ )The . study of the distribution of enzyme activity in mitochondria1 and nuclear subfractions could establish that the distribution of the enzyme family of neutral proteinases is different from the acidic proteinases, yielding further evidence for the separateness of the two groups of enzymes. Evidence was also obtained for the heterogeneity of the neutral group of proteinases ( Marks and Lajtha, 1963). Recent evidence has shown that neutraI proteinases, in contrast

PROTEIK METABOLISM OF THE NERVOUS SYSTEM

65

to catheptic activity, are associated with the lighter subfractions of mitochondria and nuclei prepared by the sucrose gradient centrifugation method, The highest activity resided in the lighter particles comprising myelin, glial cell fragments and nerve ending structures, while the lowest activity was found in the fractions containing mitochondria and lysosome-like particles. The range of activity in rat tissue homogenates in the presence of protein substrates showed brain has double the activity of muscle but only 2/3 that of liver; spleen and kidney were respectively 2 and 6-fold that of brain (Marks and Lajtha, 1963). It is likely from the available evidence that, just as several enzymes are contained in the group of acidic proteinases, more than one will be present in the fraction active at neutral pH. Further fractionation and identification of the enzymes has to be accomplished before their biological role can be established.

4. Proteinases in Peripheral Nerve The mechanism of protein breakdown and the enzymes involved are too little known to decide whether any differences exist between the central and the peripheral nervous system. However, enzymes of similar properties to those in brain have been described in nerve. The enzymes that have been studied were a peptidase that splits L-leucylalanine ( McCaman and Robins, 1959) and a neutral proteinase (Porcellati and Curti, 1960) that had similar properties to the enzymes described in brain by Ansell and Richter (1954~).The properties of a peripheral nerve cathepsin have been investigated histochemically and biochemically ( Adams and Tuqan, 1961; Adams and Bayliss, 1961) ; this thermostable, sulfhydryl-dependent enzyme was resistant to some organic solvents and was mainly associated with the lipid and proteolipid fractions of the nerve. This cathepsin appears to be localized in the myelin sheath, although its presence in the axon has not been ruled out. The close association between demyelination and proteinase activity is shown by the parallel increase of neutral proteinase activity in spinal cord and sciatic nerve with demyelination following organo-phosphorus compound poisoning, while no changes in proteinase or in myelination occur under such conditions in the brain (Porcellati et al., 1961).The possible role of myelin proteinases in degeneration will be discussed under Wallerian degeneration (Section VII. D ) . If proteins are formed in the cell body and subsequently migrate

66

ABEL LAJTHA

down the axon (see Section VII, C on axoplasmic flow), then an active mechanism is required in, or in close proximity to, the axon, capable of breaking down the proteins supplied in a continuous How. Information about the localization of these enzymes may shed further light on the mechanism of breakdown and its controlling factors, Aminopeptidases in peripheral nerve were located more in the structures around the axoplasm than in the axoplasm itself ( Aclams and Glenner, 1962).

B. MECHANISMOF BREAKDOWS It is likely that the mechanism of cathepsin action consists of h~drolysisof peptide bonds, resulting in polypeptides that are furtiler broken down to the free amino acid stage through the action of peptidases. No energy seems to be required for this type of breakdown, and anaerobiosis slightly increases activity. Present evidence points to a somewhat different mechanism in the action of at least some of the neutral proteinases. Simpson (1953) found that the release of labeled amino acids from the proteins of liver slices incubated near the physiological p H range was inhibited by anaerobiosis, cyanide, and dinitrophenol. Further support for the requirement for energy in breakdown at neutral pH came from Steinberg and Vaughan (1956) and Steinberg et al., ( 1956), who obtained inhibition of breakdown by amino acid analogs as well as anaerobiosis and dinitrophenol. Fluorophenylalanine inhibited the incorporation of amino acids into brain-slice proteins and also their release from the slices; thus, synthesis and breakdown were equally inhibited. This compound had no effect on autolysis at acidic pH, showing a further difference between cathepsins and neutral proteinases. /3-Thienylalanine, another amino acid analog, was also inhibitory at neutral pH, but S-methylcysteine was inactive. At neutral pH protein breakdown probably occurs not through hydrolysis alone, but with other intermediate steps, at least some of which require energy. There are some difficulties in unequivocal interpretation of the above results. Release of labeled amino acid from a prelabeled protein, one of the methods of estimating breakdown, is usually measured after the addition of large amounts of unlabeled amino acid to “trap” the liberated label, i.e., to prevent its reincorporation. Circumstances that affect the entry of the trapping agent into the

PROTEIN METABOLISM OF THE NERVOUS SYSTEM

67

cell would give the false impression of altered breakdown. Also, interference with the utilization of energy might have nonspecific effects and might alter protein breakdown rates only secondarily. In spite of these and other difficulties of interpretation, one can say that it is likely that protein breakdown at neutral pH is an energy requiring process. The energy might be required for the formation of cofactors or transport of products, or the need for it may mean that the breakdown involves complex intermediates and the acceptor after the splitting of the peptide bond is not water but other substances. One of the possibilities is that breakdown is a reversal of the synthetic steps. The inhibition of breakdown by the same agents that inhibit synthesis points to the close connection of synthesis and breakdown; on the other hand, findings such as that a compounds peptides could be split even though the compound is not a substrate for incorporation (Loftfield et al., 1953) suggest separate mechanisms. This latter proposition seems to be the more likely one. In this area, one of the most important tasks is to establish the relationship of the in vitro breakdown to the mechanism occurring in the living brain. In the schematic representation of protein turnover (Fig. 2 ) (Lajtha, 1961c) it is supposed that, although the pathway of breakdown may be similar to the synthetic steps, it is not a reversal of synthesis but occurs through different intermediates. These intermediates go mostly through the free amino acid stages, although (according to the scheme) turnover may be possible without going through free amino acids. (For a recent discussion of the use of catabolic derivatives other than free amino acids, such as other proteins, for protein synthesis, i.e., exogenous protein as the precursor for cellular proteins, see Walter, 1960 and Loftfield, 1962.) Each step in this scheme might consist of several parts. There is no direct evidence for the identity of the intermediates, but among the possibilities are nucleotide peptides and polynucleotidepeptides. Recently a number of such compounds have been identified from various sources, such as yeasts, algae, bacteria, and fish and mammalian tissue, but it has not been established whether they can be produced by the breakdown of proteins. There is some evidence that they participate in protein synthesis. Such intermediates would clearly show the participation of nucleic acids in protein breakdown. The specificity in protein synthesis is imparted by nucleic acids, the

68

ABEL LAJTHA

amino acid (a.a.) 1

I

i

-

amino acid

1

(activating enzymes)

(peptidases)

Intermediates (peptides)

a.a. adenylate

I

m In

.d

j

5 2

3

'F

-- ---

! (transfer enzymes)

C .d

Q *

-----' ?

'\

I

Intermediates (nucleotide peptides)

I

2

I

a.a. sRNA-template RNA

PI

I

1' (condensing enzyme) peptide chain formation a.a.-a.a. etc. RNA 5

Intermediates

f

\

(releasing enzyme)

Proteins -Proteins

FIG.2. Schematic summary of protein turnover. Left, tentative steps of protein synthesis. Not all enzymes or other participating compounds have been identified, and it is possible that other mechanisms besides the ones pictured here exist. Right, degradation part of the turnover cycle. Nothing is known of the mechanism of steps, the enzymes, and the intermediates participating in this process. Partial breakdown may result in the formation of other kinds of proteins. The location of synthesis may be separate from that of breakdown.

nucleotide peptides isolated till now make a similar role in protein breakdown unlikely at the present time. [For recent references on nucleotide peptides and their possible role see Hoagland ( 1960), Davies et al. (1961), Szafranski and Bagdenanian (196l), Wilken and Hansen ( 1961) ,and Harris and Wiseman ( 1962).] In summary, it can be said that only very scanty evidence is available about the in uico mechanism of protein breakdown at physiological pH. It seems to be more complex than hydrolysis, and it is unlikely that it represents a reversal of the synthetic steps.

PROTEIN METABOLISM OF THE NERVOUS SYSTEM

69

C. THERELATIONOF BREAKDOWN AND FORMATION If a system is in dynamic equilibrium, i.e., has an active turnover with no net increase or decrease in protein content, the rate of overall formation has to be equal to that of breakdown. This will be true for each individual protein species only if in addition there is no change in the level of individual proteins and proteins are not interconverted. If, for example, protein A is not interconverted, then its rates of formation and breakdown have to be equal; if protein A is a precursor of proteins B and C, then the rate of A formation has to be equal to the sums of the rates of breakdown of B and C. In our laboratory (Lajtha, 1961c), a number of experiments were performed to assess the connection of rates of formation and breakdown of cerebral proteins. Labeled amino acids were administered to living animals to label cerebral proteins in vivo, and the subsequent release of label and protein breakdown in vitro was studied. In short-term in vivo experiments, only the protein fractions most active metabolically will be labeled, When brains were incubated in vitro, the breakdown was preferentially but not exclusively of the labeled proteins. For the 15 minutes between administration of the label and killing of the animals, less than 1% of the cerebral proteins became labeled, it was estimated. Upon subsequent in vitro incubation at p H 7.6, six per cent of the protein was broken down and 16% of the label was liberated (Table IX) . The relatively larger liberation of label as compared with protein breakdown shows the preferential breakdown of labeled proteins; however, only a relatively small fraction of those proteins that were rapidly labeled in Vivo were broken down in the subsequent in vitro incubation. Similar results were obtained when the experimental circumstances were varied by: increasing the in vivo labeling time (causing the metabolically slow proteins to become labeled) ; increasing the in vitro incubation time up to 60% breakdown of proteins; using p H 3.8 instead of 7.6 for the in vitro incubations; using newborn instead of adult mice; using mitochondria1preparations instead of whole brain, etc. In each case, a preferential but not complete liberation of the label occurred (Table IX). A preferential splitting of proteins of a higher rate of incorporation (but only slightly greater release of label than splitting of proteins) had been previously observed in liver slices ( Steinberg et al., 1956).

70

ABEL LAJTHA

T.1BLE IX PROTEOLYSIS OF RAPIDLY LABELEDCEREBR.AL PROTEINS FROM RATS" ~

~

Incubat ion Soiirrc

PIT

Time (hours)

Addt Adult Ken-horn Adult Atlult*

3.8 7.6 7.6 3 8 T G

G 2 2 24

3

Protein hydrolyzed (7( total)

Radioactivity liberated (% total)

16 2 3

26 9 13 64 16

58 6

Rats were killed 15 minutes after the intraperitoneal administration of a mixture of labeled amino acids. During this short time mainly the brain proteins of high rates of turnowr should become labeled. Upon subsequent in vitro ineutiation there is a preferential release of label (more radioactivity liberated than protein hjdrolyzed), but all the label is not released under these conditions, that is, d l the proteins that are rapidly labeled in civo are not broken down as rapidly in titro. From Lajthn, 1961~. * 3Iitochondria instead of whole brain incubated in zdtro.

One possible explanation for this finding, still to be confirmed in cico, is that the protein fractions that are labeled most rapidly apparently do not lose their label with die same speed. It could be that a single protein species that is formed on the ribosome can be the precursor of other proteins with varying breakdown rates, and that breakdown occurs at a different part of the cell than does synthesis. Since, according to the present theory, protein synthesis occurs in the ribosomal particle, a subsequent migration of the newly formed protein into other parts of the cell has to follow. Such a movement of freshly formed protein has been obtained in other tissues (Hendler, 1957) and in the nervous system (Droz and Verne, 19S9). Chymotrypsinogen in pancreas is transported after its synthesis in the ribonucleoprotein particles into other compartments of the cell (Siekevitz and Palade, 1960), and Straub (1961) proposed that in this tissue (pancreas) an amylase precursor is formed in the ribosomes which, after being transferred into the mitochondria1 fraction, is changed into amylase. In this way one protein may be converted into other proteins, which are then broken down at times and places different from the synthesis of their precursor.

PROTEIN METABOLISM OF THE NERVOUS SYSTEM

71

A possible example of spatial separation of synthesis from breakdown in the nervous system is the axoplasmic flow (see Section VII. C ) . If proteins in the nerve cell are formed mainly or exclusively in the cell body, subsequently migrate down the axon in a continuous flow, and are degraded along the axon or at the nerve endings, then at least for the axoplasmic proteins the site of formation is clearly separated from the site of breakdown. 1. Alterations in Breakdown Rate The control of breakdown processes is of special importance because it has to be connected with the controlling mechanisms of the synthetic processes. It is obvious that any net change in proteins can be affected by influencing either the anabolic or the catabolic processes, An illustration of this point is the finding of Rittenberg et a2. (1948) that, in regenerating liver, growth is the result not of an increased synthetic rate but of a decreased breakdown. The much lower rate of breakdown in growing organisms as compared with nongrowing ones has been observed in several systems, for example, in microorganisms (Escherichia coli) (Mandelstam, 1957; Borek d al., 1958). Halvorson (1958b) found rate of breakdown at least 23 times lower in growing yeast than in resting cells; the ratio in Bacillus cereus (Urba, 1959) was 5 times lower. A possibility for a somewhat similar situation in the living brain was indicated (Roberts et al., 1959). However, local reutilization of the newly liberated amino acids, in addition to other problems, such as transport, does not make possible an unequivocal interpretation of in vivo results at present. Alterations in breakdown rates as a response to changes in the organisms were shown in a study (Brattgard et al., 1958) in which, during the so-called latent period following nerve crushing, while protein content decreased in the cells belonging to the hypoglossal nucleus, the incorporation of added lysine increased. This indicated that the net decrease of protein was the result of a greater increase in breakdown that outbalanced synthetic rate. A similar case was described in myopathic mice, where the loss of muscle proteins was the result of an approximately twofold increase in rate of protein turnover, with the higher rate of synthesis exceeded by an even higher rate of breakdown-an effect shown in muscle but not in liver (Coleman and Ashworth, 1959; Kruh et al., 1960; Simon et al., 1962).

72

XBEL L.4JTHA

The above examples clearly illustrate the importance of the control of breakdown rates in general protein metabolism. Alterations occur not only during growth but in other states, such as excitation, exhaustion, or disease. Increased proteolytic activity in pathological states as shown by analysis of the spinal fluid has been reported (Chapman and Wolf?, 1958, 1959). Upon prolonged stimulation, in addition to structural changes in proteins, an increase in proteolytic activity of over 100%and protein breakdown were observed (total protein content decreased; acid soluble fraction increased). After shorter periods of stimulation no breakdown occurred but a small increase in proteolytic activity was detected. It was proposed that short stimulation produces onIy codgurational changes in the proteins, and that such partial denaturation may activate proteinases. Renaturation takes place rapidly if the nerve is allowed to rest (Ungar et al., 1957). Although our knowledge of the mechanism of breakdown is far less than that of the synthetic steps, it is clear that the control of breakdown plays an important role in all phases of protein metabolism in health and in disease. It seems likely that the mechanism of breakdown is not a reversal of the synthetic steps, although in some aspects it may be similar. It is clear that the rate of breakdown must be closely related to the rate of synthesis, and in a system in equilibrium the h 7 0 rates have to be equal. Thus, if the two events are spatially separated in a cell, they must be connected through a control mechanism. Processes of growth or degradation show that this control can selectively increase one of the two processes, either synthesis or breakdown, in relation to the other. It might well be, then, that in protein metabolism we deal with 3 separate mechanisms -those of synthesis, of breakdown, and of the control of these two. VII. Protein Metabolism in Nerve

Perhaps mainly because of the difficulty in obtaining axoplasm uncontaminated with material from the surrounding cells, our knowledge of the metabolism of axoplasmic proteins is very scant. From the following it will be clear, however, that problems peculiar to peripheral nerve can be recognized. A. PROTELNS OF PERIPHERAL, NERVE Attempts at isolation and purification of nerve proteins for characterization have been made in several laboratories. From lobster

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nerve, fractions A (Maxfield, 1951) B, and C (Max6eld and Hartley, 1955) have been isolated, and some of their physical properties have been studied. Although fractions B and C were intimately related to nerve, they appeared to originate from blood. From squid giant nerve axoplasm, a fibrous protein has been isolated (Maxfield, 1953) which makes up the axon filaments and constitutes about 10%of the protein of the axoplasm. Physicochemical studies (Schmitt and Geren, 1950; Fernandez-Moran, 1952; Schmitt, 1957; Maxfield and Hartley, 1957), electron microscopy, ultracentrifugation, diffusion, viscosity, electrophoresis, light scattering, and spectrophotometry, showed the filament protein to be highly charged, and have uniform width, indefinite length, and a very high particle weight. The molecule could be dissociated (for review see Schmitt, 1958, 1959). The physiological role of the filaments is not yet known. The electrophoretic separation of proteins has occupied several laboratories (Deuticke et al., 1952; Keil, 1954; Missere et al., 1957; Palladin et al., 1957b; and Poliakova and Kabak, 1958); albumin , and y-globulin of nervous origin could be identified, and and /I-a-, the nerve fractions could be distinguished from those of the brain. The albumin fraction was further purified from sciatic nerve (Li and Sheng, 1957) and a collagen-like protein was isolated from the spinal cord (Roboz et al., 1958). The amino acid content of a protein fraction from lobster nerve has also been analyzed (Koechlin and Parish, 1953).

B. INCORPORATION IN NERVE The investigation of protein metabolism in the peripheral nervous system presents special problems because of the difficulty of separation of neuronal from the nonneuronal elements. The question whether or not protein synthesis occurs in the axoplasm will be discussed below. Even if the axoplasmic proteins originate from the cell body proper it is still likely that their breakdown occurs locally, and at least some of the axonal proteins are active metabolically. After the administration of labeled amino acids, the proteins in the peripheral nerve are labeled too, showing a turnover ( Waelsch, 1958; Brattgard et al., 1958; Lajtha, 1961b). The incorporation is less in the nerve than in the central nervous system, and although turnover rates have not been calculated, nerve proteins show the same heterogeneity metabolically as do brain proteins (there are some nerve proteins with higher and some with lower rate of turnover).

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Nerve proteins show similar decreases in turnover rates as development proceeds (the average turnover rate is higher in the young than in adults), There is rapid incorporation of amino acids in spinal cord and in cerebral white matter, as already discussed. Nerve fragments, which are incubated in vitro, incorporate labeled amino acids from the medium (Takahashi et al., 1961), but whether this is due to synthetic activity in the axoplasm or in its surrounding cells has not been ascertained. It is of interest that in the above study the incorporation in vitro was the same in nerve as it was in cerebral cortex, whereas in the in uivo studies (Waelsch, 1957; Palladin et al., 1957a; Lajtha, 1961b) the incorporation into the central nervous system was the higher.

C. AXOPLASMICFLOW An important question to be answered concerns the localization of the synthetic mechanism in the nerve cell. The question whether proteins are formed exclusively in the cell body and subsequently migrate down the axon, where they are degraded, or whether synthesis occurs in the axoplasm as well, has not been finally decided ( Waelsch and Lajtha, 1961; Weiss, 19611 , A continuous proximodistal growth process in nerve fibers was discovered by FVeiss and his co-workers (Weiss, 1943; Weiss and David, 1943; Weiss and Hiscoe, 1948; Weiss and Cavanaugh, 1959). When a nerve fiber was mildly constricted, the portions distal to the constriction became thinner while the portion between the cell body and the constriction became enlarged by the damming up of substances. If the constriction was removed, the thinner distal portions increased and thicker proximal portions decreased in width until they reached normal dimensions. This was explained as a continuous flow of axoplasm from its site of formation, the perykaryon, which replaces the continuously degraded materials in the axon. A number of observations have been reported since the original discovery which are compatible with such a theory. A proximodistal gradient of phosphoproteins in axons after the administration of labeled phosphate has been found (Samuels et al., 1951), and there was evidence for intra-axonic movements in the distal direction from studying the movements of Pj2 after its injection into the spinal cord (Ochs and Burger, 1958). Destroying cell bodies, or lowering the temperature reduced the flow of P32,indicating the role of an active

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process (axonal “pump”) in the control of the rate of axoplasmic flow (Ochs et al., 1962); there was evidence for a proximodistal movement of P32phospholipids, too (Miani, 1962). An accumulation of synaptic vesicles in the axonal swellings proximal to ligatures was interpreted as compatible with the moving of the vesicles with the axoplasmic flow (Van Breemen et al., 1958). The rate of flow, as will be discussed further, may not be uniform for all the proteins. The average values reported are the following: 1 mm/day (Weiss and Hiscoe, 1948), 3 mm/day (Samuels et al., 1951), 4.5 mm/day (Ochs et al., 1962) and 4-11 mm/day (Koenig, 1958b). Morphological support was derived from the observation that the proximal portion of the Ranvier node region is larger in most nerves than the distal part, perhaps because the narrower and less extensible nodal part creates a normally occurring constriction (Lubinska, 1954). Among enzymes, those of acetylcholine metabolism were studied in the greatest detail. If nerves are sectioned, acetylcholine formation drops to below 5%of normal (Feldberg, 1945; Nachmansohn c?t al., 1946; Banister and Scrase, 1950); since there is no change in acetyl coenzyme A formation, the decrease is due to the disappearance of choline acetylase (Hebb and Waites, 1956; Berry et al., 1958). In the part of the nerve distal to the section the enzyme decreases immediately and reaches 5%of the normal level in a week, whereas in the proximal portion there is an increase in the first few days, then a return to control values for a few weeks (Sawyer, 1946; Hebb and Waites, 1956). The interpretation of these findings was again that the fall in the distal part is caused by blocking of the supply from the cell body, and the increase in the proximal part is caused by accumulation of the continuously produced enzyme when it is no longer utilized ( Hebb, 1957). With lipolytic esterase a similar proximal increase and distal decrease was found, and after reunification of the portions, the highest enzyme activity passed into the upper part of the distal stump (Lumsden, 1952). Upon stimulation, more acetylcholine was lost from cut nerves, since these were deprived of the supply of fresh choline acetylase (Krause, 1955). Not all enzymes have this proximodistal gradient upon sectioning of peripheral nerve. No changes have been reported in pseudocholinesterase (Sawyer, 1946), perhaps because this enzyme is located in the Schwann cells. Acetic thiokinase and cholinekinase did not change (Berry and Rossiter, 1958; Berry et al., 1958).

The behavior of cholinesterase is not clear at present. Dale (1955) postulated that this enzyme is formed in the cell body and is subsequently transported down the axon. Further indications came from observations that during development this enzyme appears first in cell bodies (Lewis and Hughes, 1957) and that its distribution resembles closely that of the Nissl substance (Fukuda and Koelle, 1959), which is perhaps the site of neuronal protein synthesis (Porter, 1953; Palay and Palade, 1955). In a number of normal nerves, without anastomoses and of uniform length, cholinesterase activity decreased linearly with distance from the cell bodies to about 3040%lower than the most proximal part (Lubinska et al., 1962). These authors (Lubinska et aZ., 1961) found that after section and during recovery, the distal part of the axon contained more enzyme than the proximal part, which they interpreted as indicating that enzyme transport to the distal parts continues even when its production in the cell bodies is slowed down or abolished. On the other hand, after irreversible inhibition the recovery of cholinesterase was found to proceed distoproximally, i.e., activity returned first in the distal portions (Clouet and Waelsch, 1961a, b, c ) . The authors point out the complexity of the problem with, among other findings, the one that inhibition of one enzyme may disturb total protein metabolism (Clouet and Waelsch, 1963); they propose that the growth of the cndoplasmic reticulum into the axon may represent the force behind axonal flow. This hypothesis is compatible with the findings after nerve constriction and with distoproximal recovery of enzymes. Lack of evidence for a central to peripheral gradient was also reported in a study in which indications were found that axonal acetylcholinesterase synthesis may proceed independently from that in the cell body. It was proposed that this enzyme may be an exception to axoplasmic flow (Koenig and Koelle, 1960, 1961; Koelle, 1961) . In a study of succinic dehydrogenase, DPN diaphorase, and TPN diaphorase following axonal interruption ( Friede, 1959,1961) ,there was a depletion in the cell body, transport along the axons, accumulation in the terminal swelling at the site of interruption, and a disappearance in the distal part for each enzyme. If a ligature was placed proximal to the section, the accumulation of enzymes occurred at the ligature. Increasing difficulty of axonal flow was shown in thinner fibers by differences in damming at the section of mixed

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fiber populations. There were quantitative differences in the behavior of the three enzymes, which were interpreted as different increases in enzyme transport after section of the nerve. Experiments with labeled amino acids although somewhat more conclusive were by no means decisive. When C14-lysinewas injected into frogs, the sciatic nerve proteins showed a proximodistal gradient of radioactivity in agreement with the concept that the proteins are formed in the cell body (Waelsch, 1958; Lajtha, 1961b) (Table X); TABLE X PROXIMODISTAL GRADIENTOF INCORPORATION IN FROQ NERVEAS SHOWN BY THE PROTEIN SPECIFICACTIVITY~ Time in days

1

8 28 38

690 380 210

Portion of Sciatic Nerveb 2 3 350 320 210

190 240 210

4 170 240 250

Specific activity = counts/mg lysine/minute. Following the intraperitoneal administration of Cl4lysine the label is highest initially in the most proximal portion. This part decreases while the most distal part increases, giving the impression of a flow of the labeled protein in a distal direction. From Waehch, 1958; Lajtha, 1961b. b 1 = most proximal; 4 = most distal. 0

the same conclusion was drawn from autoradiographic experiments with labeled methionine and glycine (Koenig, 195813; Verne and Droz, 1960). When similar experiments were performed with CI4lysine in monkeys, with a mixture of five C14-labeledamino acids in axolotls and chickens, or with C'4-glucose in chicks, the results, although indicative, were not conclusive ( Waelsch, 1958; Weiss, 1961; Lajtha, 1961b). In a study of the metabolic activities of proteins in different areas of monkey brain (Furst et aZ., 1958), although the incorporation of label in white matter was relatively low, the specific activity of the free amino acid was even lower, and therefore the calculated turnover rate was higher than in other parts of the brain. If, however, the specific activity of the free amino acid in other brain areas was taken as the basis for calculation, the more likely lower-than-average turnover rate of white matter resulted. This finding points out the

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possibility of axoplasmic flow in the central nervous system, according to which some of the proteins in white matter may be formed at other areas and migrate into this area later. Many of the studies quoted above corroborate Weiss’ original finding, and only a few observations are in apparent contradiction with it, The finding of incorporation of amino acids into nerve fragments incubated in citro ( Takahashi et d., 1961) would point to the possibility of protein synthesis in the axon, but an alternate explanation may be a synthesis in the surrounding Schwann cells or glia. On the other Iiand, the findings of Clouet and Waelsch (1961a, b, c ) of distoproximal recovery of cholinesterase activity following its irreversible inhibition, of Koenig and Koelle (1960, 1961) of the independence of axonal cholinesterase formation from that in the cell body, and of Friede ( 1959, 1961) of the apparently different flow rates of the three oxidative enzymes studied, demonstrated that the picture may be considerably more complex than a flow of all the contents of the axoplasm at a uniform rate. The contribution of the Schwann cells to axoplasmic metabolism probably cannot be disregarded. There are other possibilities that have to be considered, among them the migration of partially assembled precursors from which products can be locally formed, or the migration of substrates with the synthetic mechanism stationary, or a constant flow of the synthetic mechanism itself, each of which may give a picture similar to that observed. It is possible that large differences will be fouiid with different substances; not only might there be quantitative difkerences in flow rates, but one compound might be formed only locally while others derive from axoplasmic flow.

D. WALLERIAN DEGENERATIOX The great number of important studies on the metabolic changes that occur in nerves during IVallerian degeneration is outside the scope of this paper; only some aspects affecting protein metabolism will be discussed here. Care has to be exercised in the interpretation of the experimental data, especially for the central nervous system. David and Brown (1961) point out that, in the first 2 weeks following a lesion in the central nervous system (cortical undercutting), the uptake of radioactive methionine by proteins in the brain is not a reliable index of the metabolic activity of the tissue. The changes are due to an in-

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creased vascular permeability, an increased penetration of free amino acids, and the presence of considerable amounts of plasma proteins with high specific activities. Specific activity ratios would not circumvent this difficulty. It seems that after section or crushing of the nerve, protein metabolism, synthesis as well as breakdown, is increased. The retrograde degenerated cells of the injured seventh nerve nucleus incorporated more S35-methioninethan the cells in the normal control nerve nucleus per unit area, and the area of the nerve cells in the denervated nucleus was greater 14 days after the section of the facial nerve on one side in rabbits (Fischer et al., 1958).After nerve crush, the nerve cells of the hypoglossal nucleus incorporated more Cl4-lysine than did the control cells (Brattgard et al., 1957; Brattgard et al., 1958). During the latent period the protein content per nerve cell decreased 30-10%, but lysine incorporation was found to increase by 708, showing that there is increased protein synthesis, which, however, cannot compensate for the even greater increase in the breakdown of proteins. In the next stage, 3-12 days after the crush, there was a 100%increase in protein per cell and a 200%increase in lysine incorporation, There were indications that some ribonucleic acids in the chromatolytic nerve cells were transformed from a less active to a more active form. Various times after sciatic nerve section or after nerve crush, the fragments upon incubation incorporated more labeled glycine or leucine than fragments from the intact control nerve. When the proteins were fractionated into 4 proteins ( soluble, salt-extractable, alkali-extractable, and residual), all fractions from the nerve regenerating after crushing incorporated more than the fractions from the control nerve (Takahashi et al., 1961). At least some of the proteinases (cathepsins) were found to be intimately bound to the lipid-protein layers of myelin in peripheral nerve, and it was postulated that this enzyme plays an important part in the process of demyelination because its activity increased during the first few days of Wallerian degeneration, probably through a liberation of the enzyme ( Adams and Tuqan, 1961; Adams and Bayliss, 1961; Adams, 1962). Neutral proteinase is also affected during Wallerian degeneration. By day 15 following the section of sciatic nerve in hens, activity increased to 1%, but by the 21-28th day there was a 50%decrease as compared with control (Porcellati et aZ., 1961). Increase of a

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peptidase has been observed in a study of enzyme activity during Wallerian degeneration (McCaman and Robins, 1959). Of the 12 enzymes studied, five (among them peptidase) increased and two decreased in both the nerves studied (tibia1 and optic nerve in rabbit following transection), while the changes in other enzymes were different in the two nerves. The processes of degeneration and regeneration not only offer great possibilities for studying processes involved in the metabolism of proteins, but also may shed light on the controlling mechanism of protein metabolism in the central as well as in the peripheral nervous system.

E. NERVEGROWTH FACTOR A growth-promoting factor, a protein that is capable of influencing nerve growth, was studied by Levi-Montalcini and her colleagues ( Levi-Montalcini and Hamburger, 1951; Levi-Montalcini and Cohen, 1960; Levi-Montalcini and Booker, 1960b; for a recent summary see Levi-Montalcini and Angeletti, 1961a, b) . More information about such factors may be of great sigdcance, since it may explain some of the mechanisms responsible for the control of protein metabolism. Beyond yielding information about factors participating in the growth, differentiation, and maintenance of certain portions of the nervous system, it may shed light on mechanisms of general biological function. The growth-promoting factor has been isolated from mouse sarcomas, snake venoms, submaxillary salivary glands, connective tissue, and from sympathetic nerve cells from various species (including human material). The biological activities of these factors from the various sources are very similar. In uitro, they all enhance the production of sensory and sympathetic nerve fibers in ganglia explanted from the chick embryo; in uivo, they cause an overgrowth of the sympathetic ganglia (sometimes also of the sensory ganglia), and a hyperinnervation of the viscera. Sympathetic ganglia can increase up to 6 times the volume of control ganglia, because of an increase in both cell number and cell size. No effects were found in other parts of the nervous system or in other organs. The experimental animals appeared as healthy as the control ones. Some of the factors have been purified (Cohen, 1959, 1960). The growth factor is nondialyzable and heat labile. It is inactivated by proteolytic enzyme, but not by ribonuclease treatment, and it has a typical

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protein-like amino acid distribution pattern after acid hydrolysis. It seems to act directly on the nerve cell, and its activity is not associated with enzymes. It increases adenine incorporation into RNA and lysine incorporation into proteins. This lysine incorporation is inhibited by fluorophenylalanine, and the inhibition is reversed by phenylalanine. The factor from mouse submaxillary glands, upon further purification by cellulose column chromatography and paper electrophoresis, yielded two fractions, one dialyzable, the other nondialyzable. The two fractions were inactive when separate but regained activity with recombination ( Schenkein and Bueker, 1962). The most likely explanation for the presence of the factor in salivary glands (and snake venom) is that these glands represent storage depots. The extirpation of the salivary glands of mice did not result in any effect on their sympathetic systems. The factor is probably produced in tissues of mesenchymal origin and is utilized by the sympathetic nerve cells, although its production in the nervous system cannot be ruled out. The specific protein factor also seems to be present in sympathetic nerve cells. The factor is not species-specific; it has similar properties from the different species, and it is found in the same cellular types. One important support for a biological role of these factors, beyond their presence in the living organism, is the effect of an antiserum to the factor (Levi-Montalcini and Booker, 19SOa). The antiserum, prepared by an injection of a purified factor with the Freund adjuvant into the pad of rabbits, had two effects: ( 1 ) it inactivated the factor, since no growth-promoting activity could be detected in the precipitate of the immunoreaction (some of the active antigenic sites may be identical with the growth promoting sites); ( 2 ) the antiserum injected into control newborn animals of different species (mice, rats, rabbits, cats) resulted in a 90 to 99%destruction of the sympathetic nerve cells; in adult mice the destruction amounted to about 60%.There was a slight volume reduction of the sensory ganglia, but no other effects on other organs or structures. The organism showed a tolerance to this treatment in that newborns developed apparently normally and adults showed no adverse effects. It is of interest that with the sixfold increase, or the 99% decrease, in the sympathetic nerve cells, there were not other detectable effects on the animals. The vulnerability of the cells of the ganglia in vim to

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the growth factor antiserum both in newborn and adult animals is good evidence for the significant role of this factor. The control of protein metabolism in all its aspects is a most important problem, It is related to processes of growth, differentiation, and degeneration, as well as maintenance of the cells and their protein complement. The growth factor discussed above may affect one or more of these processes. This factor is specific for a part of the nervous system, but controlling factors of decisive influence may be operative that are specific for other parts of the nervous system, or specific for a cell type in other organs. The knowledge of the mechanism of action of such factors will shed light on the control mechanisms of cellular metabolism. VIII. Conclusions

The great advances made in the past in our knowledge of protein metabolism in general, and of proteins and their metabolism in the ilervous system in particular, constitute but the first step in understanding the role protein metabolism plays in the functioning and malfunctioning of the nervous system. Only a brief survey of some of the most pertinent aspects of the problem was attempted here and many important areas and implications were neglected, especially areas that have been discussed recently elsewhere. The renewal of whole cells occurs at a considerably lower rate in the nervous system than in many other organs; some cells may not be renewed at all, but it seems that at least part of the proteins in most or all of the cells in the nervous system turn over at rates which are commensurate with the metabolic rates of other organs. The rates of protein metabolism are perhaps as heterogeneous as the proteins themselves; there are proteins with very rapid turnover and others that may be stable throughout the life of the organism. The more stable fraction seems to constitute only a minor portion of such organs as the brain. There are differences between the protein composition and protein turnover of the central and peripheral nervous system, also between various cerebral areas, perhaps between cells; each protein may have its own characteristic rate of metabolism. Indeed, there are problems characteristic to a cell type or area, such as axoplasmic flow.

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A great deal has been learned about the mechanism of protein synthesis in recent years, but our knowledge about the mechanism of protein breakdown is very scant. It seems that the mechanism and the site of breakdown are not the same as in synthesis, although controlling mechanisms exist that keep a dynamic equilibrium of the two processes. The metabolic pattern does not seem to be completely stable. It changes during development, the turnover rates in young brain generally being higher than in adult. It may change with the various activities of the functioning nervous system, although some changes may be too subtle to be detected with our presently available methods. REFERENCES Aboul-Nour, B., and Webster, G. C. (1960). Exptl. Cell Research 20, 226. Acs, G., Neidle, A., and Waelsch, H. (1961). Biochim. et Biophys. Actu 50, 403. Acs, G., Neidle, A., and Schneiderman, N. (1962). Biochim. et Biophys. Actu 56, 373. Adams, C, W. M. (1962). In “Neurochemistry” ( K . A. C. Elliott, I. H. Page, and J. H. Quastel, eds.), 2nd ed., p. 85. Charles C Thomas, Springfield, Illinois. Adams, C. W. M., and Bayliss, 0. B. (1961). J . Histochem. and Cytochem. 9, 473. Adams, C. W. M., and Glenner, G. G. (1962). J. Neurochem. 9,233. Adams, C. W. M., and Tuqan, N. A. (1961). J. Neurochem. & 334. Aelony, Y., Logothetis, J., Bart, B., Morrell, F., and Bovis, M. (1962). ExptZ. Neurol. 5, 525. Allen, E. H., and Schweet, R. S. (1960). Biochim. et Biophys. Actu 39> 187. Allen, E. H., and Schweet, R. S. (1962). J. Bid Chem. 237,760. Allen, E. H., Glassman, E., and Schweet, R. S. (1960). 3. BWZ. Chem. 235, 1061. Allfrey, V. G., Mirsky, A. E., and Osawa, S. (1955). Nature 176, 1042. Allfrey, V. G., Meudt, R., Hopkins, J. W., and Mirsky, A. E. (1961). Proc. Nutl. Acud. Sci. U.S . 47, 907. Altman, J. (1962). Science 135, 1127. Amaducci, L. A. (1961). In “Regional Neurochemistry” (S. S. Kety and J. Elkes, eds.), p. 99. Pergamon Press, New York. Amaducci, L. (1962). J . Neurochem. 9, 153. Ansell, G. B., and Richter, D. (1954a). Biochem. J . 57, 72. Ansell, G. B., and Richter, D. (195413). Biochim. et Biophys. Actu 13, 87. Ansell, G. B., and Richter, D. ( 1 9 5 4 ~ )Biochim. . et Biophys. Actu 13, 92. Appel, K. R., Appel, E., and Maurer, W. (1960). Biochem. 2. 332, 293. Askonas, B. A,, Campbell, P. N., Godin, C., and Work, T. S. (1955). Biochem. J . 61, 105.

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PATTERNS OF MUSCULAR INNERVATION IN THE LOWER CHORDATES By Quentin Bone

The Marine Biological Association, Citadel Hill, Plymouth, England

I. Introduction . . . . . . . . . . . . . . . . 99 A. The Pattern of Muscular Innervation in Frog and Mammal . . 100 B. The Swimming Muscles of Fishes . . . . . . . . . 105 C. The Muscle Fibers of the Myotomes . . . . . . . . 106 11. Motor and Sensory Innervation of Myotomal Muscles of Lower Chordate Groups . . . . . . . . . . . . . . . 108 A. Acrania . . . . . . . . . . . . . . . . 108 B. Agnatha . . . . . . . . . . . . . . . . 110 C. Elasmobranchs . . . . . . . . . . . . . . 116 D. Various Osteichthyes, Excluding Teleosts . . . . . . . 127 E. Teleosts . . . . . . . . . . . . . . . . 128 111. Discussion . . . . . . . . . . . . . . . . 133 A. Functional Significance of the Dual Motor System in Fishes . . 134 B. Mode of Action of the Two Systems, and Gradation of Contracture in Fast and Slow Motor Systems . . . . . . . . . 135 C. The Problem of Proprioception in Fishes . . . . . . . 139 D. Concluding Remarks . . . . . . . . . . . . . 140 References . . . . . . . . . . . . . . . . 141

I. Introduction

Our picture of the innervation of vertebrate striated muscle is almost entirely derived from mammals and amphibia (see review articles by Hinsey, 1934; Tiegs, 1953; Couteaux, 1960; and for the frog, Gray, 1957). Only a very small proportion of the work mentioned in these reviews is concerned with fish, reptiles, or birds. Recently (Barets, 1961), an admirable review dealing with the innervation of teleost muscle has appeared, but so far as the other groups of lower chordates are concerned, there is little or no recent information except as regards the Agnatha, where a number of 99

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workers have studied the histology and physiology of the muscular innervation. The present review discusses the innervation of the swimming muscles of different groups of fish in the light of this recent work, and of unpublished observations upon elasmobranchs, lungfish, and Pdgpterus. First of all, the pattern of muscular innervation in the frog and the mammal may be recalled briefly. A. THE

PATTERN OF h4UsCULAR

INNERVATIOS IN FROG AND MAMMAL

In the frog and toad, there are two histologically distinct types of muscle fiber in the striated muscles (Gray, 1958; Peachey and Huxley, 1962), which receive a different pattern of motor innervation. The end-formations upon the two types of muscle fiber are entirely distinct histologically (Gray, 1957), and the muscle fibers themselves have different physiological properties, Tasaki, 1942; Tasaki and Mizutani, 1945; Tasaki and Tsukagoshi, 1945; Kuffler et al., 1917; Kuffler and Vaughan Illilliams, 1953a, b ) . One type of muscle fiber, innervated by fine axons ending in a characteristic manner, is polyneuronally innervated, does not conduct muscle action potentials, and gives tonic responses. The second type, supplied by thicker axons, which end in the so-called Buissons of Kiihne, shows typical propagated muscle action potentials and gives phasic responses. The two types form, respectively, the slow and fast motor systems, the former being apparently concerned with postural activity, the latter with rapid locomotory movements. In frogs, the slow muscle fibers are not different in color from the fast fibers, but in toads (Stendel, 1959) the slow fibers are red, the fast fibers white. A few of the slow muscle fibers resemble the fast fibers in that they conduct propagated muscle action potentials (Burke and Ginsborg, 1956; Shamarina, 1962), but the histology and innervation of this possible third class of muscle fiber in amphibia has not yet been investigated. A recent study by Orkand (1963) does not support Shamarina's observations. In mammals, the situation is more complicated than in amphibians, and it is difficult to summarize shortly, since there is at present some conflict of opinion between different workers. There is a dual motor system in mammals, as there is in the frog, but the distinction between the two types of muscle fiber and their patterns of inner-

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vation is less clear cut than it is in frog. Typical mammalian slow tonic fibers are red (Ranvier, 1873; Denny-Brown, 1929), and in transverse section show a characteristic “Feldenstruktur” (areal pattern) of myofibrils (Kruger, 1929, 1952). In these and other characters, they contrast with the typical fast phasic muscle fibers which are white (since they are poor in myoglobin), and show a “Fibrillenstruktur” ( fibrillar pattern) in section. Both types of muscle fiber are twitch fibers, propagating muscle action potentials [thus resembling the slow and fast fibers of birds (Ginsborg, 1960)l; this is a very different situation from that found in amphibia. It has been suggested by Haggqvist (1938) and by Kriiger (1960) that, on the slow tonic fibers, there are end-formations of the “en grappe” ( traubenformigen Endigungen ) type, while upon the fast phasic fibers there are end-formations of the “en plaque” ( Endplatten) type. These two types of end-formation are derived from axons of different diameter, the smaller supplying the tonic muscle fibers (Haggqvist, 1938, 1959). It has been found in conformity with this, that the axons supplying the tonic muscle fibers are slower than those supplying the phasic muscle fibers (Eccles et at., 1958; Steg, 1962). Haggqvist ( 1959, 1960, 1962) extended his observations upon the two types of end-formation upon the slow and the fast fibers, by providing evidence for an enzymatic differentiation between the two. He found that, at the “en grappe” endings upon the tonic fibers, there was butyrylcholinesterase (but see Csillik, 1961), while at the “en plaque” endings upon the phasic fibers there was acetylcholinesterase. Although other workers have not accepted that there is either an enzymatic or an histological dichotomy of end-formations in mammalian muscle (for reasons which will be adduced below), several other lines of evidence suggest a duality of motor innervation in the mammal. A number of wurkers (Granit d aZ., 1956; Granit et aE., 1957; Eccles et al., 1957, 1958) have provided evidence for a distinction between “phasic” and “tonic” a-motoneurons in the spinal cord, supplying fast and slow muscle fibers, respectively, Buller et al. (1958, 1960) have further shown by cross-union experiments that the type of motor supply determines the physiological characteristics of the muscle fiber, for by such experiments, slow muscle fibers could be converted to fast muscle fibers. Giacobini (1959) and Giacobini

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and Holmstedt (190) have shown that there are two types of motoneuron (presumably equivalent to the phasic and tonic types of the electrophysiologists) which can be recognized by their content of acetylcholinesterase. Those with a higher acetylcholinesterase activity were found (in rat) to supply extensor muscles. In the muscles themselves, Kawakami (1954a, b ) has shown that there are slow and fast types of neuromotor units (his “tonic” and “kinetic”), as had been assumed by Tasaki and Tsukagoshi and by Eccles et al. A similar conclusion was suggested by Norris and Gasteiger’s (1955) observations of spike amplitudes in human muscles. It has been shown that the neuromuscular junctions upon tonic and phasic muscle fibers differ in their responses to various drugs (e.g., Jewel1 and Zaimis, 1954). The work summarized above, all suggests a duality of motor innervation in mammalian muscle, but it is by no means certain that the division between slow and fast fibers, and their innervation is as simple as it is in the frog, As Austin (1962) remarks in another connection, “it is essential, here as elsewhere, to abandon old habits of Gleichschaltung, the deeply ingrained worship of tidy-looking dichotomies.” With regard to the enzymatic division between tonic and phasic neuromuscular junctions, a number of workers have reported both types of enzyme at the same neuromuscular junction. Thus, Denz (1953, 1954) found both enzymes on the fibers of the rat diaphragm; Holmstedt (1957) found both at the endplates upon fibers of biceps fernoris in cat; and Giacobini and Holmstedt (1960) apparently found both types in other muscles of the cat. PecotDechavassine (1961) has found both types at the neuromuscular junctions upon fast fibers in the frog. It is worthy of note also that Giacobini and Holmstedt found acetylcholinesterase ( and little butyrylcholinesterase ) in both of their a-motoneuron types in the rat spinal cord (i.e., the enzymatic division between motoneurons which was perhaps impIied by Ilaggqvist’s observations does not seem to occur). It would be of much interest to see investigations along the lines of those carried out by Haggqvist, upon the rat-tail muscle preparation discovered bv Steg, such a preparation seems ideally suited to provide a conciusive decision on this matter. At present, most workers would seem to support the view that acetylcholine is the transmitter substance at the neuromuscular junctions on both phasic and tonic fibers ( Koelle, personal communication, 1963).

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With regard to the muscle types, it seems that there are intermediate types of fiber between the classical ‘%eldenstruktur” type and the classical “Fibrillenstruktur” type ( see Peachey, 1961a; Boyd, 1962); it is not yet possible to harmonize the histology of these intermediate fiber types with their physiological characteristics. Granit et al. (1956) point out that the division between phasic and tonic motoneurons is not an absolute one, i.e., there are also intermediate types of neuron. It therefore appears that in the mammal it is not possible to divide striated muscle fibers into a single fast phasic type and a single slow tonic type, different in kind; the two types differ in degree, and there are intermediate types. The protagonists of the enzymatic and histological differentiation of two types of motor endformation seem to have ignored this point, and their conclusions, though of great interest, must therefore be accepted with reserve, as yet, until further investigations have been performed. One point of importance which has not so far been considered, is that a proportion (sometimes a large proportion) of the muscle fibers in any given muscle may receive more than one end-formation, the different end-formations being in some cases derived from different motoneurons (Hunt and Kuffler, 1954; Kupfer, 1960). Muscle fibers polyneuronally innervated in this way bear some resemblance to the slow muscle fibers of birds which are innervated at several points along the fiber (Ginsborg and MacKay, 1960). It will be seen later that there is a good deal of uncertainty whether or not some types of muscle fiber in lower chordates are polyneuronally innervated. As in the case of the mammal, electrophysiological investigation is more likely to lead to a definite answer than histological investigations. The observations discussed briefly above have been made upon extrafusal muscle fibers. The type and innervation of intrafusal muscle fibers in mammals and amphibia are at present the subject of dispute. On the one hand, there is good physiological evidence for two types of muscle fiber in toad spindles (Eyzaguirre, 1957), while only one type has yet been demonstrated physiologically in the mammalian spindle (Eyzaguirre, 1960a, b; but see footnote in Boyd, (1962). The intrafusal fibers of the mammalian spindle studied by Eyzaguirre were found to conduct propagated action potentials, but it was not excluded that tonic fibers (which did not conduct action potentials) might also be present. The work of Jansen and

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Matthews (1962) on the dynamic responses of cat spindles supports the view that there are two types of intrafusal muscle fiber in the rnammalian spindle. On the other hand, it has been variously suggested on histological grounds that mammalian intrafusal fibers are of: ( a ) two types (Boyd, 1958, 1962; Haggqvist, 1961; Kupfer, 1960; Hess, 1961); ( b ) fast or slow type depending upon the type of the extrafusal fibers among which the spindle lies (Kriiger, 1960); ( C ) a single type not readily equated with either of the types of extrafusal fiber (Barker, 1961). These possibilities are discussed by Boyd, but the discussion in Barker (p. 271) should be consulted, and also the recent contribution by Bridgman et al. ( 1962). Histological findings upon the innervation of the two suggested types of intrafusal fiber have also been conflicting. Although it does secm that the weight of the evidence is that there are two types of v-eff erent innervation supplying two types of muscle fiber, there is not complete agreement that this is the case. Confirmation has yet to be obtained for Haggqvist’s claim for an enzymatic differentiation of the two types of innervation. Boyd finds that there are probably several different types of muscle fiber within the general framework of his nuclear-bag and nuclear-chain divisions and, as when considering the extrafusal fibers, it is necessary to recall Austin’s injunction. The most striking feature of the sensory innervation of striated muscle in the frog and mammal is the richness of the sensory terminations associated with the muscle. Indeed, the muscles of these animals are highly developed sensory organs, rivalling in complexity the organs of special sense, as Ranvier (1878) recognized when he spoke of the striated muscle fiber as “l’Csclave du systAme nerveux central.” Lambertini (1956) also emphasizes this point, and reviewing the sensory innervation of mammalian muscles writes “il muscolo, compenetrato di recettori nervosi, e dunque anche un organo di senso.” A number of morphologically different sensory endings have been described from mammalian muscle (see review by Cooper, 1960); of these, the most studied have been the neuromuscular spindles and the Golgi tendon organs. Since the first recognition of the spindles as sensory structures (Ramon y Cajal, 1888, in the frog; R~iffini,1892, 1898; Sherrington, 1894, in mammals) there has been a large body of work dealing with spindle structure and function (see, e.g., reviews by Granit, 1955; Cooper, 1960; and in Barker, 1961). The significance of the motor innervation of the

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spindle has become clearer, and it is now seen that neuromuscular spindles are highly complicated organs, playing an extremely important role in the gradation and initiation of muscular contraction, together with the Golgi tendon organs (Golgi, 1880). It is not surprising that preventing feedback from the musculature by the cutting of all dorsal roots should stop locomotor activity (Gray and Lissmann, 1940, 1946). Sensory terminations of one kind or another are found associated with the striated musculature of all terrestrial vertebrates; neuromuscular spindles are found in amphibia, reptiles, and birds, as well as in mammals (see Barker, 1958). The pattern of motor and sensory innervation of striated musculature in the frog and mammal, briefly described above, is somewhat like that found in fish, but there are important differences in the innervation of fish muscle from that of the frog and mammal. In the present review, attention is chiefly directed to the innervation of the swimming muscles, for it is these muscles which have been most studied, and which are comparable in the different fish groups. Before discussing the innervation of these muscles, it is necessary first to consider briefly the way in which they are arranged in fishes.

B. THE SWIMMING Musms OF FISHES All the various fishes swim in essentially the same manner by oscillating a flexible tail, thereby producing a back-thrust upon the water, and so driving the fish forwards. Many studies have dealt with the hydrodynamic and mechanical aspects of this process (e.g., Chevrel, 1913; Gray, 1933a, b, 1953; Taylor, 1952; Bainbridge, 196l), and in general terms, the mechanics of the process are fairly clear. Detailed analysis is for various reasons, exceptionally difficult, but for our purpose it is sufficient to notice that, with few exceptions, the swimming pattern is similar in all the lower chordates. The exceptions are the fishes which use one or another of the sets of fins to provide the propulsive force, as for example the flattened rays or the more specialized teleosts such as the sea-horse (Hippocamps).It is not surprising that the method of swimming should be identical in all the lower chordates (although these are animals of very different dimensions, and radically different internal organization), for the way in which they all swim is certainly the result of the conditions imposed by the aquatic medium, as Gray (1953) emphasizes. First, the density of the animal's body approaches that of water. Many teleosts have achieved neutral buoyancy [as some

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elasmobranchs have very nearly ( Harrison-Matthews and Parker, 1950; Corner et uZ., 1963)], but even where the fish is slightly heavier than water, the effect of gravity upon it is much less than that upon a land animal. Second, the resistance of the medium is of great importance, whereas in land animals, except during very high speed movements, this is of little importance. Conditions such as these have determined the selection of a more or less streamlined form in all the lower chordate groups and have determined the way in which they swim. It is no accident that ichthyosaurs resembled fish in form and in the way in which they swam. When one or another of the sets of fins are used for propulsion, other considerations may apply (as in many of the more specialized teleosts ) . However, the more primitive teleosts such as the salmonids and clupeids swim by oscillating the tail, and are typically “fish-shaped.” It would seem that this swimming method appeared early in chordate evolution and, except in some eIasmobranchs and teleosts, has remained throughout the aquatic gnathostonies. The power for oscillating the tail is provided by the serial contraction of the segmented blocks of myotomal muscle along the trunk and tail. It is with the innervation of the fibers of the myotomes that we are concerned. Each myotome is separated from its adjacent neighbors by a connective tissue myoseptum (myocomma ) upon which the muscle fibers of the myotomes insert. The muscle fibers run roughly parallel to the main axis of the animal, but since the myosepta are-attached obliquely to the axial skeleton ( notochord or vertebral column), their contraction is transmitted to the axial skeleton and not to neighboring myotomes (see Nursall, 1956). The myotomes are complicated in shape; from a simple series of roughly rectangular blocks, they become folded as ontogeny proceeds-in side view appearing V- or W-shaped (see Maurer, 1913). Not only are they folded, but they come to lie one upon another to form a series of overlapping cones; a section through the tail of a fish, tlierefore, will show parts of several myotomes. The functional aspects of the shape and arrangement of the myotomes have been considered by Nursall (1956); Willemse ( 1959) and Jannan ( 1961). OF THE MYOTOMES C. THEMUSCLEFIBERS

It was early discovered that the muscle fibers in the myotomes of various groups of fishes were two M e r e n t sorts ( a t least), for,

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as in mammals (Lorenzini, 1678), two sorts of fibers are usually differentiated by their color. Thus, Agassiz and Vogt (1845) observed red muscle in the salmon; Arloing and Lavocat (1875) described red and white muscle fibers in a number of different teleosts; Stirling (1885) figured red and white muscle fibers in the herring, haddock, and whiting; Low (1813, but writing before 1795) mentions that the flesh of the basking shark (Cetmhinus)is partly like halibut and partly like beef; Ranvier (1873) found red and white muscle fibers in the ray as did Arloing and Lavocat (1875); and Daniel (1922) described red and white muscle from the myotomes of other elasmobranchs. In other lower chordates, it was found that the muscle fibers differed in their histology although their color might be similar. Stannius (1851,1854) found two different types of muscle fiber in lampreys, as did Grenacher (1867); the latter author also found them in hagfish-an observation confirmed by Maurer ( 1894). The two types of muscle fiber found in the myotomes of different fishes differed from one another in such characters as color, size, myogbrillar array (Lansimaki, 1910), and so forth, much as do the slow and fast muscle fibers of mammals. In some groups, however, the distinction between the two types of fiber seemed to be an absolute one, as in amphibia. In lampreys, for example, one type of muscle fiber was found to be thin, circular in section, and relatively scarce in the myotome, whereas the second, more abundant type was extremely large and, being flattened and sheet-like, was in transverse section much like the leaf of a book (see also Peters and Mackay, 1961) . Ranvier investigated the responses to stimulation of the red and white muscle fibers in rays, and showed that these were distinctly different. His pioneer investigations were not extended until Takeuchi (1959) studied the red and white muscle fibers of the fin musculature in teleosts. The earlier writers upon the motor and sensory innervation of the myotomal muscle fibers, however, (for example, Ciaccio, 1877; Retzius, 1892; Giacomini, 1898a,b; and Supino, 1898) did not appreciate that the myotomal muscles which they were studying contained two different types of muscle fiber which, as in the frog, are innervated by separate motor systems with different kinds of end-formation upon each type of muscle fiber. There was, therefore, a certain amount of disagreement between the different authors as to their findings, and a good deal of difficulty in deciding which

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endings were motor and which, if any, were sensory. Thus, Giammini described endings which we now know to be motor, as sensory, for he observed in the same muscles other more characteristic motor terminations upon a different type of muscle fiber. The investigations at the turn of the century suggested that in teleost and elasmobranch myotomal muscles, at any rate, there were abundant sensory endings of a peculiar type, and a motor innervation not unlike that of the mammal. Little more work was done during the next five decades, and it is only recently that the innervation of the myotomal musculature of the lower chordates has been reinvestigated, with the result that we now possess a fairly good idea of the general pattern of myotomal innervation in the various groups and also that some progress has been made in understanding the function of this system. Most of this recent work has been concerned with teleosts (e.g., Barets, 1953, 1961; Takeuchi, 1959; Pecot-Dechavassine, 1961) , and with cyclostomes (Jansen and Andersen, 1960; Peters and hilackay, 1961; Andersen et al., 1962); there is still little or no information upon the innervation of the muscles of the other lower chordate groups. The state of prescnt knowledge is considered in the following sections. II. Motor and Sensory Innervation of Myotornal Muscles of lower Chordate Groups

h.

AaUNIA

The Acrania are the most primitive living chordates and, for this reason, have long attracted the attention of histologists. Their small size (adult amphioxus is some 23 inches long) has perhaps led to their neglect by physiologists, though there is much need for physiological investigation of many of the points raised by the histology of the neuromuscular system. It was early shown by Grenacher (1867) that the muscle fibers in the amphioxus myotome are quite exceptional in form, for they are flattened sheets, some 1p thick and 12 ,u wide, the whole myotome being built up of stacks of these sheets. As far as is known, all the muscle fibers of the myotome are of this peculiar kind, and there have been no reports of a second type of muscle fiber in the acroniate myotome. In this respect, as in the shape of the muscle fibers, the acraniates are unlike other lower chordates.

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There is some disagreement upon the mode of innervation of these curious muscle fibers. It was initially suggested by Rhode (1888) that fine nerve fibers passed out of the ventral roots, to merge eventually with the substance of the muscle fiber. Ayers (1921) seems to have held this view. Retzius (1892) figured branching varicose fibers among the myotomal muscles, but it was van Wijhe ( 1893) who first observed the characteristic spade-shaped end-formations on the muscle fibers, an observation later confirmed by Heymans and van der Stricht (1898), Dogie1 (1903), Boeke (1908), and Kutchin (1913). Boeke’s account is the most detailed. This kind of end-formation is extremely abundant in the myotome, the impression being that each muscle plate has upon it a single end-formation which may be near the middle of the fiber or towards either end. The cells of origin of the large axons passing to these spade-shaped end-formations are arranged in the spinal cord in small segmental groups. The axons branch within the spinal cord before issuing from the ventral root (branching of the ventral root fibers is rare in the myotome itself) , it appears that each motoneuron supplies a large number of muscle fibers (Bone, 1960). In addition to these spade-shaped end-formations, Boeke (1908) described other terminations derived from fine nerve fibers, which he regarded as sensory, A special bundle of fine nerve fibers has been found within the ventral root nerves (Ayers, 1921; Bone, 1960, Fig. 47). These may perhaps connect with this second type of endformation observed by Boeke. Ayers (1921), who first described the fine fiber component of the ventral root nerve, regarded it as “sympathetic,” destined for the walls of blood vessels, The discovery of the atrial nervous system (Holmes, 1953; Bone, 1961)has disposed of this view; in fact, these fine nerve fibers almost certainly pass to the myotome. If they in fact connect with the endings described by Boeke (which have not been observed by other workers), either these are sensory (in which case it is rather surprising that they emerge from ventral roots and that there are so many of them-some 200300 fine fibers are visible with the light microscope in each ventral root) or they are motor, supplying an as yet undescribed second type of muscle fiber in the myotome. Peachey (1961b) has studied the myotomal muscles with the electron microscope and does not report a second type of mus-

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cle fiber, but it is conceivable that it might have escaped observation. With the light microscope, all the muscle fibers of the myotome appear to be of the same type. It is worthy of note, however, that the endings found by Boeke (1908) lie near the surface of the myotome, i.e., where if there are other sorts of muscle fibers, it would be expected that they would be found by analogy with other lower chordates ( see below). The swimming of amphioxus has been described by various authors (e.g., Couch, 1865; Franz, 1923; Ten Cate, 1938a, b); all agree that it is rapidly exhausted, which would suggest that there is only one type of muscle fiber in the myotome. At present, we cannot decide between the alternatives mentioned above, and further investigation is required before the pattern of myotomal innervation in acraniates is understood. Cholinesterase investigations have not ,@en much result with amphioxus as yet (Mackay and Peters, 1961), except to show that the so-called myotendinous reaction is apparently absent in the myotomes. The significance of this observation is not clear, for it is not known why cholinesterase should be present at the myotendinous junctions of higher forms (see Gerebtzoff, 1956; Schwarzacher, 1961). On the whole, the pattern of innervation of the amphioxus myotome, insofar as it is known, is not very like that in craniates; it is with the next group of lower chordates, the Agnatha, that the craniate pattern, albeit in a specialized form, is first found.

B. AC.S.~THA Lampreys and hagfish have a very peculiar arrangement of muscle fibers in their myotomes, not found in any other group. There are two types of muscle fiber (Flood and Storm Mathisen (1962) have provided some evidence for the existence of a third type of muscle fiber in the hagfish myotome) which are arrayed in muscle units or, as Stannius called them, “Muskelkastchen.” These units were described by Stannius ( 1851, 1854), Grenacher (1867), Maurer (1913), and by Cole (1907). In lampreys, the units are made up of some sheet-like large muscle fibers extending the whole length and breadth of the myotome (which may be termed central fibers, following Grenacher ) , surrounded except on the inner face by a single row of thin, more or less circular, parietal fibers. The myotome is built up of a series of such units stacked one on top of another (as is seen in Fig. 2 ) .

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In myxinoids, the arrangement is essentially the same, but the thick central (150-2OOp) fibers are not flattened and sheet-like, being similar in shape to the thinner (50-70 p ) , less abundant parietal fibers (Fig. 1). The situation is simpler in Myxine than it is

FIG.1. Diagram of innervation and arrangement of the two types of muscle fiber in the myotome of Myzine (hagfish). In this, and other diagrams in the figures, no attempt has been made to indicate the complex folding of the myotomes actually found; myotomes are here represented as simple cubes.

in petromyzonts, so we shall consider Myxine first. The two types of muscle fiber in Myxine differ not only in size, abundance, and capillary supply (Cole, 1907), but also in the amount of mitochondria they contain ( Andersen, personal communication, 1961) , and most significantly, in their motor innervation. The innervation of the myotomal musculature of Myxine was first described by Retzius (1892), who figured large basket-like endings of thick nerve fibers on or near the myoseptal ends of some of the muscle fibers and also thinner axons running along some other muscle fibers. He did not specify which ending lay upon which type of muscle fiber and, indeed, figured two instances of an axon branching to give rise to two endings of Werent type. This was an oversight, for the two

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motor systems are quite separate (Anderson et al., 1963; Bone, 1963a). The basket-like endings are found upon the thicker central fibers, while the smaller parietal muscle fibers are supplied by thin axons passing along them. The motor axons emerge from the large bundle which (in the ventral portion of the myotome) runs down its medial face at the level of the myoseptum, and then pass to the muscle fibers of the two adjacent myotomes. Each parietal muscle fiber is innervated by two thin s o n s passing onto the fiber at each of its myoseptal ends, so that the single parietal fiber is supplied by two Werent axons, emerging from the cord through two different ventral roots. It at first appeared that the central fibers were innervated also by two separate axons, that is, that upon either myosepta1 end of a central fiber, there was a basket-like end-formation. This was the view of Jansen and Andersen (1960),but later work led them to conclude (upon physiological grounds) that the central fibers were innervated only at one of their myoseptal ends (Andersen et al., 1963). Support for this conclusion has come from histological observations using cholinesterase techniques (Storm Mathisen, personal communication, 1962) and methylene blue staining (writer’s observations). Previous workers upon muscle fibers [in other lower chordate groups, and in amphibia (Mackay and Peters, 1961)]which are innervated in a similar manner to the central fibers of Myxine, have concluded that such fibers are innervated by basket-like terminations at both myoseptal ends. This point will be discussed further in a later section. The axons supplying the central and parietal muscle fibers in one Myxine myotome also pass to similar fibers of the adjacent myotome (as shown in Fig. 1);each axon probably supplies some 15 muscle fibers in the two adjacent myotomes. Thus, excitation of muscle fibers in one myotome will be accompanied by excitation of at least some muscle fibers in the two neighboring myotomes. The physiological properties of the two types of muscle fiber in Myxine, were considered by Cole (1907), who concluded that the central fibers were probably concerned with sluggish contractions, the parietal fibers with rapid contractions. In fact, as Jansen and Andersen (1960),and Andersen et al. (1963) have shown, the reverse is the case. The thick muscle fibers conduct action potentials and give typical twitch responses, while the thinner parietal fibers do not

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conduct action potentials, and are typical phasic fibers. As these workers point out, their results suggest that the two different types of muscle fiber in Myxine have properties corresponding to the fast and slow muscle fibers of the frog. It is, however, difficult to think of the “slow” parietal fiber system in Myxine as concerned with the maintenance of posture, as it is supposed to be in the amphibian. In other lower chordates, as will be seen, it is very probable that the similar systems of “slow” muscle fibers are concerned with a particular type of sustained swimming pattern, but we do not know if this type of swimming is shown by Myxine. In captivity, at any rate, hagfish lie on the bottom of their container and rarely move unless disturbed or stimulated by the presence of food particles in the water near the tentacles. Myxine is of interest because it is the only lower chordate (with the exception of the elasmobranchs) in which presumably proprioceptive terminations have been found associated with the myotomal musculature (Bone, 1963a). Fibers which may in same instances be traced to their origin in the dorsal root ganglia, end in branching terminations just under the connective tissue sheet covering the visceral face of the myotome, at the level of the myoseptum. Presumably such endings will respond to the strain set up in the connective tissue as the myotomes contract during swimming. However, they have not been investigated physiologically, which would not be a difficult matter as the spinal nerves are readily accessible in cyclostomes. An observation which has had a wide circulation in the literature, is that of Allen (1917), who reported neuromuscular spindles in the caudal heart muscle (which consists of parietal type fibers); Barker (personal communication, 1960) and the present author have made a special search for these without success. It is most probable that Allen (who did not use a staining method for nerve fibers) observed small blood vessels and interpreted them as spindles. In the other subclass of the Agnatha, the Petromyzontia, the information at present available regarding the innervation of the myotomal musculature is somewhat confusing. The muscle units are more clearly defined than they are in myxinoids (Fig. 2 ) . Each consists of some 4 sheet-like central fibers surrounded except at the medial face, by a layer of thin parietal fibers. The innervation of

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/

FIG.2. Diagram of innervation and arrangement of the two types of muscle fiber in the myotome of Lompetra planeri (brook lamprey). There is some dispute about this arrangement ( see text).

these units has been something of a puzzle for a considerable time, and has been investigated by a number of workers, of whom Fusari (1901, 1905) gave the first correct account, largely c o n k e d by Gerebtzoff (1956) and by Peters and Mackay (1961) investigating the small Lampetra fruoiatilis and L . p h m r i . The latter workers showed that the thinner parietal fibers were innervated by axons which entered the myotome between the muscle units and ramified over the surface of the units upon the parietal fibers. From this plexus, fine branches were given off which passed along the individual parietal muscle fibers. One axon might supply miiscle fibers in several adjacent myotomes, since there is a greater overlap than in myxinoids. These observations are in substantial agreement with those made by Fusari (1901, 1905). The larger central fibers are innervated in quite a different manner. No nerve fibers pass into the middle of the muscle units, and according to Peters and Mackay (1961), the central fibers are innervated by large diameter axons which sweep around the edges of the myotomes into the myosepta, where they branch to give rise to end-formations not unlike those found upon

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the central fibers of Myxirze, at the myoseptal edges of the sheet-like central fibers. These end-formations are apparently found at both ends of the central fibers. This description is not quite like that given by Fusari (1905) and Gerebtzoff (1956), nor does it accord with the writer‘s own observations. Fusari described special formations on the inner margins of the muscle sheets, and the writer can confirm that in adult L. pZuneri at any rate, large diameter axons give rise to elongate end-formations along the inner edges of each central fiber, apparently not at their myoseptal ends. Gerebtzoff (1956) comes to the same conclusion from his cholinesterase preparations (“La jonction myoneurale se trouve donc A I’extremitk centrale de chaque bande large”). Peters (personal communication, 1962) agrees that the central fibers are innervated along their inner edges, but maintains that they are also innervated at their myosepta1 ends. Further investigations on this problem are in progress. In the larger sea lamprey (Petromyzm mrinus), the innervation of the muscle units (Fig. 1; Plate I, 1) would appear to be slightly different, and perhaps more closely resembling that in Myxine (see Bone, 1963a). It is evident that the innervation of the central fibers of the lamprey myotome is not fully understood, probably there are specific differences. In essentials, however, the pattern of innervation would appear to be similar to that of Myxine, and we may infer that the roles of the two types of muscle fiber are similar in both animals. No suggestion has yet been made to explain the curious muscle unit arrangement found in Agnatha but not in other craniates. As regards the sensory innervation of petromyzont muscles, there have been a number of reports of various proprioceptive terminations, although no neuromuscular spindles have been observed (Baum, 1900; Stefanelli, 1932). Johnston (1908) found some fine fibers in the myosepta, and suggested that these were sensory. As he said, “These fibers seem to be in the proper position to serve the muscle sense.” Tretjakoff (1927) was unable to confirm this observation. Stefanelli worked only with various muscles of the head region, in which he observed (in P. marinus), endings very similar to those which the writer has found in the myotomes and interpreted as motor. These endings Stefanelli regarded as homologous with the Golgi tendon organs of higher forms. He also reported another type of sensory ending amongst these muscles, which

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Razzauti (1914) and Tretjakoff had previously supposed to be motor. It seems improbable that Stefanelli’s interpretations (based upon gold chloride preparations) can be accepted, but in view of the rarity of reports of proprioceptive terminations in lower chordates, it would be worth reinvestigating the muscles in which he claimed to have observed such endings. In my own view, it is likely that petromyzonts possess the same type of proprioceptive termination as is apparently found in Myxine; possibly Johnston’s fine fibers in the myosepta may belong to terminations of this sort. Cyclostomes show for the first time in the chordate series a dual motor system, in some ways analogous to that of amphibia. In Myxine, these systems have been more fully investigated than in any other lower chordate except the teleost. C. ELASMOBRAXCHS

The earlier workers, such as Trinchese (1885, 1892); Ciaccio ( 1877, 1882, 1889); Pansini (1888); Retzius (1892); Giacomini (1898b); Poloumordwinoff ( 1898, 1899) and Cavalie ( 1902), described a variety of nerve endings in or associated with elasmobranch muscles. Many of these workers studied Torpedo, but rays and dogfish were also examined. Some of the endings which they observed were interpreted as motor, some as sensory, but there was disagreement about which should be placed in either category. The motor end-formations were variously described as “piastre a corimbo,” “piastre implicata,” grappolini spargoli,” “terminaisons en ombelle” and so on. Three types of sensory ending were described. The first, the basket-like “terminazione nervosi a paniere” of Giacomini (the “en panier” terminations of many later authors), lay at the myoseptal ends of the fibers resembling the sensory endings found at the ends of the eye-muscle fibers of mammals by Dogie1 (1906). Second, Ciaccio (1889) described end-organs among the tendons of the tail of the ray, which he equated with the Golgi tendon organs of higher forms. Last, Poloumordwinoff ( 1898, 1899) described elongate sensory endings lying between the muscle fibers of Torpedo, rays and Trigon, which he compared with the neuromuscular spindles of terrestrial forms. These latter terminations were also observed in Torpedo by Cavalih. “

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It soon became clear that the first of these three types of sensory termination might really be motor. Perroncito (1902) who found basket-like endings in lizard muscle which resembled those described by Giacomini, supposed them to be motor, and suggested that it was not unlikely that Giacomini’s endings in fish might prove to be motor also. Murray (1924) described rather similar endings from the muscles of the pectoral fin of Squulus, and stated that they were motor. Couteaux (1950) gave good reasons for supposing that these basket-like endings were motor, and later (Couteaux, 1955) showed that cholinesterase was found in them. Similar endings in teleosts were interpreted as motor by Barets (1952) on histological grounds, and then Cuypers and Fessard (1954) gave physiological evidence for this conclusion. Despite this, Tiegs, (1953) and Barker ( 1958) still follow Giacomini’s interpretation, which is certainly incorrect. The disagreement between the various authors upon the organization of the motor innervation of elasmobranch muscle was in part due to the fact that different types of muscle fiber in the same animal are innervated in a different manner. The only indication to be found of the way in which the conflicting observations of the earlier workers are to be reconciled is due to Couteaux (1950), who points out that “les innervations motrices ne sont pas d’une type uniforme dans tout0 la musculature strike de ces Poissons (rays),” and goes on to state that in Torpedo myotomes where red and white muscle fibers lie side-by-side, the former possess end-formations of the classical type (i.e., “en plaque”), while the latter are supplied by basket-like end-formations at their myoseptal ends. It is, therefore, necessary briefly to summarize conclusions reached from a study of the myotomal musculature of various elasmobranchs (Bone, 1963b). In dogfish ( Scyliorhinus, Squalus, Dean&), in the flattened Squatina and Torupedo, in the larger sharks (Cetorhinus, Carcharinus), and probably in most sharks, the myotome is divided into a deep portion and a superficial portion. In some sharks ( Braekkan, 1959), the superficial portion may become infolded into the myotome to lie under part of the deep portion. The muscle fibers of the superficial portion are thin [SCr-W p in Raia (Ranvier, 1873) 1, rich in myoglobin, and plentifully supplied with blood capillaries. In contrast, the muscle fibers of the deep portion are thicker [150-180p in Raia (Ranvier, 1873)], poor in myo-

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globin, and poorly supplied with blood capillaries. In fresh specimens, the distinction between these two types of muscle is very marked (Daniel, 1922), for the superficial portion is bright red, while the deep portion is white. Figure 4 shows sections across the

C

A

E

FIG. 3. Cross sections (not to scale) of tail region in different elasmobranch, showing the. relative proportion of red (stippled) and white muscles in the myotomes. A: Scyliorhinus. B: Squalus. C: Torpedo. D: Squatina. E: Cetorhinus. ( Section of Cetorhinrn drawn from photograph kindly loaned by Dr. F. C. Stott.)

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body of various elasmobranchs illustrating the relative proportions of the two components of the myotomal muscle. The red and white muscle fibers in the shark myotome correspond to the parietal and central fibers of cyclostomes, respectively, as will be seen from their physiological characteristics, and from their innervation. Since, however, they are not arranged in muscle units, the terms “central” and “parietal” are not descriptive in elasmobranchs, and they will be termed “fast” and “slow” fibers; the mass of fast fibers in the deep portion of the myotome being termed the “fast motor system” and the slow fibers of the superficial

Fast fibers Aberrant fost fibers

Skin

I

FIG.4. Diagram of innervation and arrangement of the two types of muscle fiber in the common dogfish (Scyliorhinus). A sensory corpuscle is shown in the myoseptum. Inset: the innervation of the three types found in the myotomes of Torpedo, and perhaps also in Scylimhinus.

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portion, the “slow motor system.” The evidence upon which this nomenclature is based will be given below. The fast muscle fibers are innervated by large diameter axons, which run in the myosepta, and give rise to basket-like end-fonnations upon the myoseptal ends of the muscle fibers (Figs. 3 and 4; Plate I, 3 and 4).These were almost certainly seen by Pansini ( 1888, Plate VI, Figs. 9 and 10) who, however, like Giacomini a decade later, interpreted them to be sensory endings. One axon supplied a number of different muscle fibers in both of the myotomes adjacent to the myosepta in which the axon runs (Fig. 3 ) . The impression gained is that the fast muscle fibers are innervated at both their myoseptal ends. Giacomini (1898b) stated definitely that each muscle fiber of the myotome was embraced at each of its ends by a hasket-like nerve ending. Hinsey ( 1934) following Giacomini) remarks that “each (fish) muscle fiber is provided at each end with a “terminaison en panier,” going on, however, to add, “in addition to the somatic-motor ending which is near the middle of the fibre”! The impression that these fibers are innervated at both ends is one which it is not easy to be quite certain about, for in a wellstained methylene-blue whole mount, for example, basket-like terminations are certainly seen at either edge of the myotome, and although it looks much as though individual fibers have two such endings, one at either end, it is not so simple to prove that this is, or is not the case. As we have seen, the observations in Myxine by Andersen et al. (1963) have suggested that fast muscle fibers innervated by basket-like endings, receive such endings at one end only. The writer has made special efforts to discover muscle fibers in methylene blue whole mounts of the fast motor system of Torpedo which have endings at both ends, but he has not succeeded. A11 the fast fibers which have been dissected out complete seem to have a basket-like ending at one end only. It is, therefore, probable that in elasmobranchs, as in Myxine, at least the majority of the fast muscle fibers receive only one end-formation, at one myoseptal end of the fiber. Gerebtzoff inclines to this view also, and (in dogfish) states “il est probable que la fibre nerveuse ne se trouve qu’A une extremitb d e chaque fibre musculaire.” He regards such fibers as slow fibers, the polyneuronally innervated fibers as

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fast fibers. In some elasmobranchs, and perhaps in all, the basketlike end formations at the end of the fast muscle fiber are derived from two nerve fibers, which seem not to be merely branches of a single axon. That is, it appears that two separate axons terminate at the single end-formation upon an elasmobranch fast muscle fiber. Further investigations of this curious situation are in progress. In Torpedo, Couteaux (1950) observed certain muscle fibers which were innervated not exactly at their myoseptal ends, but in the neighborhood of the myoseptal ends. These, he stated, looked much like the basket-like terminations at the myoseptal ends of other muscle fibers in the same animal, and represented an intermediate type between the “myoseptal” innervation, and that which is found upon the fibers of the slow motor system, (The last is described below.) It is possible that the fibers which Couteaux described were of an intermediate type, but there are certainly some muscle fibers lying in the superficial zone of the deep portion of the Torpedo myotome, which are innervated in a more strikingly different manner from the majority of the fibers of the fast-motor system, and in a different manner from those of the slow-motor system. These fibers are supplied by bundles of nerve fibers passing out among the midregions of the muscle fibers, which give off as they course through the superficial zone of the deep portion of the myotome, large end-formations (Fig. 2; Plate I, 2 ) resembling those found in the musculature of the fins. Each muscle fiber in this region receives one end-formation near the middle of the fiber, and each endformation supplies one muscle fiber (although it may occasionally overlap onto an adjacent fiber). Similar “aberrant” fibers have not been found (though they may well occur) in the fast-motor systems of other elasmobranchs, but they are found in some teleosts (see Barets, 1952,1961), where their innervation is of exactly the same pattern as it is in Torpedo myotomes. For the present, these aberrant fibers, with a different pattern of innervation from that of the typical fast fibers or the slow fibers, may be considered as a part of the fast-motor system. It will be recalled that there is some evidence for an infrequent third category of muscle fiber in Amphibia, but this is supposed to belong to the slow-motor system, rather than to the fast-motor system. However, if there is a third type of fiber in amphibia, nothing is known of its

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innervation, except that it can give rise to a propagated muscle action potential, as we may assume the aberrant elasmobranch fibers can also do. The innervation of the slow motor system of muscle fibers in the superficial portion of the myotome is very different from that found in the fast motor system. Each muscle fiber receives a number of terminations, derived from at least two separate axons; these endformations are of the “en grappe” type. In different species they vary somewhat in form, and may occur at any point along the length of the fiber, except at its myoseptal ends. The thin axons which supply these end-formations either pass onto the slow fibers at both their myoseptal ends from the nerve bundles of the myoseptal region, or they may enter the muscles there and pass onwards across the surface of the muscle bundles giving off side branches to innervate a number of slow muscle fibers. The pattern of innervation of these two systems of muscle fibers in elasmobranchs is, from an histological viewpoint, not unlike that of the fast and slow motor systems in amphibia. Preliminary physioIogicaI investigation of the slow and fast muscle fibers of dogfish ( Pennycuick, 1962) has yielded very interesting results, which go Plate I. 1. Termination of a large diameter axon upon the central fibers of a muscle unit in Petromyzon marinus (sea lamprey). Parts of two adjacent muscle units are seen, The finer axon seen partly in the focal plane to the right of the thick axon (upper right) passes to the parietal fibers of the unit. 2 . Innervation of the aberrant muscle fibers in the superficial zone of the fast motor system in Torpedo. The end-formations lie on the midregion of the muscle fibers. 3 and 4. Basket-like terminations from the fast motor system of Torpedo, the Iower figure showing them more or less in side view, the upper more or less as seen from the myoseptum. 5. Stretch-receptor from the pectoral fin of Torpedo. 6. Sensory ending (perhaps equivalent to that of no. 5) from the surface of the tail myotomes in the ray ( R . clauuta). 7. Stretch receptors from the pectoral fins of the ray. Note that this is to same scale as Fig. 5. The muscle fibers have been partially dislocated from one another to show the ending ( a multiple one in this case) lying freely between the fibers. 8. Corpuscle of Wunderer from the surface of the myoseptum of Squulus. 9. Basketlike end-formations upon fast muscle fibers in Protopterus. Note apparent absence of any termination from middle 6ber. 10. Two “en grappe” end-formations upon a slow muscle fiber of Polypterus. All to same scale except nos. 6, 9, and 10. Nos. 9 and 10 from silver-impregnated polyester wax sections, remainder from fixed methylene-blue whole mount preparations.

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far to explain the occurrence of the two motor systems in the myotomes. Pennycuick (1962) found that the slow fibers apparently did not conduct action potentials, whereas the fast fibers did, but the importance of his observations lay in his discovery that when the dogfish was swimming slowly electrical activity was found only within the superficial portion of the myotome; no activity was found in the fast motor system under such conditions. Only during active movements resulting from strong stimulation could muscle action potentials be picked up from the fast motor system in the deep portion of the myotome. It, therefore, appears that the slow motor system is used during normal slow cruising, and that the fast motor system, inactive during cruising, is only operative when bursts of speed are exhibited by the fish. That muscles used for prolonged and sustained effort are of the red type was suggested by Lankester (1871) and by Ranvier (1873), but it was Arloing and Lavocat (1875) who first suggested that the slow and fast muscle fiber systems of fishes were used during different types of swimming. Their conclusions have been overlooked by recent workers, and deserve quotation in full. They state (p. 193): “Lorsque le Poisson nage tranquillement, les mouvements, lents, soutenus et gracieux de sa queue, repondent l’action des muscles foncks de ligne lathrale; lorsqu’il est agit6, poursuivi, ses mouvements brusques et saccad&, indiquent suffisament l’activite des muscles p Ales; c’esta-dire, I’activitk de la plus grande partie de Pappareil musculaire.” Their conclusions, which are those reached independently by modern workers, were based on a study of the muscles of rays and various teleosts. Support for this view of the role of the slow and fast motor systems is given by a consideration of different elasmobranchs of varying habit. Those (such as the common dogfish, Scyliorlzinus) which spend the greater part of the time immobile (in aquaria), and only cruise about searching for food at night, have a much smaller proportion of slow muscle fibers in their myotomes than do those fish (such as the spur dogfish, Squalus) which are more active and swim continuously (in aquaria). The basking shark (Cetorhinus) which cruises about feeding upon plankton, has a high proportion of slow muscle fibers in its myotomes (Stott, personal communication, 1962). As will be seen in the next section, considerations of this sort led Boddeke et al., (1959) to come to similar conclusions with regard to teleost slow muscle fibers.

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The histological features of the slow and fast muscle fibers in the myotome would, indeed, suggest a conclusion, for the thin, polyneuronally innervated slow fibers, rich in myoglobin, would be expected to perform continuous work, whereas the thick, white, fast muscle fibers innervated by large diameter axons would be expected to be "twitch" fibers suited only for bursts of activity. It is important to notice that Pennycuick's unpublished observations upon the activity of the different portions of the myotome during swimming are the only direct observations upon the function of the slow and fast systems in the living fish. Further investigations, in teleosts as well as in elasmobranchs, are much needed to confirm the view suggested first by Arloing and Lavocat ( 1875). As regards the sensory innervation of the muscles, it is in elasmobranchs alone among the lower chordates, that a suitable nervemuscle preparation has been found, and where it has been shown that there are stretch-receptive endings in parallel with muscle fibers. Fessard and Sand (1937), who have examined the responses of these endings, showed that they were analogous to the neuromuscular spindles of higher forms. They found them in the wing of the ray (where, they point out, their responses would be capable of regulating the swimming rhythm), and in various (unspecified) muscles of the dogfish. The work of Poloumordwinoff (1898, 1899) and Cavalik (1902) upon sensory endings in the muscles of Torpedo and rays was not known to Fessard and Sand; it was Barets (1956) who reinvestigated these endings in Raia, and demonstrated that Fessard and Sand were probably working with endings of the type first found by Poloumordwinoff. These endings (Plate I, 5 and 7) lie between the muscle fibers and consist of an elongate mass of fine beaded fibers, derived from a thick nerve fiber, The characteristic beaded appearance of the fibers is seen in silver as well as in methylene blue preparations (Barets, 1956); perhaps if these bulblike expansions be not artefacts, they may function as the mechanoelectrical transducers of the system, as Katz (1961) suggests for the similar expansions found in the sensory fibers of the frog neuromuscular spindle. Barets at first failed to find similar endings in the myotomal or fin musculature of dogfish, although Fessard and Sand reported similar sensory discharges from dogfish muscles to those from the pectoral fins of rays. He later (Barets, personal communication, 1962) found them among the muscles of the fins where the

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writer has also occasionally observed them. It is worthy of note that these endings are, in my experience, much more easily demonstrated in some species than in others; thus in Torpedo, they can be found in every methylene blue preparation of the muscles of the fins; in Raia in the majority of preparations; whereas in Scyliwhinus fins, they are only exceptionally stained. That they have not yet been observed in the myotomal musculature of any elasmobranch (with the possible exception of rays; see below) may, therefore, be explained as a failure of technique (though the author does not believe that this is probable). There are also other proprioceptive endings in elasmobranchs, which have been investigated physiologically. These are large coiled endings (possibly first observed in the ray by Purvis, 1890, and later illustrated in various species by Wunderer, 1908),which lie amongst the connective tissue at the bases of the fins (Plate I, 8 ) . Lowenstein (1956) investigated the responses of these endings in dogfish fins, and concluded that they were sensitive to pressure, but also functioned as second-order proprioceptive endings, for as the fin was bent, so the connective tissue in which the endings lie was stretched or compressed, thus giving rise to a discharge from the receptor. These endings are also found in the edges of the myosepta, just under the skin (personal observations), in dogfish, not apparently, in rays. The endings on the surface of the tail myotomes in rays (Plate I, 6 ) have not been fully investigated; in the writer’s own methylene-blue preparations, they rather resemble the endings described by Purvis and by Poloumordwinoff than those figured by Wunderer (Plate I, 8 ) ; possibly they should be placed in the former category. It is possible that Ciaccio (1889) observed endings of this type. The writer has not seen the Golgi tendon-like organs which he figured from the tendons of the tail of the ray. In Ciaccio’s figures they are not very like the endings which the author has observed in his methylene blue preparations. In dogfish the coiled endings have been found only in the superficial edge of the myosepta, and towards the bases of the fins. Wunderer described “pinselfonnige” endings from the pectoral fins of Torpedo, which the author has also observed; these resemble the endings found at the bases of teleost fins (see below), and were probably seen earlier by Pansini.

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D. VARIOUSOSTEICHTHYES, EXCLUDING TELEOSTS

So far as the author is aware, there is nothing to be found in the literature concerning the histology of the innervation of the muscle fibers of Holostei, Chondrostei, Dipnoi, or Crossopterygii. Little information can be gathered, moreover, about the types of muscle fibers in the myotomes of these groups, In Amia (Holostei), Polypterw (Branchiopterygii) and Protopterus (Dipnoi), it seems that conditions are much like those found in elasmobranchs. In all three, the myotomal muscle is divided into a superficial portion consisting of thin muscle fibers, and a deep portion made up of thicker muscle fibers. In all three, there is a smaller proportion of the former in the myotomes than in the common d o g s h which is not surprising if we are correct in assuming that the thinner fibers are concerned with cruising swimming, for all three forms are predators behaving in much the manner of the pike (Esox)-lurking in shelter and then making a short sortie for prey-and in the pike, there are no thin muscle fibers in the myotome (Boddeke et al., 1959). Boulanger (1900) quotes an amusing account of the swimming behavior of the African lungfish (Protopterus). It swims rapidly for only short distances, and then remains more or less in the same position; it does not cruise about. In P r o t o p t e r n and Polypterm' (the only fish in which the author has been able to examine the myotomal innervation) , the thick muscle fibers of the deep portion of the myotome (which may be assumed to be the fast motor system) are innervated by basket-like endings at their myoseptal ends in the same manner as in elasmobranchs (Plate I, 9). These end-formations are derived from large diameter axons passing from nerve bundles in the myosepta. It is not yet possible to say whether the basket-like endings occur on only one end of the thick fibers, or upon both myoseptal ends. It is probable that there are aberrant fast fibers in the superficial zone of the deep portion of the myotome, as in Torpedo and some teleosts. The thinner muscle fibers in the small superficial lateral muscle are

' The writer has recently found that

in Acipenser (Chondrostei), there are

two types of muscle fiber in the myotome, the larger fibers receiving basket-like

end-formations at their myoseptal ends.

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innervated much as are the thin slow muscle fibers of elasmobranchs. In Polgpterus, the end-formations upon these fibers are much like those in dogfish, and are apparently derived from at least two separate axons (Plate I, 10). In Protopterus, the terminations upon the thin muscle fibers seem to be not exactly the same as in Polypterus and elasmobranchs; they are somewhat like those found in frog slow muscles (Couteaux, 1960, Fig. 3 ) ; in silver-impregnated material appearing as fine fibrils running longitudinally along the muscle fibers from the point at which they are given off from the axon branch supplying the fibers. Several of these terminations seem to occur upon one thin muscle fiber. Sensory terminations have not been described from the muscles of these fishes, and there do not appear to be muscle spindles in Protopterus, so far as the author has been able to examine the myotomal musculature. Holmes (personal communication, 1956) has observed branching sensory terminations associated with the pectoral fins (these fins are thread-like, and capable of very delicate movements). Further investigations of the myotomal and eye-muscle innervation of this animal are in progress. Nothing has as yet been reported concerning the innervation of the muscles of the surviving crossopterygian fish, Latimerria, but to judge from illustrations of the transverse sections given by Millot and Anthony (1958a, b ) , if a superficial lateral portion of the myotome exists, it is even smaller in proportion than in Prutopterus. 3lillot states that there are two types of muscle fiber in the animal (one red, one yellow in color), but it is not known if the former are present in the myotomal musculature.

E. TELEOSTS In tcleosts, where Giacomini (1898a) described the basket-like terminations for the first time in fishes, and where it was first realized that there were two types of muscle fiber within the myotomes, the pattern of the innervation of the myotomal musculature is more complicated than in any of the groups hitherto considered, for in different teleosts, there are striking differences in the innervation of the thick muscle fibers of the fast motor system. The earlier workers described various types of nerve terminations in teleosts (see also Racah, 1932); none observed the basket-like myoseptal end-formations which Giacomini (1898a) described from the eel ( Anguillu) and the tench ( Tinca). More recently, Kirsche ( 1948), working upon Lebistes, de-

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scribed two types of nerve fibers passing to the myotomal muscles, one of which he interpreted as sensory. It was, however, left to Barets (1952,1955,1961) to provide the definitive description of the innervation of teleost myotomal muscle, and to reconcile the conflicting observations of the earlier workers; showing that in teleosts there are slow motor systems in many respects equivalent to those found in amphibia. In his admirable later account (196l), Barets gives the results of his examination of 26 species of teleost (from 12 different orders), dealing in most detail with the tench, and the catfish ( A m e i w ) .In this account Barets summarizes the results of earlier workers, which will, therefore, not be considered here. He found from his own observations, that in all the various teleosts which he examined, the thin muscle fibers of the superficial lateral portion of the myotome, were innervated in essentially the same way, in a manner not unlike that described above for the slow motor system of elasmobranchs. Fine nerve fibers passed across the thin muscle fibers more or less at right angles to them, giving off twigs ending in small rings or loops (seen in silver preparations). In cholinesterase preparations, these small rings and loops were seen to be the site of the synaptic junctions (at which as in lampreys, the postsynaptic membrane does not exhibit the complex folding seen in higher forms), Each thin muscle fiber receives several endformations derived from different axons, distributed more or less regularly along its length. Certain muscle fibers in the superficial lateral portion of the myotome are innervated in quite a different manner, by arborescent terminations recalling those on frog fast muscle fibers. Each muscle fiber of this type generally receives one such termination, and each termination of this type is generally restricted to one muscle fiber, although occasionally the termination may overlap onto an adjoining fiber, This pattern of innervation is also found (in the catfish) in the superficial zone of the deep portion of the myotomal muscle, and Barets concludes that the “aberrant” muscle fibers of the superficial portion of the myotome are rightly considered as belonging to the superficial zone of the deep portion of the myotome, occurring accidentally in the superficial portion. In the deep portion of the lateral muscle of the catfish, there are two types of muscle fiber, each with a distinct type of innervation. The superficial zone of the deep portion consists of fibers innervated in the same manner as the aberrant superficial fibers discussed above. We have seen that fibers innervated in a similar manner, are

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found in the same region in Torpedo and probably also in Polypterm and Protopterm The majority of the muscle fibers of the deep portion of the myotome are, however, innervated by basket-like terminations similar to those found in elasmobranchs (Fig. 5 A ) . Barets states that these

Fast flbers Aberrant fost flbers

Slow

flbers

171

Skin A

Skin

B

FIG. 5. Diagrams of the two types of myotomal innervation in telcosts. (Redrawn after Barets, 1961.)

terminations may be found either at one or at both ends of the deeper fibers; this observation requires further investigation in the light of the work upon Myxine central fibers, Interestingly enough, basket-like end-formations of this type were only found by Barets in the catfish, the eel (Anguilla) and Conger eel (Conger). He did not observe them in any other of the teleosts he examined, and stated that Giacomini was mistaken in his report of basket-like terminations in the tench myotome. In the tench, indeed, the deep

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portion of the myotome consists of fibers innervated in a quite different way, in a manner like that of the superficial muscle fibers (Fig. 5B). Each fiber is innervated by a number of different axons, terminating in small rings and loops like those found in the catfish superficial muscle fibers. In other fishes, such as the perch (Pmca), Barets found upon the muscle fibers of the deep portion of the myotome terminations varying between those found in the superficial zone of the deep portion of the catfish myotome, and those found in the deep portion of the tench myotome. The two types mentioned above were, therefore, considered as the two extreme types of innervation of this portion of the muscle in teleosts, the basket-like terminations being rare in the group. Barets concluded finally that there was not an absolute structural difference between the terminations found in the superficial and the deep portions of the myotome, the sole difference being that those in the superficial portion belonged to a single type only, whereas those in the deep portion varied between wide limits. Acetylcholinesterase is the only enzyme found at the neuromuscular junction upon both slow and fast fibers in teleosts ( Siou, 1955; Pecot-Dechavassine, 1961). Histologically, therefore, the situation is more complicated in teleosts than it is in other fishes, and although the basket-like terminations found in elasmobranchs, lungfish, and P o l y p t m s are found in some teleosts, this type of ending seems to be rare in the group. Barets extended his histological observations by investigation of the physiology of the superficial and deep muscle fibers (Barets and Le TouzB, 1956; Barets et al., 1956; Barets, 1961) . It was found that the former had a membrane potential of some 55mv as compared with some 90mv for the latter, and that, at least in the catfish, typical propagated muscle action potentials were obtainable from the fibers of the deep portion of the myotome. The superficial portion of the myotome was not studied in detail but it appeared that, since single stimuli never produced a response, while repetitive stimuli gave rise to a slow sustained contraction which increased in amplitude as the frequency of stimulation was increased, it was likely that propagated muscle action potentials are not found in the superficial muscle fibers. The situation is similar to that found in elasmobranchs and in amphibia. In the tench, where each muscle fiber in the deep portion of the myotome receives several “distributed” end-formations, local potentials were obtained, equally distributed, and accompanied by a

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twitch response of the muscle fiber. Propagated action potentials could also be obtained, but these were not regarded as being typical for various reasons (e.g., the polarization of the muscle membrane does not invert as it does in a typical “spike,” such as is found in the catfish deep muscle fibers). Similar findings were reported by Takeuchi (1959) in teleost pectoral fin muscles. These results, as Barets recognized, are certainly very remarkable, for it is most surprising to find fast muscle fibers in vertebrates which are innervated polyaxonally and which do not normally conduct propagated action potentials, twitch responses of the muscle fiber following local distributed action potentials. Barets remarks “la jonctions neuro-musculaires de la Tanche rapellent celles des Crustacks. Le muscle lateral profond des TeleostCens assure ainsi la transition entre les CrustacCs et les Batraciens, en ce qui concerne certaines caracteristiques des jonctions neuro-musculaires, et constitue de ce fait un type particuli6rement interessant de systbme moteur “rapide”.” While some of the further generalizations drawn by Barets in his comparison of the crustacean and vertebrate type of innervation of twitch fibers (see Barets, 1961, pp. 175-176) may not be acceptable, there is at the least a great degree of similarity between the tench rapid muscle fibers and certain crustacean fibers. As regards the function of the two motor systems in teleost muscle, there have been no direct experiments (as there have been in elasmobranchs ) but it seems highly probable from the comparison by Boddeke et al. (1959) of various teleosts of different habits that, as in elasmobranchs, the superficial portion of the myotome is used in cruising while the deep portion is active only in bursts of rapid swimming. The relative amounts of the superficial and deep portions of the myotome in various teleosts are also illustrated by Le Danois (in Grasse, 1958), and were noticed by Arloing and Lavocat. Curiously enough, Braekkan (1956) who remarked upon the general correlation between the amount of red muscle in the myotomes, and the activity of the fish, concluded that “the main purpose of the red muscle is not muscular work, but that it functions as an organ.” Earlier workers had noted the high proportion of fat in the red muscle of different teleosts (e.g., Greene, 1913; Wilson, 1939) but had not suggested its function apart from fat-storage. Sensory terminations associated with the myotomal musculature have never been found in teleosts. None were observed by Barets,

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who has examined teleost muscles in more detail than any previous worker. The writer has, however, observed what appear to be simple branching sensory endings in the connective tissue surrounding the bases of the “walking” fin rays of gurnards (Trigla sp.) but none within the muscles operating these fin-rays. Ciaccio (1889) described somewhat similar endings from the tendons of the pectoral fin muscles in the tench and the carp. Baum (1900) did not find sensory endings in the muscles of the pectoral fins of pipe-fishes (Syngnuthus). Neuromuscular spindles have been searched for in teleost muscle, but none have been observed (Giacomini, 1898a; Baum). Gerebtzoff figures an “intra-fusal” fiber (!), but he certainly did not observe neuromuscular spindles. It is, of course possible if there are sensory endings in teleosts, such as those found in Myxine, associated with the connective tissue of the myotome, that these have escaped observation. The results of physiological investigations, however, (e.g., Gray, 1936) have not, on the whole, supported the view that proprioceptors associated with the myotomes play a significant role in the swimming of teleosts. Ill. Discussion

The investigations surveyed above have established the following facts: a. In different groups of fish (except the Acrania) there are in the myotomal swimming muscles two histologically different types of muscle fiber; usually one type is red, the other white. b. Where the two types of fiber have been investigated physiologically it has been found that they are similar to the two types of muscle fiber found in amphibia, i.e., in the presence or absence of propagated muscle action potentials, in differences of resting membrane potential, and in their response to stimulation of the nerve fibers supplying them. c. As in amphibia, these two types of muscle fiber are innervated in a different manner. The pattern of innervation of each type is essentially similar throughout the lower chordate groups (apart from the Acrania) , d. In certain teleosts, however, the patterns of innervation of the two types of muscle fiber have something in common, and neither type of fiber exhibits normal propagated action potentials. e. In some elasmobranchs and teleosts, and probably in other

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groups, a small proportion of the myotomal muscle fibers are innervated in a manner different from the two main types of fiber in the myotome. f. There are no neuromiiscular spindles in any group of fishes, and only in Myrine and elasmobranchs is there evidence for proprioceptive terminations in, or associated with, the swimming muscles. These facts raise questions of several different kinds. In what follows, it is important to notice that the suggestions put forward require a good deal of further investigation before they can be accepted or rejected. Few physiologists have yet turned their attention to the lower chordates, and histologists have disagreed about the anatomical basis of the pattern of innervation in the myotomal muscles, so that it is hardly surprising that few firm conclusions can be drawn from the evidence reviewed above. A.

FUNmiOwAL

SIGNIFICANCE OF THE DUAL MOTORSYSTEM

IN FISHES

Fishes live in a dense medium. In order to increase their speed of swimming, they must generate a relatively very large amount of extra power-to bring into use, therefore, a relatively very much greater mass of muscle than is required for normal cruising speed. Bainbridge (1961) points out that doubling the weight of muscle available for propulsion can only raise the speed of the fish by a factor of 1.32 (even when the most favorable assumptions regarding laminar flow over the body surface are made). It is understandable that a dogfish (in which, as we have seen, some 90%of the myotomal muscle is inactive during cruising) might require for rapid locomotion 10 times the amount of muscle needed for cruising, and that this muscle might be specialized for the purpose of rapid contraction. In the great majority of gnathostome fishes, the slower muscle fibers lie in the superficial zone of the myotome, i.e., outside the main mass of the fast motor system. In this position, the slow motor system may be supposed to be at the greatest mechanical advantage for bending the trunk via the pull upon obliquely set myosepta. It should, however, be mentioned that in some species (for example, in Torpedo and Squatina), the superficial lateral muscle is folded among the deep portion of the musculature to some extent, possibly as a result of the development of the flattened body form in these fishes. In others, such as the salmon (Boddeke et d., 1959),

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the thin fibers not only constitute the superficial lateral muscle, but are also scattered throughout the deep lateral muscle. In Agnatha, the functional significance of the curious arrangement of muscle units is not clear, for although some experiments have elucidated the physiology of the two types of muscle fiber in Myxine, it is not certain under what condition either or both types are active. We may assume that the parietal fibers are used for cruising and the central fibers for active swimming, yet this does not explain why these two types should be organized in so strange a manner. Investigation of the electrical activity of the slow and fast motor system during slow and rapid swimming in different forms is much needed, along the lines of the experiments carried out by Pennycuick (1962) in dogfish.

B. MODEOF ACTION OF THE Two SYSTEMS,AND GRADATION OF CONTRACTURE IN FAST AND SLOWMOTOR SYSTEMS Before considering the problems raised by the different mode of innervation of the two motor systems and by the variation in the mode of innervation in the fast motor system in teleosts, it is necessary first to consider the problem of gradation in the swimming muscles of fishes. Few investigations have given evidence for gradation of the myotomal motor systems other than in teleosts, for [as in the various studies of the swimming of dogfish (Gray and Sand, 1936; Le Mare, 1936)l the existence of two separate motor systems has not been taken into account, the myotomal musculature being considered as a single system. However, for some of their results, it is often possible to deduce that one or the other of the motor systems was responsible for the phenomena observed, and to find evidence for gradation of the slow motor system in elasmobranchs. Such experiments as that recorded by Gray and Sand (1936, Text-fig. 1) appear to show that gradation does take place within the slow motor system. Gray and Sand found that light touch upon the surface of the skin of a swimming “spinal” fish-a fish in which the spinal cord is transected behind the medulla, in which, therefore, the brain does not modify the behavior-produces a %!%increase in amplitude, and a 40% increase in frequency as compared with the normal “spinal” rhythm. These responses are quite different from those (illustrated in their Text-fig. 2) resulting from the application of much stronger stimuli, when the fast motor system is

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evidently active. It is almost certainly correct to assume that the typical dogfish “spinal” swimming rhythm and its alteration by light touch are manifestations of the activity of the slow motor system, hence that this system at least is capable of grading its contracture. Support for this view comes from other experiments (Gray and Sand, 1936), in which it was found that the initially violent response (to the attachment of a clamp) does not appear in the fatigued “spinal” fish, although a spontaneous slow rhythm is still to be found. It would appear that the fast motor system (responsible for the violent response) fatigues more rapidly than the slow-motor system, and independently of it, (as we should expect from the histology of the two types of muscle fiber), the slow motor system being capable of sustained activity, as is found in the swimming rhythm of the “spinal” fish. Gradation of the slow motor system can evidently also take place in teleosts, for Barets has shown that the amplitude of an isotonic contraction of the fibers of the slow motor system in catfish depends upon the frequency of stimulation. As far as the writer is aware, however, although it is natural to suppose that the catfish does grade its slow motor system, there have been no experiments on the catfish along the lines of those on the spinal dogfish, which actually proce gradation. It would, of course, be surprising to find that teleosts could not cruise slowly at different speeds. Turning now to the fast motor system, it is less clear that this can be graded. One explanation of the rapid fatigue of what would appear to be the fast-motor system in the experiments of Gray and Sand might be that all the muscle fibers in the system were contracting at one time, indeed, if our view of its function as an emergenw power supply be correct, it would be curious if all the fibers did hot act to give maximal power when the fish was strongly stimulated. As Prosser (in Bourne, 1960) remarks, “Gradation of movement in noniterative muscles has been indicated as due largely to variation in number of motor units. In iterative muscles, and to a very slight degree in noniterative muscles, gradation is by variation in frequency of stimulation.” We have seen above that in the iterative slow-motor system of the catfish, Barets (1961) has shown that gradation takes place by variation in frequency of stimulation, and we might expect that in the noniterative fast motor system, grada-

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tion would take place by recruitment of separate motor units within the system. In the fast motor system of the dogfish, it is probable that each axon supplies some 20 muscle fibers but not quite certain that this neuromotor unit is equivalent to that of the frog or the cat. (This point will be considered below.) Gray (1936) is the only author who has considered the possibility of the recruitment of muscle groups in fishes; he speaks of “extensive recruitment from inactive muscle groups.” Gray worked upon the resistance reflexes of the eel; and investigating the question of whether the rhythmic responses involved all the musculature of the fish, he concluded “. . . even when swimming very actively, an eel is utilizing only a small percentage of its total available musculature . . . .” In resistance reflexes, Gray found that the tension developed was some 15 times that developed during “active” swimming. This does not, however, directly indicate the relative fractions of the muscle fibers active since the force exerted by the muscle depends upon its velocity (Hill, 1949). At first sight, it would appear that such experiments demonstrate that gradation by recruitment may take place within the fast motor system, since during “very active swimming the fish would presumably use the fast-motor system. However, the question is still open to experiment, for it is not quite certain that the active swimming of the normal eel is not the result of the maximal activity of the slow motor system. The larger scombroids which possess a relatively large slowmotor system (Kishinouye, 1923) cruise at high speeds for long periods. In such fishes the term “slow motor system” is inappropriate; it would be more reasonable to speak of the “sustained motor system.” Eels migrate over large distances, and it may be that their slow motor system is, likewise, capable of giving rise to active swimming. It is, therefore, possible to interpret Gray’s results in terms of the activation of an hitherto inactive fast motor system, rather than by the recruitment of motor units within that system. We may at this point consider a further possibility, not to be ignored, regarding the equivalence of the neuromotor unit in the fish fast motor system and in the frog or mammal. Although in dogfish each axon probably supplies around 20 fast muscle fibers in the fast motor system, this is not to say that such a neuromotor unit is like that of the mammal, for it may be that all the fast motoneurons

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of the particular segment supplying the myotome of that segment normally fire at one time. The rapid through-conducting pathways from large reticular and spinal neurons (e.g., the Mauthner, Muller and Rhode systems of cells) are conspicuous elements in the ventral fasciculi of the spinal cord of fishes. These pathways probably link the fast (phasic) motoneurons of the cord, since they all consist of large-diameter rapid-conducting fibers, and where these have been investigated, they seem to mediate the rapid escape movements by fishes (e.g., Berkowitz, 1956); movements resulting from the activity of the fast motor system. It is, thus, worth considering the possibility that the fast motoneurons of the fish spinal cord are normally activated by such rapid through-conducting pathways, and that the neuromotor unit is normally the entire mass of fast muscle fibers in the myotome. Further investigations are required before it will be known whether the fast motor system can be graded in those fishes where the fast fibers are innervated by basket-like terminations, and where they conduct propagated action potentials. It is obviously important to know in this regard whether the fibers are innervated at one or both of their myoseptal ends. It is very probable, however, that in the majority of teleost fishes [where the fast fibers are polyneuronally innervated, and where in the tench (Barets, 1961) the amplitude of the twitch response of the muscle fiber increases following successive stimuli], the fast motor system can be graded. It is, the writer thinks, fairly clear from the distribution of the basket-like type of innervation of the fast fibers in different fishes, that this type of innervation is the primitive mode of innervation of fast fibers in lower chordates, and that the polyneuronal innervation of fast fibers found in certain teleosts is a secondary development. Not only are the basket-like endings found in all groups except the teleosts (and in teleosts, in the less advanced catfish), but they are found in Polypterus, which has been supposed to be related to the Paleoniscid ancestors of modern teleosts (see Goodrich, 1928). This view would be strengthened by evidence of basket-like endings from the more primitive teleost groups, such as the salmonids and clupeids (which are at present under investigation: three species of clupeids possess these endings but Salmo sular and S. friittu do not). This conclusion differs somewhat from that of Barets (1961), who considered that the basket-like type of innervation was not “de

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nature differente, mais comme un localisation exceptionnelle de l’innervation motice.” Until it is shown that the fast motor system in those forms where the fast muscle fibers are innervated by basketlike endings can or cannot be graded, hypotheses concerning the advantage of a pattern of innervation (in teleosts) which evidently allows gradation; or views on the real or only apparent distinction between these two types of motor innervation, are premature. C. THEPROBLEM OF PROPRIOCEPTION IN FISHES

Finally, we may consider the situation as regards the sensory innervation of the musculature of fishes. If the observations yet made represent the real state of affairs, the swimming muscles of fishes are strikingly different from the locomotor muscles of arnphibia, reptiles, birds, and mammals, for they do not, as a rule, possess proprioceptors regulating their contraction. When sensory endings are found in association with fish muscles, they occur not in the myotomes, but in such muscles as those of the fins of dogfish, of the pectoral fins of Torpedo, around the bases of the fins in some teleosts, in the eye muscles of dogfish [where Holmes (personal communication, 1961) has found spiral endings resembling those described from mammalian eye muscles by Cooper et al. (1955)], and in the pectoral fins in lungfish. These muscles and fins are all capable of delicate adjustments. The only sensory endings associated with the swimming musculature yet found are those found by Poloumordwinoff (1898) in the pectoral fins of rays, the coiled endings found in the superficial zone of the myosepta in dogfish, and the presumed sensory endings in the connective tissue covering the inner faces of the myotomes in Myxine. None of these sensory endings are associated with special muscle fibers, and all are therefore very much less complex in function than the neuromuscular spindle. There has been a good deal of discussion concerning the possible role of afferent information in the swimming patterns of different fishes (see review by Healey, 1957). Its importance is suggested by data from elasmobranchs, whereas workers upon teleosts (where no proprioceptive terminations have yet been observed) have generally concluded that sensory stimuli from proprioceptors play no role in normal locomotion. Lissmann (1946a, b ) demonstrated that the “spinal” dogfish did not swim if all dorsal roots were cut ( a similar result to that ob-

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tained in amphibia by Gray and Lissmann, 1940, 1946). He concluded that afferent information was essential for the swimming pattern. This pattern was, of course, the slow swimming of the “spinal” fish, which the writer has attributed to the slow motor system, in which gradation can take place. In other elasmobranchs, such as Squatina and Raja, the fish cannot swim after the spinal cord is cut anteriorly (the writer’s observations; see also Campbell, 1931), however Torpedo can do so. The presence or absence of the “spinal rhythm” does not allow inferences about the existence of proprioceptors, but depends upon the organization of the spinal cord itself, probably upon the presence or absence of large throughconducting elements within the cord, since these are present in dogfish and absent in rays (the writer’s observations). Investigations of the discharges of ventral and dorsal root nerves during swimming are much needed in teleosts and, indeed, in elasmobranchs. Until these are performed, it is difficult to say whether the absence of proprioceptors in the former group is the result of failure of histological techniques, or whether it represents a real difference between the two groups. In both groups, of course, there is a lateral line system, which as Hoagland (1935) has pointed out, “. . . may perform a tonic re-inforcing role in postural and swimming reflexes. . . .” At present, however, it seems unlikely that the lateral line system is to be implicated in the regulation of contraction of the myotomal muscles in fishes, for in the “spinal” dogfish (where the cord is transected below the level of entry of the lateralis fibers) the fish still swims.

D. CONCLUDISG REMARKS The study of the pattern of innervation of the swimming muscles of fishes has clearly shown the truth of Hoyle’s (1963) dictum that “The vertebrate physiologist is no longer justified in complacency regarding the universal applicability of the data obtained from the ordinary (phasic) muscles of frog and cat.” In general terms, the fast and slow systems in the fish myotome are much like the fast and slow systems of the amphibian, and it does seem that mammals and birds are set apart from all other chordates in that their fast and slow fibers are twitch fibers conducting propagated action potentials. The “typical” vertebrate pattern of muscular innervation is thus not found in the mammal, where the division between slow and

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fast fibers does not seem to be so clear as it is in the lower chordates. In certain respects, the lower chordate pattern of innervation is unlike the amphibian pattern. In teleosts, at least, the axons supplying both fast and slow fibers are of equivalent diameter (Barets, 1961) ; there is no butyrylcholinesterase at the neuromuscular junctions (Pecot-Dechavassine, 1961), and there is a wide variation in the type of innervation of the fast muscle fibers. The significance of these differences is not clear as yet, but there is also a much more important differencethe absence of neuromuscular spindles from the fish muscle. The outstanding problems raised by the investigations of the innervation of the swimming muscles of fishes are certainly the existence of proprioceptors and the role which they play in swimming, and, linked with this, the question of the gradation of the fast motor system in fishes where the fast muscle fibers are innervated by basket-like endings. These problems, no doubt, appear strange to the vertebrate physiologist accustomed to the frog and cat. That such problems can be stated at all underlines the differences between our knowledge of the innervation of the lower chordate swimming muscles, and of the muscles of the more familiar laboratory animals. ACKNOWLEDGMENTS I am indebted to Prof. J. Godeaux, and Dr. H. G. Vevers for their kindness in supplying me with specimens of Protopterus; to Mr. E. Hamblyn for a specimen of PoZypterus; and to Mr. J. Hill for sending specimens of Petromyzon. Dr. J. S . Alexandrowicz has given helpful criticisms of the typescript, and 1 have been much assisted by discussions with Dr. E. J. Denton, Dr. R. Miledi, and Mr. B. Roberts.

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THE NEURAL ORGANIZATION #OFTHE VISUAL PATHWAYS IN THE CAT'

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By T h o m a s H Meikle. Jr and J a m e s M. S p r a g u e Deportment of Anatomy. Cornell University Medical College. New York. New York and Department of Anatomy and Institute of Neurological Sciences. University of Pennsylvonia. Philadelphia. Pennsylvania

I . Introduction . . . . . I1. Historical Preview . . . 111. Primary Optic Terminations A . Optic Nerve . . . . B. Optic Chiasm . . . C . Retinohypothalamic Tract D. Supraoptic Commissures E . Optic Tract . . . . F. Thalamus . . . . G. Pretectum . . . . H. Superior Colliculus . . I. Accessory Optic Tract . IV. Mesencephalic Projections . A . Pretectum . . . . B. Superior Colliculus . . C. Reticular Formation . . V. Thalamic Projections . . A. Lateral Geniculate Nucleus B. Lateral Geniculate Nucleus C. Other Thalamic Nuclei . VI . Neocortical Projections . . A. Lateral Gyms . . . B. Suprasylvian Gyms . . C. Other Cortical Areas .

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I. Introduction

Since the last comprehensive review of the mammalian visual system and its central neural connections ( Clark, 1942), considerable anatomical and physiological data derived from new, more refined experimental techniques have been reported and have suggested additional concepts in the organization of this system. It is the purpose of this report to review these new data, to attempt to correlate them with previous observations, and to summarize the results in a schema which seems to represent best the presently recognized visual centers and their neural pathways. Because of the authors’ primary experimental interest in, and experience with the domestic cat, and because so much of the data in this field has been derived from investigations of this species, the visual system of the cat will be evaluated primarily; other species will be discussed only in pertinent reference. This review will not consider the cat’s peripheral visual apparatus, which has been described by Thieulin (1927), by Kennedy (1939), and by Walls ( 19421, or the cat’s retina, which has been discussed by Vincent ( 1912), and by Polyak ( 1941 ) . The neural elements of the retina, although integral parts of the central nervous system, also will be omitted from this review which will consider the terminations of optic nerve fibers within the brain and the principal neuronal projections arising from these termini. The accompanying illustrations attempt to represent schematically the most probable neural organization of the cat’s visual system. II. Historical Preview

The development of knowledge concerning the neural aspects of vision began with the work of German physiologists during the last half of the nineteenth century. Several early observers had noted diminution in the size of the contralateral superior colliculus (Gall and Spurzheim, 1825) and optic thalamus (Panizza, 1855) following eruption of the eye in various mammals; but the first systematic investigation of the visual pathways began with the experimental work of Gudden and his students in Munich. Gudden

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perfected methods of operating on newborn kittens and, in 1874, reported that following enucleation each optic nerve projected bilaterally to the brain, where gross atrophy occurred in the superior colliculus, lateral geniculate nucleus, and adjacent thalamus, and the transverse peduncular tract ( Gudden, 1874, 1879). Gudden’s report was the first definitive description of what are now recognized to be the primary optic terminations within the brain. It was followed, in 1879, by Munk‘s demonstration, using spohisticated behavioral tests, that total ablation of the occipital region in dogs resulted in permanent blindness whereas partial, unilateral ablations resulted in homonymous field defects. To explain his findings, Munk (1879) postulated that, in the dog, optic nerve fibers from the temporal one-fourth of each retina did not cross at the optic chiasm, but continued into the ipsilateral optic tract. His illustrations of this schema were widely used during the period. In 1882, Ganser, using the cat, discovered the origin of optic nerve fibers from retinal ganglion cells, and, in the same year, von Monakow ( 1882), for the first time, described microscopical atrophy in the ipsilateral lateral geniculate nucleus, pulvinar, superior colliculus, and transverse peduncular tract following occipital cortex lesions. Later, von Monakow (1889) summarized his detailed investigations of systematic lesions in various parts of the visual pathway, including the optic nerve, optic tract, superior colliculus and visual cortex. In this report, he also postulated the presence of internuncials ( “Schaltzellen”) in the lateral geniculate nucleus relay and the origin of centrifugal fibers to the retina from the superior colliculus and possibly from the lateral geniculate nucleus. With the perfection of the Marchi stain, Pick (1894) traced the course of optic nerve fibers after small retinal lesions and established the retinotopical organization of these fibers within the cat’s optic nerve and optic tract. In 1898, Colucci (see von Monakow, 1905) first reported Marchi degeneration in the pulvinar, lateral geniculate nucleus, and superior colliculus following enucleation of the eyes in dogs, thus essentially confirming earlier studies which had relied on gross or microscopical atrophy. Probst (1900), using the same technique, confirmed Colucci’s findings; and, in 1901, he described corticofugal projections from the visual cortex (posterolateral and posterior suprasylvian gyri) of the cat to the superior

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colliculus, lateral geniculate nucleus, pulvinar, and the lateral thalamic nucleus, but none to the pretectum, thereby confirming von Monakow’s earlier work. Although during the next few years Campbell (1905) and Brodinann (1906) histologically defined the striate area within the mammalian cortex, it was not until 1910-1911 that Minkowski (1911) first demonstrated that the cortical visual area, as previously determined with behavioral techniques by Munk, was essentially COextensive with the histological striate area. Later, Minkowski ( 1913) also first showed a precise point-to-point degeneration relationship between the striate cortex and the lateral geniculate nucleus; and he postulated a more encompassing, precise retinothalamocortical projection system. This same investigator later ( 1920) described the systematic termination of crossed and uncrossed optic tract fibers within distinct laminae of the lateral geniculate nucleus of the cat and monkey, Since these early studies of the visual pathways, many important and significant contributions have been made by outstanding investigators, some of whose work will be indicated in the following review. Nevertheless, in retrospect, it is remarkable to note the accuracy and precision of these early, self-critical, and systematic workers, who firmly established the basis for future work in this field. 111. Primary Optic Terminations

The extensive course which optic fibers may take to their ultimate synaptic destinations comprises the primary visual pathway. This consists of a number of consecutive neuronal pathways beginning with the optic nerve and continuing through the optic chiasm, optic tract, brachium of the superior colliculus, and accessory optic tract. The optic nerve arises from the posteromedial aspect of the eye, passes posteriorly to the optic chiasm where the two optic nerves meet and from which the optic tracts continue posteriorly and dorsalIy to the posterior thalamus where, in the cat, a majority of optic fibers terminate. The remaining optic fibers continue through the stratum zonale of the thalamus, some leaving this structure to synapse in the pretectum and the remainder passing through the brachium of the superior colliculus to the tectum where most of these fibers terminate. A few optic fibers, however, continue

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without synapse beyond the tectum and course through the accessory optic tract to other mesencephalic nuclei where they synapse. Thus, along this variously-designated, but continuous pathway, optic fibers leave to reach their primary synaptic terminations within the diencephalon and mesencephalon, as illustrated in Fig. 1. A. OPTICNERVE

In the cat, the optic nerve is composed chiefly and perhaps exclusively of the axons of retinal ganglion cells, presumably on a 1:1 relationship as suggested by k e y and Gore ( 1942) for the dog. Nevertheless, the possibility of centrifugal fibers passing from the brain through the optic nerve cannot be excluded by either these studies or those reported by Bishop (1953), who counted 127,000 optic nerve fibers and 125,000 retinal ganglion cells in the cat. Other reported counts of the cat’s optic nerve fibers are within the same range, considering experimental error: 119,000 (Bruesch and Arey, 1942); 120,000 (Bishop et al., 1953a). Apparently, all optic nerve fibers in the cat are myelinated, and Bishop and Clare (1955) have found a size range from 0.5-12.0p with a maximum number in the 1 . 0 ~range; although, using the electron microscope, they have . noted myelinated optic nerve fibers as fine as 0 . 2 ~Electrophysiological work by Hubel and Wiesel (1962) has even suggested that some unmyelinated fibers may be present. Bishop and Clare (1955) have also suggested a rather even distribution throughout the nerve of fibers of different size but were unable histologically to confirm the four fiber groups described in their electrophysiological studies. On the other hand, Chang (1952) and Lennox (1958), using electrophysiological techniques, have independently reported three chief fiber groups in the cat’s optic nerve; whereas anatomical studies by Bishop et al. (1953a) have described two principal fiber groupings at 1.0-1.5 p and 4.0-4.5 p with over 80%of all fibers having a diameter of less than 3p. In addition, these latter workers were able to correlate their histological findings with a bimodal distribution of conduction velocities of optic nerve fibers which occur at about 20 meters/second and 40 meterdsecond. The retinotopical organization of fibers within the optic nerve of the cat has been illustrated by Overbosch (1927), from Marchi studies, with nasal retinal fibers in the medial half of the nerve and temporal fibers in the lateral half of the nerve. Fibers from the

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FIG. I . Primary optic fiber terminations indicated (Iower left) on dorsal v i e w of the thalamus and techim, adapted from Thuma (1928), and (lower

right) on transverse sections of the thalamus and midbrain at levels A-5 and A-2, adapted from Jasper and Ajmone-Marsan (1954). AOT: accessory optic tract. BSC: brachium of the superior colliculus. Dors.Nucl.AOT: dorsal nuclcus of the accessory optic tract. LGNd: lateral geniculate nucleus, pars dorsalis. LGN,: lateraI geniculate nucleus, pars ventralis. Lat.Nucl.AOT: lateral nucleus of the accessory optic tract. LP: lateralis posterior nucleus. Med.Nucl.AOT:

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upper retina generally lie above and arch over, both medially and laterally, fibers from the lower retina, as illustrated in Fig. 2.

B. OPTICCHIASM The optic chiasm is a complex interchange of optic nerve fibers with fibers from the nasal portions of each retina decussating into the contralateral optic tract, the most anterior of these decussating fiber bundles detouring slightly into the contralateral optic nerve (Polyak, 1957, Fig. 192). Fibers from the temporal third of each retina, lateral to a meridian through the area centralis, do not cross at the optic chiasm but enter the ipsilateral optic tract. No precise count of the number of crossed and uncrossed optic tract fibers in the cat has been reported, but Polyak (1957) estimates that about 50-70% of these fibers cross. Walls (1942), Doty (1958), and Hayhow ( 1958) estimate that about two-thirds cross. Computations by the Newton-Muller-Gudden law ( Apter, 1945) would suggest that about 65%of optic nerve fibers cross at the cat’s optic chiasm.

C. RETLNOHYPOTHALAMIC TRACT From the visual pathway at the rostra1 part of the optic chiasm, Bliimcke ( 1958) has recently reported a direct retinohypothalamic tract in the cat consisting of finely myelinated fibers which proceed within the lamina terminalis to its dorsal border where these fibers terminate in the hypothalamus, the infundibulum, and the posterior hypophysis. This pathway, which has also been reported by Knoche (1957) in the rabbit, dog, and man, differs from a previously reported pathway to the hypothalamus described by Frey (1937) in many species including carnivores, and considered by many investigators to represent aberrant optic chiasmal fibers (Jefferson, 1940; Clark, 1942). Gerard et al. (1936) and Massopust and Daigle ( 1961) have also recorded photically evoked potentials in the suprachiasmal regions of the hypothalamus. However, Hayhow (1958) and Altman (1962) in Nauta-stained material from the cat and Polyak (1957) from Marchi studies in the rat, have been unable to medial nucleus of the accessory optic tract. OT: optic tract. Post.: posterior nucleus of the thalamus. Pret: pretectum. Pulv.: pulvinar. S. Coll.: superior colliculus. St. Gris. Int.: stratum griseum intermediale. St. Gris. Sup.: stratum griseum superficialis. St. Opt.: stratum opticum.

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confirm the existence of a direct retinohypothalamic tract which, if present, could be of paramount importance in the regulation of light-influenced visceral functions.

D. SUPRAOPTIC Co~~rsscms As demonstrated by many early workers, in addition to fibers originating from the retina, the optic chiasm of the cat also contains decussating fiber systems which connect the two sides of the brainstem, These decussating fibers comprise a dorsal supraoptic commissure (commissure of Ganser) lying above and behind the optic chiasm, and a ventral supraoptic commissure ( commissures of Meynert and of Gudden) lying within, above, and behind the optic chiasm. As described by Magoun and Ranson (1942) and by Bucher and Biirgi (1953), the fibers forming the dorsal snpraoptic commissure arise in the medial midbrain, or more posteriorly, pass forward in the medial midbrain, run through the subthalamus, decussate above and behind the optic chiasm, pass laterally above the optic tract, pierce the internal capsule and terminate in the thalamic reticular nucleus at tlit level of the lateral geniculate nucleus (Bucher and Biirgi, 1953). Some fibers also continue to the pretectum and superior colliculus. The ventral supraoptic commissure includes both tectopetal and tectofugal fibers, according to Bucher and Biirgi (1953). The tectopetal fibers arise from the lateral midbrain, or more posteriorly, ascend in the lateral tegmentum, pass through the cerebral peduncle, run forward in the optic tract, decussate in the ventraI supraoptic commissure, and descend in the opposite optic tract to terminate ventrally to the medial geniculate nucleus or to terminate within the pretectum or superior colliculus. The tectofugal fibers arise from the superior colliculus ( Altman and Carpenter, 19611, pass forward in the optic tract, decussate, and descend in the opposite optic tract to terminate near the opposite medial geniculate nucleus or more posteriorly within the lateral tegmentum (Bucher and Burgi, 1953). Thus, the supraoptic commissures contain fibers which connect various brainstem centers with neurons bordering the contralateral primary optic centers ( Cragg, 1962f . The possible relationship of these fibers to retinopetal fibers will be discussed later.

E. 0m1c TRACT From the optic chiasm, the optic tract passes posteriorly and

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dorsally toward the thalamus where it divides into medioventral and dorsolateral components as it approaches the lateral geniculate nucleus, whose caudal extent is encapsulated by the tract. More caudally, the remaining optic fibers pass through the stratum zonale of the thalamus to form the brachium of the superior colliculus which extends to the mesencephalon. Optic nerve fibers from the nasal portion of the contralateral retina and from the temporal portion of the ipsilateral retina intermingle to form the optic tract ( Hayhow, 1958), maintaining, however, a general retinotopical organization. Minkowski ( 1920) and Overbosch (1927) have reported that fibers originating from the lower part of the retinas lie dorsolaterally in the optic tract and fibers from the upper part of the retinas lie ventromedially, as illustrated in Fig. 5. Bishop et al. (1953a) state that in the cat’s optic tract, the smaller fibers lie dorsally and the larger fibers lie ventrally, as determined both histologically and electrophysiologically. However, Hayhow (1958) more recently failed to find histological support for this finding. In addition to retinal fibers passing posteriorly in the optic tract, other fibers pass rostrally within the tract toward the supraoptic commissures. Although not confirmed histologically, Bishop and Clare ( 1955) electrophysiologically demonstrated 4 groups of fibers with different conduction rates within the optic tract of the cat and stated that these different groups of fibers are distributed to different synaptic regions. Thus, the two groups of faster fibers were found to synapse in the lateral geniculate nucleus, the intermediate group in the pretectum, and the smallest fibers in the midbrain, a finding in agreement with Harman and Berry (1956). They also found that within the lateral geniculate nucleus, the larger fibers synapsed in the upper two laminae of this nucleus (A, Al), and the smaller fibers in the lowest lamina ( B ) . Others, including Harman and Berry (1956) and Altman and Malis (1962), have also found longer latencies of photically-evoked potentials to the superior colliculus than to the visual cortex despite the geniculate synapse in the cortical pathway, However, it has also been demonstrated anatomically by Barris d al. (1935), OLeary (1940) and Glees (1941) that many large fibers entering the lateral geniculate nucleus have fine collaterals extending into the brachium of the superior colliculus and that many small fibers entering the brachium of the superior colliculus

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JAMES

M. SPRAGUE

have collaterals leaving the optic tract at almost right angles and ending in the lateral geniculate nucleus, Thus, some optic nerve fibers may innervate both mesencephalic and diencephalic structures by means of collaterals, as has recently been emphasized by Doty ( 1961).

1. Lateral Geniculata Nucleus (Pars Dmsalis) In the cat, the primary thalamic termination of optic fibers is the lateral geniculate nucleus which consists chiefly of a trilaminated pars dorsalis and a small, ventrolaterally placed pars ventralis. Thuma (1928) has described 3 principal cellular laminae of the pars dorsalis which he designated A, Al, and B, dorsal to ventral, and from transneuronai atrophy studies ( Minkowski, 1920; Barris, 1935; Silva, 1958), from Nauta studies (Hayhow, 1958), and from microelectrode electrophysiological studies ( Cohn, 1956), it has been established that crossed optic tract fibers terminate exclusively in laminae A and B whereas uncrossed optic tract fibers terminate exclusively in the intervening lamina A, of each lateral geniculate nucleus. Laminae A and A, receive predominantly optic tract fibers of large size and lamina B is supplied almost exclusively by optic tract fibers of small diameter (Bishop and Clare, 1955). Individual optic tract fibers end within a single cellular layer and in the adjaocnt interlaminar margin ( O’Leary, 1940). Each individual laminar cell probably makes synaptic contact with several different optic fibers, as shown by OLeary (1940), and each optic tract fiber of the cat probably terminates with extensive overlap in an area containing about 10 laminar or principal cells (Glees, 1941), as opposed to the situation in the monkey where there is apparently little or no overlap of terminals of different fibers (Glees and Clark, 1941). This overlap may provide in the cat a neuroanatomical basis for great sensitivity in low illumination. Between the laminae and along the medial border of the pars dorsalis are interlaminar cellular zones [nucleus interlaminaris centralis and nucleus interlaminaris medialis (Thuma, 1928) ] where an overlap between crossed and uncrossed optic tract fibers has been demonstrated histologically ( Hayhow, 1958; Fillenz, 1961) , and where binocular interaction has been shown electrophysiolog-

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ically (Bishop and Davis, 1953; Erulkar and Fillenz, 1960; Hubel and Wiesel, 1961). The microelectrode studies of Griisser and Saur (1960) have emphasized that the number of binocularly influenced geniculate neurons is small. Nevertheless, on this basis, it has been postulated that binocular interaction with fusion may occur at a thalamic level in the visual system of the cat. Determination of the numerical relationship between fibers of the optic tract and neurons within the lateral geniculate nucleus could have important functional implications concerning possible modifications to transmitted visual information at the thalamic level ( Brindley, 1960). However, experimental data are not available to indicate what percentage of optic tract fibers or their collaterals terminate within the lateral geniculate nucleus and what percentage continue to the midbrain, although von Monakow (1905) has estimated that about 80% of optic tract fibers terminate in the lateral geniculate nucleus. On this basis, in the cat about 95,000 optic fibers would be expected to synapse in the lateral geniculate nucleus, where Bishop (1953) has counted about 450,000 cells. This contrasts to similar studies in man (Bruesch and Arey, 1942; Balado and Franke, 1937), in the monkey (Bruesch and Arey, 1942; Clark, 1941; Chow et d.,1950), and in the rat (Lashley, 1939), where the number of optic tract fibers greatly exceeds the number of lateral geniculate neurons. In the cat, the relatively large number of lateral geniculate neurons may be accounted for by the presence of numerous short-axon cells, as described by Ram6n y Cajal (1904) and by OLeary (1940). These may serve as internuncials and provide the anatomical basis, through a reverberating circuit activated by recurrent collaterals from the optic radiations, for repetitive firing, as described in this nucleus by Bishop et al. (1953b). These shortaxon neurons apparently also persist in the lateral geniculate nucleus after striate decortication in the cat (Minkowski, 1911), whereas total lateral geniculate atrophy occurs in the rat (Lashley, 193413) and in the monkey (Polyak, 1957) after similar lesions. Marchi degeneration studies reported by Overbosch ( 1927) indicate a precise retinotopical organization of optic terminals within the pars dorsalis of the lateral geniculate nucleus as illustrated in Fig. 2, with the lower retinal quadrants represented dorsal to the upper and the temporal quadrants represented lateral to the nasal (see Brouwer, 1923).

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THOMAS H. MFXlUJ3, JR.AND JAMES M. SPRAGUE

VISUAL

DORSAL

L AT.

M ES

POST.

FIG. 2. RekinOtQpiCaI organization of the visual system of the cat (chiefly from Apter, 1945; Hoessly, 1947, Overbosch, 1927, Talbot and Marshall, 1941). It should be pointed out that the data on the optic nerve, optic tract, and lateral geniculate nucleus are derived from anatomical studies, and those on the striate cortex and the superior colliculus are derived chiefly from electrophysiologica1 work. The representation of all 4 retinal quadrants in each lateral geniculate nucleus is explained by the fact that Overbosch divided the retina by means

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2. Lateral Geniculate Nucleus (Pars VentraZis) Although Marchi studies (Barris et d.,1935), transneuronal atrophy studies ( Barris, 1935), and silver-stain degeneration studies (Glees, 1941; Silva, 1956; Altman, 196Z), all failed to find optic terminations within the pars ventralis of the lateral geniculate nucleus of the cat, Hayhow (1958) has demonstrated termination of both crossed and uncrossed optic fibers within this nucleus utilizing the Nauta stain, confirming earlier studies by Ram6n y Cajal ( 1904), Minkowski ( 1920), OLeary ( 1940), and Polyak ( 1957). O'Leary (1940) described terminations within the pars ventralis of collaterals from thin optic tract axons which synapsed in the pars dorsalis. Altman and Carpenter (1961) have shown that this nucleus also receives afferent input through ascending fibers from the superior colliculus, and Altman (1962) has described afferents to this nucleus from the pars dorsalis of the lateral geniculate nucleus. 3. Other Thalumic Nuclei

Considerable conflicting data have been reported for many mammalian species regarding the termination of optic fibers in thalamic nuclei other than the lateral geniculate nucleus, particularly in the pulvinar and its related nucleus, the lateralis posterior. Negative findings have been reported, utilizing the Marchi stainby Barris et al. (1935) in the cat; by Clark (1931) and by Lashley (1934a) in the rat; by Packer (1941) in the phalanger; and, utilizing the Nauta stain, by Hayhow et al. (1962) in the rat. On the other hand, positive findings have been reported by Minkowski (1913, 1920) in the cat on the basis of transneuronal atrophy, by Polyak (1957) in the rat from Marchi studies, and by Nauta and Van of lines drawn through the optic disc rather than through the area centralis. Had he used the area centralis, presumably each lateral geniculate nucleus would receive fibers from only 2 retinal quadrants, as is shown in this figure for the optic tract and the striate cortex. Transverse sections: (1,l') retina; (2,2') optic nerve; (3,3') optic tract; (4,4') rostra1 part of lateral geniculate nucleus; (5,s)medial part of lateral geniculate nucleus; (6,6') caudal part of lateral geniculate nucleus. Lat.: lateral view of left cerebral hemisphere. Mes.: mesial view of left cerebral hemisphere. Post.: posterior view of cat's brain with midbrain transected below the inferior colliculi. Sup. Coll.: dorsal view of superior colliculi. Vis. Cort.: visual cortex indicated on dorsal view of cerebral hemispheres.

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THOMAS H. ME[E;LE, JR. AND JAMES M. SPRAGUE

Straaten (1947) in the rat from Nauta studies. Polyak (1957) was uncertain, from his Marchi work, whether optic fibers terminate in the pulvinar of the cat. However, recent studies utilizing the Nauta technique ( Altman, 1962) and electrophysiological methods ( Bishop and Clare, 1955) have described a small number of optic fibers terminating in a nuclear mass just medial to the pars dorsalis of the lateral geniculate nucleus. This region, which lies in the same general area of the pulvinar and the lateralis posterior nucleus, includes the cellular masses designated the posterior nucleus of the thalamus in the cat by many investigators (Rioch, 1929; Ingram et al., 1932; JimenezCastellanos, 1949; Jasper and Ajmone-Marsan, 1954). This same thalamic area appears to be involved in the transmission of photically evoked potentials recorded in the associational cortex of the suprasylvian gyrus by Buser d al. ( 1959) and Vastola (1961). These findings, taken together, seem to justify including the nucleus posterior as a primary optic termination in the cat. G.

PRETECruM

Medial to the lateral geniculate nucleus, optic fibers, mostly of fine caliber and including collaterals, pass through the brachium of the superior colliculus and through the stratum zonale of the thalamus dorsal to the pulvinar toward the pretectal region terminating chieffy in the large-celled nucleus of the optic tract (nucleus lentiformis mesencephali, Ingram, Hannett and Ranson, 1932) and, to a lesser extent, in the small-celled pretectal nucleus of the cat (Altman, 1962). Optic fiber terminations, mostly from the contralateral retina, have been described in this region by Barris et al. (1935) and by Minkowski (1920) in the cat, as well as by Tsai (1925) in the opossum, by Lashley (1934a) and by Nauta and Van Straaten (1947) in the rat. Retinal fiber terminations within the large-celled nucleus of the optic tract have been specifically noted in this region by Nauta and Van Straaten (1947) and by Hayhow et al. (1962) in the rat; whereas Barris et al. (1935) found Marchi degeneration in this nucleus only after visual cortex lesions in the cat. There is no evidence in the cat of optic fiber terminations in the nucleus of the posterior commissure or the oculomotor nuclear complex.

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13. SUPERIOR COLLICULUS

Caudal to the pretectum, the stratum zonale of the thalamus becomes continuous with the brachium of the superior colliculus in which optic fibers, lying dorsal to the more ventral corticotectal fibers, course toward the tectum where most enter the stratum opticum of the superior colliculus. The fibers of the brachium of the superior colliculus form an arc around the rostra1 border of the superior colliculus and enter the stratum opticum in a radial manner, forming medial and lateral branches of the brachium when viewed in cross section. Upon entering the stratum opticum, both crossed and uncrossed optic fibers terminate within this layer and within the adjacent gray layers of the superior colliculus (stratum griseum superficialis and stratum griseum intermediale ) , as shown anatomically by Altman ( 1962) and electrophysiologically by Altman and Malis ( 1962). This pattern of distribution has been demonstrated also in the rat (Tsang, 1936; Nauta and Van Straaten, 1947; Hayhow et al., 1962), in the rabbit ( Minkowski, 1913), and in the opossum (Bodian, 1936). No optic fibers decussate at this level. Both anatomical data (Barris et d., 1935; Altman, 1962) and physiological data (Apter, 1945; Altman and Malis, 1962) indicate that many more optic fibers originating from the contralateral retina than from the ipsilateral retina terminate within the superior colliculus of the cat. Apter ( 1945) has demonstrated electrophysiologically a systematic projection of the temporal retina upon the ipsilateral superior colliculus and the nasal retina upon the contralateral superior colliculus with the homonymous lower part of the retinas represented medial to the homonymous upper part of the retinas, as seen in Fig. 2. These results have been confirmed anatomically in the cat by Hoessly (1947), and are similar to Marchi studies in the rat (Lashley, 1934a), in the rabbit (Brouwer et al., 1923), and in the opossum (Bodian, 1936).

I. ACCESSORYOPTICTRACT Although early figures by Gudden ( 1881) clearly illustrated atrophy in the tractus peduncularis transversus (posterior accessory optic tract) following enucleation in the cat, most subsequent

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THOMAS H. MJ!XKLE, JR. AND JAMES M. SPRAGUE

studies of this animal failed to find experimental evidence of an accessory optic tract system (Probst, 1900; Minkowski, 1913; Barris et al., 1935; Gillilan, 1941), in contrast to rodents and marsupials where such a tract has long been established (Munzer and Wiener, 1902; Clark, 1932; Lashley, 1934a; Gillilan, 1941; Nauta and Van Straaten, 1947; Hayhow et al., 1960). However, recent work by Hayhow (1959), using the Nauta stain, has clearly demonstrated an accessory optic tract in the cat consisting of finely myelinated fibers arising from the contralateral retina. These fibers separate from the brachium of the superior colliculus, where some almost immediately end in the dorsal terminal nucleus of the accessory optic tract; others proceed ventrally to the lateral border of the cerebral peduncle where more fibers terminate in the lateral terminal nucleus, and the remainder transversely cross the lateral and ventral surfaces of the peduncle to terminate in the medial terminal nucleus, just rostra1 to the attachment of the ocuiomotor nerve. This accessory optic tract system together with its terminal nuclei represent the presently recognized final terminations in the mesencephalon of optic fibers from the retina of the cat. No additional data on this system are presently available for the cat, but Giolli (1961) has identified an accessory optic tract in the rabbit; and, using the Nauta stain, he has reported that only onehalf to two-thirds of the fibers within this tract originate from the retina. According to this study of the rabbit, at least S10%of the fibers comprising this tract were found to originate from the region of its medial terminal nucleus (nucleus of the transpeduncular tract) and pass forward into the ipsilateral optic tract. No fiber degeneration, however, was reported in the optic nerves. An anterior accessory optic tract has been described arising from the main optic tract at the posterior extremity of the optic chiasm and passing over and through the cerebral peduncle to end in the subthalamic nucleus (Clark, 1932). In the cat, as in the rat and phalanger, any optic fibers which take this course are now thought to terminate in the medial terminal nucleus of the (posterior) accessory optic tract (nucleus opticus tegmenti) . Thus, Lashley (1934a) and Packer (1941) consider these fibers to be aberrant fibers from the (posterior) accessory optic tract, and Hayhow d al. (1960) designated these fibers as the inferior fasciculus of the (posterior) accessory optic tract.

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IV. Mesencephalic Projections

A. PRETEC~UM As described above, the pretectal area receives its primary afferent supply from optic fibers of intermediate size (Bishop and Clare, 1955). In the cat and monkey, Hare et al. ( 1935) have traced optic nerve fibers mediating the pupillary light reflex into the pretectum where these fibers synapse. In addition to these retinal fibers, some fibers from the ventral supraoptic commissure terminate in the pretectum (Bucher and Biirgi, 1952), and the pretectum may also receive finely myelinated fibers from the dorsal supraoptic commissure which pass through this region to the reticular nucleus of the thalamus. The termination of ascending fibers from the superior colliculus within the pretectum has been described by Altman and Carpenter (1961) in Nauta-stained material, and Banis (1936) has demonstrated that fibers arising from a restricted area of the cat’s cerebral cortex on the inferior part of the posterolateral gyrus pass mainly to the pretectum, and to the superior colliculus and lateral pontine nucleus. According to Banis (1936), electrical stimulation of either the pretectum or of this small cortical region produces pupillary constriction. Barns et al. (1935) and Polyak (1927), using the Marchi method, found no corticofugal supply to the small-celled nucleus of the pretectum from the striate or parastriate regions in the cat, but Altman (1962), using the Nauta stain, has recently described the termination of fibers from these cortical areas within this nucleus. Both Barris d al. (1935) and Altman (1962) have described the termination of fibers from these same cortical areas within the large-celled nucleus of the optic tract. Clark (1932) has also described Marchi degeneration in the pretectum of the rat after cortical lesions. Bilateral destruction of the pretectum permanently abolishes the pupillary light reflex (Magoun and Ranson, 1935), whereas after destruction of the superior collicuIus, the pupillary reactions to light remain normal and unimpaired (Magoun, 1935). The majority of pupillary constrictor fibers originating from the pretectal nucleus decussate in the posterior commissure (Magoun et al., 1935) or by other commissures ventral to the central gray matter to reach the Edinger-Westphal division of the oculomotor nucleus. However, in the cat, some fibers do not cross but synapse in the

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THOMAS H. MEMLE, JR. AND JAMES M. SPRAGUE

ipsilateral nucleus ( Magoun and Ranson, 1935). From the EdingerWestphal nucleus, fibers of third-order neurons pass to the ciliary ganglion through the oculomotor nerve. Barris (1936) has traced efferent pretectal fibers into the white matter underlying the posterolateral gyms of the cat (Brodmann areas 19, 36), and Papez and Freeman (1930) also observed corticopetal fibers from the pretectum of the rat but were unable to determine their cortical destinations in Marchi studies. In addition to receiving afferent fibers which form the ventral supraoptic commissure, the pretectum also supplies fibers which course through this commissure in the reverse direction (Bucher and Burgi, 1953). Other efferent connections of the pretectal nucleus include a pretectotegmental fascicle which passes to the substantia nigra and has been described in the opossum (Tsai, 1925; Bodian, 1936), in the hedgehog (Clark, 1932), and in the cat (Bucher and Burgi, 1952). Although the pretectum topographically is a transitional region between the diencephalon and mesencephalon, its cortical projection reveals its diencephalic affinity.

B. SUPERIOR COLLICVLUS Excluding the deeper-lying periaqueductal gray matter, the mammalian superior colliculus consists of 7 laminae, 4 predominantly fiber layers (strata zonale, opticum, album intermediale, and album profundum ) separating and alternating with 3 intervening cellular laminae (strata griseum superficialis, griseum intermediale, and griseum profundum), as described by Huber and Crosby ( 1943) and Burgi ( 1957). The stratum lemnisci variously represents groupings of the deeper layers of the colliculus, most commonly the stratum griseum intermediale and stratum album intermcdiale, whereas the stratum griseum profundum and stratum album profundum are frequently referred to as the “stratum profundum,” which is intimately related to the underIying outer part of the central gray matter. Afferent connections of the superior colliculus of the cat enter chiefly through the stratum opticum or the stratum album intermediale (stratum lemnisci) . Through the stratum opticum pass retinotectal fibers dorsally, corticotectal fibers ventrally, and fibers arising from the contralateral brainstem which cross in the ventral supraoptic commissure (Huber and Croshy, 1943; Burgi, 1957). The retinotectal fibers enter the adjacent gray layers (stratum griseum

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superficialis and griseum intermediale), but do not rise to the more superficial stratum zonale. This latter layer contains only corticotectal fibers which also ramify in the deeper-lying layers (Probst, 1901; Polyak, 1927; Mettler, 1932; Barris et d.,1935; Gobbell and Liles, 1945; Beresford, 1961; Altman, 1962). Additional input to the superior colliculus includes ascending fibers from the brainstem and spinal cord through the spinotectal tract, collaterals from the ventral secondary trigeminal tract, and fibers from the reticular systems, all entering through the stratum album intermediale and ramifying in the adjacent gray layers (Huber and Crosby, 1943; Morin et al., 1951; Burgi, 1957; Nauta and Kuypers, 1958; -4nderson and Berry, 1959). Other afferent connections which have been reported include a nigrotectal tract (Rioch, 1930), an incertotectal tract (Rioch, 1930; Burgi, 1957), a habenulotectal tract (Nauta, 1958), and fibers from the adjacent central gray matter (Nauta, 1958). The efferent connections of the superior colliculus of the cat have been studied utilizing the Marchi technique (Rasmussen, 1936; Marburg and Warner, 1947; Bucher and Burgi, 1950; Pearce and Glees, 1957), and utilizing the Nauta technique ( Altman and Carpenter, 1961). The latter authors have reported three principal descending tectofugal fiber bundles. The largest descending bundle consists of laterally placed, uncrossed efferent fibers which terminate chiefly within the dorsolateral pons. From this region, Brodal and Tansen (1946) have reported fibers passing to the same cerebellar bermal regions in which Snider (1950) has found photically evoked electrophysiological potentials. An intermediately placed, mostly uncrossed, tectofugal bundle descends rather diffusely to end chiefly within the mesencephalic reticular formation. Medially, descending tectofugal fibcrs consist of a crossed predorsal bundle, partially terminating within the nucleus of Darkschewitsch and the nucleus of Cajal, and the medial reticular formation, then continuing ventral to the median longitudinal fasciculus to supply the paramedian reticular formation of the medulla, and finally forming the tectospinal tract distributed to anterior horn regions as far as the lower cervical segments. Thus, descending tectofugal fibers in the cat distribute chiefly to the reticular formation-as has also been reported in electrophysiological studies by Pearce (Jefferson, 1958)and to the pontine nuclei. These projections are illustrated in Fig. 3. Ascending tectofugal fibers are grouped to form dorsal, medial

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THOMAS H. MEIKLE, JR. A S D JAMES M. SPRAGUE

5 u pro o p t IC

To

C o $71m

Cortex--’

Retic Form

R e t i c Form To C e r e b e l l u m

P o n t Nucl

TECTOSPINAL TRACT

FIG.3. Mesencephalic fiber projections related to the visual system indicated on dorsal view of the brainstem (after Thuma, 1928) with optic tracts, optic

nerves and eyeb. Interrupted lines indicate postulated pathways not conclusively established. LGNa: lateral geniculate nucleus, pars dorsalis. LGN,: lateral geniculate nucleus, pars ventralis. LP: lateralis posterior nucleus. MGN : medial geniculate nucleus. Pont. Nucl. : pontine nucleus. Pret: pretectum. Pulv: pulvinar. Retic. Form.: reticular formation. Subthal.: subthalamus. Sup. Coll.: superior colliculus. Supragen: suprageniculate nucleus. Supraoptic Comm.: Supraoptic Commissures.

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and ventral bundles (Altman and Carpenter, 1961) which pass forwa.rd to terminate in the pretectum, suprageniculate nucleus, magnocellular portion of the medial geniculate nucleus, pulvinar, and the lateralis posterior nucleus of the thalamus. The dorsal ascending bundle also terminates within the ventral part of the lateral geniculate nucleus. The ventral ascending bundle passes rostrally in the optic tract to decussate in the ventral supraoptic commissure. Additional ascending tectofugal fibers in the cat have been reported by Bucher and Biirgi (1950) to the centrum medianum nucleus and Field H, and by Marburg and Warner (1947) to the habenula and substantia nigra. No tectoretinal fibers have been reported in the cat, and no tectofugal fibers have been found to terminate directly upon any cranial nerve nuclei (Rasmussen, 1936; Marburg and Warner, 1947; Hiirgi, 1957), the dorsal part of the lateral genicdate nucleus, or the cerebral cortex.

c. &,TICULAR

FORMATIOX

With the recent electrophysiological work on the functional significance of the brainstem reticular formation, considerable data now suggest that the reticular formation is a region of convergence and centrifugal regulation of specific sensory information (Starzl et al., 1951; Magoun, 1952; Amassian and Devito, 1954; Scheibel et al., 1955; French, 1960). Collaterals from the specific sensory systems, including tactokinesthetic, auditory, and olfactory, as well as visual, converge on and are presumably integrated in the reticular formation, which, it has been postulated, discharges a centrifugal feedback to these same specific sensory systems as a means of regulating information arriving from peripheral receptors. Convergence of visual information and other sensory information in the reticular formation has been suggested mostly by the work of Hernhdez-Pe6n and his associates. They have reported that light-evoked potentials are diminished in the optic tract when cats behaviorally attended to stimuli other than visual ( HernAndez-Pe6n et al., 1957), and that touch-evoked potentials are diminished in the spinal tract of the trigeminal nerve when cats attended to visual stimuli (Hernlndez-Pe6n, 1962). Although, in the cat, there is no evidep.ce of direct termination of retinal fibers within the brainstem reticular formation, this region does receive a rich input of fibers descending from the superior colliculus ( Altman and Carpenter,

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THOMAS H. MEIKLE, JR.AND JAhlES M. SPRAGUE

1961), a primary optic terminus. In addition, the terminal nuclei of the accessory optic tract system may also provide internuncials between optic fihcrs and the mesencephalic reticular formation. Thus, there is firm anatomical evidence for considerable visual input to the reticular formation where visual information may converge and interact with information from other sensory systems. Centrifugal control of visual information by means of specific fibers to visual synaptic sites in the retina, lateral geniculate nucleus, and visual cortex has been disputed for many years. However, present data seem to suggest this type of control in the visual system as in other sensoiy systems (Livingston, 1959; Desmedt, 1960). 1. Retina

Anatomicnlly, in Golgi material, Rarn6n y Cajal (1904) demonstrated fine optic nerve terminations among amacrine cells in the retina of the dog and he postulated that these were centrifugal fibers to the retina. This finding was later confirmed by Polyak (1957) in the chimpanzee. In addition, Elinson (1896, cited in Brindley, 1960) has reported Wallerian degeneration in the distal as well as the proxima1 stump of optic nerves sectioned in dogs and cats, and Fillenz and Glees ( 1961) in the cat and Wolter and Liss ( 1956) in man have reported intact optic nerve fibers in the proximal stump of sectioned optic nerves. These anatomical &dings, suggesting centrifugal optic nerve fibers, differ, however, from the negative findings reported by Bodian ( 1936) in the opossum and by Iless (1958) in the fetal guinea pig. More recently, Cragg (1962) has found, after crushing the rabbit’s optic nerve and utilizing Glees and Nauta stains, small numbers of terminal boutons as well as fine degenerated fibers in the retina and optic nerve stump which he has cautiously interpreted as being centrifugal. Like Brindley and Hamasaki ( 1961), Cragg noted that longer than usual Nauta degeneration times were required to demonstrate degenerating optic fibers distal to the crush. In an attempt to identify the source of these possible centrifugal fibers, which he has suggested may arise as collaterals from fibers decussating in the supraoptic commissures, Cragg destroyed portions of the superior colliculus, dorsolateral thalamus, and mesencephalic reticular formation, with resultant degeneration in various fiber components of the supraoptic commissures. However, he concluded that his ana-

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tomical evidence only suggested that fibers within the supraoptic commissures sent collaterals centrifugally to the retina through the optic nerve. Nevertheless, if centrifugal fibers to the retina do exist, it seems likely that they may originate from the reticular formation and pass through this well-recognized fiber system of the cat. Physiologically, Granit (1955) has reported in the cat that electrical stimulation of the mesencephalic reticular formation can potentiate or inhibit retinal ganglion cell sensitivity to light, and he postulated centrifugal control to these cells although he did not compIetely exclude, as the basis of his results, the possibility of antidromic effects on other retinal neurons or retinal vascular alterations produced by sympathetic fibers from the reticular formation. (See Tucker and Beidler, 1956 for such effects in the olfactory system.) Dodt (1956) recorded from the rabbit's retina while stimulating the contralateral optic tract and found, in addition to the expected antidromic responses, spikes with longer latencies which he identified as being centrifugal in origin. Miiller-Limmroth (1954) has reported that if one eye of the guinea pig is illuminated, a slow electrical response in the contralateral eye is abolished if the lateral geniculate nuclei are destroyed, thereby presumably eliminating centrifugal control. Finally, Jacobson and Gestring (1958) have shown that the effects on the electroretinogram of cats and monkeys caused by several different central nervous system stimulants and depressants can be abolished by section of the optic nerve, again suggesting centrifugal fibers to the retina which mediate these electrical changes. Considered together, the anatomical and physiological evidence, although not conclusive, is suggestive of centrifugal retinal control. 3. Lateral Geiiicuhte Nucleus

In addition to possible centrifugal control of visual information at the retinal level, considerable data now suggest that the brainstem reticular formation exerts centrifugal control of sensory transmission through the lateral geniculate nucleus, and an anatomical basis for a projection to the lateral geniculate nucleus from the reticular formation has been provided by the Golgi studies of Scheibel and Scheibel (1958). Physiologically, Chavaz and Spiegel ( 1957) have demonstrated rhythmic potentials within the lateral geniculate nucleus both before and after section of the optic nerve,

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and a “second input” (in addition to optic fibers) to this nucleus from the reticular formation has been postulated by Arden and Soderburg (1962) who have inhibited evoked potentials in the lateral geniculate nucleus of the rabbit by direct stimulation of the inesencephalic reticular formation. Hubel ( 1960), recording from the lateral geniculate nucleus of unrestrained cats, also found that behavioral arousal could modify activity of single neuronal units in this nucleus. Diminution or habituation of evoked responses in the lateral geniculate nucleus of the cat has been reported by HernLndez-Pebn and his associates ( Hernhndez-Pe6n et al., 1956; HernLndez-Pebn et al., 1958), and the work of Dumont and Dell (1958) also suggests that the lateral geniculate nucleus can be influenced by pathways other than the optic tract. From their work, Arden and Soderberg ( 1969) have suggested that reticulogenicd a t e influences are responsible for most of the resting activity within the lateral geniculate nucleus and for most of the alteration in visual information as it is transmitted through this nucleus.

3. Vimd Cortex *41though visual cortex responses altered by stimulation of the reticular formation (Lindsley, 1958) may be secondary to alterations within the lateral geniculate nucleus (Bremer and Stoupel, 1959), the work of Jung and his collaborators (1958, 1962) suggests that there is convergence of geniculate, vestibular and nonspecific thalamic efferents upon some visual cortex neurons [as is also suggested by the findings of Dumont and Dell (1958)l. Fox and OBrien (1962) have shown that caudate nucleus stimulation, like stimulation of the reticular formation, can alter the photically evoked potentials recorded in the visual cortex. Thus, both specific and nonspecific afferents have been found important to maintain normal activity of the visual cortex. Visual cortex neurons can maintain normal activity if either the specific geniculate or the nonspecific brainstem influence is disrupted, but visual cortex activity drops almost completely when both influences are eliminated. Thus, retinal, lateral geniculate and visual cortex neurons all seem capable of being influenced presumably by centrifugal pathways from the reticular formation where convergence of visual information occurs with information from other sensory systems.

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V. Thalamic Projections

A. LATERAL GENICULATE NUCLEUS(PAM DORSALIS) Although the primary afferent supply to the lateral geniculate nucleus consists of optic fibers, both principally and as collaterals from fibers passing to the mesencephalon (Barris et d.,1935; OLeary, 1940; Glees, 1941 ) , considerable electrophysiological data suggest that the mesencephalic reticular formation also projects to the ipsilateral lateral geniculate nucleus. In addition, this nucleus receives corticofugal fibers from the striate area of the cerebral cortex [ Reresford, 1961; Nauta (personal communication cited by Wid& and Ajmone-Marsan, 1961); Altman, 19621, as has also been reported in the rat by Nauta and Bucher ( 1954). From the lateral geniculate nucleus, efferent fibers pass chiefly through the occipital radiations to the cerebral cortex where their principal terminations lie within the histologically defined striate area of the lateral gyrus. From retrograde degeneration studies (Minkowski, 1913), the projection fibers from the lateral geniculate nucleus terminate in this area in a rather precise manner, the medial part of this nucleus projecting to the medial part of the striate area, the lateral part of the nucleus to the lateral striate area, and the rostral and caudal parts of the lateral geniculate nucleus to the rostral and caudal parts, respectively, of the striate area. Efferents from the lateral geniculate nucleus terminate outside the striate area, however, as was early proposed by von Monakow (1882) and demonstrated by Minkowski ( 1913) and Polyak ( 1927). Electrophysiological evidence in the cat (Gerard et aZ., 1936; Marshall et al., 1943; Hunter and Ingvar, 1955; Harman and Berry, 1956; Buser et al., 1959) has also suggested an extensive visual projection to the cortex which, in addition to the striate area, includes the remainder of the lateral and posterolateral gyri and most of the middle and posterior suprasylvian gyri. Indeed, Doty (19%) has reported photically evoked potentials of greatest amplitude in the cortex in a region (“A”) of the lateral gyrus lying outside the striate area, and ablation of this region failed to produce atrophy in the lateral geniculate nucleus. In addition, complete ablation of the striate area in the cat apparently does not result in total degeneration of the dorsal part of the lateral geniculate nucleus (Minkowski, 1911),

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unlike the monkey (Polyak, 1933) and the rat (Lashley, 1934b). Dov (1961) has found that complete atrophy of the lateral geniculate nucleus of the cat results when the splenial and posterior suprasylvian gyri are ablated in addition to the striate regions of the lateral and posterolateral gyri, and the results of Smith (1938, 1939) tend to confirm this. The persistence of lateral genicuIate neurons in the cat after striate removal most likely indicates a cortical projection from the lateral geniculate nucleus that is more extensive than the striate area. However, it may also reflect an alternate afferent input to lateral geniculate neurons from the mesencephalon or from internuncials within the nucleus. Nevertheless, in the cat, the fastest-conducting fibers from the lateral geniculate nucleus terminate in the striate area and the immediately adjacent paraperistriate areas, and Bishop and Clare (1955) have suggested that these fibers arise from layers A and A, of the lateral geniculate nucleus. From layer B, these authors have described postsynaptic responses occurring within the lateral nucleus of the thalamus, suggesting an intrathalamic associational pathway for visual impulses from the lateral geniculate nucleus to the lateral thalamic nucleus. It should be noted, however, that neurons in layer B do degenerate following cortical lesions (Minkowski, 1913). In lower mammals, Clark (1932) has stated that intrathalamic connections exist between the lateral geniculate nucleus and the ventral thalamic nucleus, and Tsai (1925) postulated associational pathways between the lateral geniculate nucleus and the ventral and lateral thalamic nuclei of the opossum. On the other hand, Barris et al. (1935) found no Marchi evidence of an intrathalamic projection from the lateral geniculate nucleus of the cat after Frevious long-standing enucleations had caused complete degeneration of optic tract fibers. However, more recently, Altman ( 1962), using the Nauta method, has demonstrated intrathalamic projections from the dorsal part of the lateral geniculate nucleus to the ventral part of the same nucleus, to the posterior nucleus, to the suprageniculate nucleus, and to the lateralis posterior and lateralis dorsalis nuclei. Furthermore, it may be significant to note that the primary cortical projection from the lateral thalamic nucleus of the cat is the middle suprasylvian gyrus (Waller and Barns, 1937) where photic stimuli have been shown to evoke responses of longer latency than in the striate area of the lateral gyrus (Doty, 1958;

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Vastola, 1961). Similarly, Buser et al. (1959) have postulated that visually-evoked suprasylvian responses originated from the posterior or lateralis posterior nucleus of the thalamus which, in turn, was innervated by fibers from the lateral geniculate nucleus. Thus, present data seem to suggest that visual information may be relayed directly to the striate and immediately adjacent parapenstriate areas from the lateral geniculate nucleus producing short-latency highamplitude cortical potentials. An indirect relay from the dorsal part of the lateral geniculate nucleus through other thalamic nuclei to paraperistriate regions and other cortical areas, including the cingulate gyrus (Ingvar and Hunter, 1955; Harmari and Berry, 1956) may produce the more widespread, longer latency, lower amplitude cortical “photic” potentials. Only by future combined anatomical and physiological investigations of these various thalamic nuclei and their cortical projections can these relationships be clarified. Projections from various thalamic nuclei are shown in Fig. 4. Although Barns et al. (1935) failed to find anatomical evidence of direct geniculotectal pathways in the cat, Altman (1962) has indicated the existence of projections from the dorsal part of the lateral geniculate nucleus to the superior colliculus and to the pretectum, confirming earlier findings in the rat by Clark (1931). Harman and Berry (1956) have also interpreted their electrophysiological data in the cat to be suggestive of a direct geniculotectal connection.

B.

LA?ERAL

GENICULATE NUCLEUS (PARS VEhTRALIS)

The presently recognized afferent supply to this nucleus consists of retinal fibers from the optic tract (Polyak, 1957) including collaterals from fibers terminating in the dorsal part of the lateral geniculate nucleus ( OLeary, 1940; Hayhow, 1958) and ascending projections from the superior colliculus ( Altman and Carpenter, 1961). In addition, Altman ( 1962) has reported considerable projection to the pars ventralis from the pars dorsalis of the lateral geniculate nucleus and has found that combined lesions of the pars ventralis and pars dorsalis resulted in degeneration in the centrum medianum nucleus, the thalamic reticular nucleus, and the subthalamus, areas in which no degeneration was found after lesions limited to the pars dorsalis. These projections from the pars ventralis may provide an anatomical basis for the photically evoked potentials in

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FIG. 4. I’halaniic fiber projections rclated to the visual system indicated on dorsal view of the thalamus and midbrain (after Thuma, 1928) and on dorsal and lateral vie\vs of the cerebral cortex of the cat. The striate area (after O‘I,cary, 1941) is cross-hatched on the dorsal view of the right hemisphere, and visiinl areas V, and ?,’ (after Woolsey, 1962) are cross-hatched on the lateral view of the lcft hemisphere. ( 1) lateral gyrus; ( 2 ) posterolateral gyrus; ( 3 ) middle suprasylvinn gyrus; ( 4 ) posterior suprasylvian gyrus; ( 5 ) ectosylvian gyrus. CM: centrum medianum nucleus. LGNe: lateral geniculate nucleus, pars dorsalis. LGN, : lateral genicnlate nucleus, pars ventralis. LP: lateralis posterior nuckus. Post.: posterior nucleus of the thalamus. Pret.: pretrctum. Pulv: pulvinar. Retic. Nucl.: rcticular nucleus of the thalamus. S. Coll.: superior collicuh~s. Subthal.: subtliala~nus.Supragen.: supragc,~~in~latc.lt~ nucleus. \’,: visual area 1. V:: visual area 2 .

the thalamic reticular nucleus reported by Negishi and Verzeano (1961) and in the centrum medianum nucleus reported by Ingvar and Hunter (1955). Waller and Barns (1937) reported no retrograde degeneration in the pars ventralis of the lateral geniculate nucleus after large cortical ablations. Hence, the available evidence suggests that this nucleus has no direct cortical projections.

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C. OTHERTHALAMIC NUCLEI

As stated above, from their studies of retrograde thalamic degeneration following cortical lesions in the cat, Waller and Banis (1937) have indicated that the lateral thalamic nucleus projects to the rostra1 part of the middle suprasylvian gyms and the pulvinar projects to the more caudal part of this same gyrus; from both of these cortical areas photically evoked potentials can be recorded. In addition, Altman (1962) has described projections from the pulvinar to the superior colliculus which, in turn, projects back to the pulvinar ( Altman and Carpenter, 1961) . VI. Neocortical Projections

A. LATERAL GYRUS

In the cat, the striate area, characterized by the prominent Stria of Gennari in lamina 1% (Brodmann, NOS), is confined to the dorsoniedial part of the posterior third of the cerebral cortex within the lateral, posterolateral, suprasplenial, and splenial gyri from the base of the splenial sulcus to the middle of the lateral gyrus rostrally and to the base of the posterolateral sulcus caudally (Campbell, 1905; Brodmann, 1906; h/Iinkowski, 1911; Rambn y Cajal, 1922; Gurewitsch and Chatschaturian, 1928; OLeary, 1941; Harman and Berry, 1956). Numerous other investigators, including Talbot and Marshall ( 1941), Marshall et aZ. ( 1943), Garol ( 1942), Doty ( 1958), and Uoty and Grimm (1962), however, have demonstrated that, in the cat, the primary visual projection area exceeds the boundaries of the striate cortex since short-latency, photically evoked potentials can be recorded in cortical regions outside of but adjacent to the histologically defined striate area (Fig. 4). A highly localized retinotopical organization from the dorsal part of the lateral geniculate nucleus to the primary visual cortex has been established histologically by Minkowski ( 1913), and electrophysiologically by Talbot and Marshall (1941), and Hubel and Wiesel (1962). This retinotopical projection in the cortex is apparently limited to the shortlatency responses, in contrast to the more diffuse distribution of the long latency potentials (Doty, 1958). The primary afferent supply to the striate area of the lateral gyrus in the cat includes the optic radiations from the dorsal part of the lateral geniculate nucleus (Minkowski, 1913; Polyak, 1927;

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Waller and Barris, 1937), which, according to Ram6n y Cajal (1922), Polyak ( 1927), O’Leary (1941), and Scholl (1953, 1955), pass into Gennari’s line, there to ramify extensively. Scholl (1955) concluded that each geniculate fiber may influence up to 5000 neurons within a volume of 0.1 nun3 of cortex. Other afferents to this region arise as associational fibers from nearby parts of the lateral gyrus, and from adjacent suprasylvian, ectosylvian, and sylvian gyri, and as commissural fibers passing through the posterior corpus callosum from these same cortical areas in the contralateral hemisphere (Polyak, 1927; Mettler, 1932; Beresford, 1961). From the lateral gyrus, efferents are distributed to adjacent areas within the same gyms and to the ipsilateral suprasylvian gyrus near the middle ectosylvian gyrus (Clare and Bishop, 1954; Marshall ct nl., 1943), and to ectosylvian gyri, as well as to the lateral and suprasylvian gyri of the contralateral hemisphere ( Polyak, 1927; Beresford, 1961) . Polyak ( 1927) reported no direct connections from the lateral gyrus to the frontal sensorimotor regions. Efferent projections from the lateral gyrus are illustrated in Fig. 5. Subcortically, efferents from the lateral and posterolateral gyri enter the retrolenticular part of the internal capsule and pass through the medial part of the superior thalamic radiation eventually reaching the brachium of the superior colliculus. Barris et al. ( 1935), using the Marchi technique, reported degeneration after lateral gyrus lesions in the superior colliculus, nucleus of the optic tract, and in the pons but no degeneration in the lateral geniculate nucleus or other thalamic nuclei. Utilizing the Nauta stain, Beresford ( 1961) and Altman (1962) have reported subcortical projections from the lateral and posterolateral gyri to the ipsilateral superior colliculus, entering laterally the deep part of the stratum opticum (and to a slight extent the stratum zonale) and terminating in the superficial and intermediate gray strata. Other terminations reported were the nucleus of the optic tract, the pretectum, the lateral geniculate nucleus (layers A and Al, and the intervening interlaminar area) and the posterior nucleus of the thalamus. Additional apparent sites of termination of corticofugal fibers were the suprageniculate nucleus, the reticular nucleus of the thalamus, and the pontine nuclei. R’idkn and Ajmone-Marsan (1961) have cited evidence by Nauta that the lateral gyrus in the cat sends fibers to both dorsal and especially ventral parts of the lateral geniculate nucleus. In the rat, Nauta and Bucher (1954) have reported cortico-

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FIG. 5. Ncocortical fiber projections related to the visual system indicated on dorsal view of the cerebral hemispheres and brainstem (after Thuma, 1928).

Cross-hatched area on the dorsal view of the right hemisphere outlines the striate area (after O’Leary, 1941). ( 1 ) lateral gym; ( 2 ) middle suprasylvian gyrus; ( 3 ) anterior ectosylvian gyrus; ( 4 ) middle ectosylvian gyrus; ( 5 ) posterior ectosylvian gyms. LGNd: lateral geniculate nucleus, pars dorsalis. LGN,: lateral geniculate nucleus, pars ventralis. LP: lateralis posterior nucleus. Nucl.OT: nucleus of the optic tract. Pont. Nucl.: pontine nucleus. Post. Nucl.: posterior nucleus of the thalamus. Pulv. pulvinar. Retic. Form.: reticular formation. Retic. Nucl.: reticular nucleus of the thalamus. Sup. CoU.: superior colliculus; Supragen.: suprageniculate nucleus.

fugal projections to the lateral geniculate nucleus, particularly the ventral part, as well as to the lateralis posterior nucleus, pretectum, subthalamus, superior colliculus, and pons.

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Niemer and Jimenez-Castellanos ( 1950), utilizing strychnine neuronography, have described corticofugal responses ipsilaterally from stimulation of the posterior one-third of the lateral gyrus, chiefly in the ipsilateral superior colliculus. Less frequently, responses were recorded in the ipsilateral periaqueductal gray matter, subthalamus, medial geniculate nucleus, pulvinar, and the anterior part of the lateral geniculate nucleus. Strychninization of the medial surface of the posterolateral gyms produced strong responses in the dorsal part of the ipsilateral lateral geniculate nucleus, pulvinar, and superior colliculus. Wid6n and Ajmone-Marsan ( 1961) have also reported definite corticofugal effects upon the lateral geniculate nucleus after stimulating a small area between the middle and posterior parts of the lateral and suprasylvian gyri. The active points lay outside of the area from which well-developed potentials were elicited by stimulating the optic nerve. In a similar study, AjmoneMarsan and Morillo (1961) found driving of lateral geniculate neurons from stimulation of either striate cortex.

B. SUPRASYLVIAN GYF~US The middle and posterior portions of this gyrus in the cat include the secondary visual projection areas as illustrated by Rose and Woolsey (1949) and Woolsey (1962), or areas 18 and 19 as designated by Brodmann (1906), or visual associational areas as termed by Buser et al. (1959). Anatomical evidence has shown that this gyrus receives afferents from the ipsilateral and contralateral suprasylvian and lateral gyri, and the sylvian and ectosylvian gyri (Polyak, 1927; Mettler, 1932; Beresford, 1961).The caudal part of the middle suprasylvian gyms obtains its principal thalamic supply from the pulvinar, and the rostra1 part of this gyms from the lateral thalamic nucleus ( Waller and Barris, 1937), and the posteromedial extent of this gyrus from the lateral geniculate nucleus (Polyak, 1927). This last observation agrees with the findings reported by Marshall et al. (1943) that short-latency responses in this area disappear after ablation of the striate cortex, allowing time for degeneration of the lateral geniculate nucleus. A small area along the lateral bank of the middle suprasylvian gyrus receives a short-latency input evoked by photic or optic nerve stimulation (Marshall et aE., 1943; Clare and Bishop, 1954; Doty, 1958). These responses were thought by Clare and Bishop to be

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relayed through the lateral gyrus, but Marshall and co-workers found that they persisted after ablation of both striate cortices. Electrophysiological evidence for input to the medial lip of this gyrus from the lateral geniculate nucleus has been presented by Vastola (1961) and, since retrograde lateral geniculate atrophy presumably does not occur after lesions in this region (Polyak, 1927), collaterals from lateral geniculate fibers may project to this area directly, or an intrathalamic relay may be involved (Buser et al., 1959). The principal efferent projections from the middle suprasylvian gyrus, as determined anatomically, include fibers to the remainder of the ipsilateral suprasylvian gyrus, lateral gyrus, and upper ectosylvian gyrus as well as to homologous but smaller contralateral cortical regions. Other projections pass to the ipsilateral caudate nucleus, to the upper and middle layers of the ipsilateral superior colliculus through the optic radiation, and to the pons through the cerebral peduncle (Polyak, 1927). Niemer and Jimenez-Castellanos ( 1950) have traced projections electrophysiologicaly from the suprasylvian gyrus to the pulvinar and lateralis posterior nucleus chiefly, but also to the lateralis anterior nucleus, caudate nucleus, subthalamus, medial geniculate nucleus, substantia nigra, midbrain tegmentum, and the pons. The projections from the suprasylvian gyrus to the caudate nucleus may be involved in the effect described by Fox and O’Brien (1962) in which stimulation of the caudate nucleus can facilitate or inhibit the photically driven responses recorded in the visual cortex.

C. OTHERCORTICAL AREAS Certain other cortical regions have projections to the visual system in the cat, both at cortical and subcortical sites. Mettler (1932) has reported a detailed Marchi study following lesions limited to various ectosylvian and sylvian gyri and has described projections from (1) the anterior ectosylvian gyrus to the lateral gyrus of the same side and to the lateral, suprasylvian and splenial gyri of the opposite side, ( 2 ) the middle ectosylvian gyrus to the lateral, suprasylvian, and splenial gyri ipsilaterally, ( 3 ) the posterior ectosylvian gyrus to the lateral, posterolateral, suprasylvian, and splenial gyri both ipsilaterally and contralaterally, ( 4 ) the anterior sylvian gyrus to the lateral, suprasylvian, and splenial gyri of the

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same side, and (5) the posterior sylvian gyms to the lateral, posterolateral, suprasylvian, and splenial gyri of the same side, and to the lateral, suprasylvian, and splenial gyri of the opposite side. Mettler further reported degenerating fibers to the intermediate strata of the ipsilateral superior colliculus from the middle and posterior ectosylvian gyri through both capsular and tegmental routes. Beevor and Horsley ( 1902) have described Marchi degeneration to the superior colliculus from the posterior sigmoid gyrus. Niemer and Jimenez-Castellanos ( 1950), using strychnine on the posterior sigmoid and ectosylvian gyri, did not succeed in evoking responses from any part of the presently recognized visual system. Barris (1936) found Marchi degeneration in the pretectum, superior colliculus and pons after small lesions in areas 19 and 36, just ventral to the inferior border of the postero-lateral gyrus. VII. Recapitulation

In a final reconsideration of the experimental evidence which supports the visual pathways reviewed in this paper, it is important not only to indicate the paucity of significant supportive evidence in certain areas within this field but also to indicate the many important advances which have been made in this field during recent years. New anatomical studies, using various silver preparations to stain degenerating fibers, have measurably expanded our knowledge of the visual pathways in the cat and seem to have resolved many controversial points. Thus, the data available from classical Marchi studies have been considerably extended and refined, and these new silver stains have helped to clarify the termination of optic fibers ( I ) in the various laminae of the dorsal part of the lateral geniculate nucleus and in the interlaminar regions where fibers from the two eyes project; ( 2 ) in a nucleus just medial to the lateral geniculate nucleus, probably a part of the posterior nucleus of the thalamus; ( 3 ) in the large-celled nucleus of the optic tract within the pretectal region; (4)in the superficial strata of the superior colliculus; and ( 5 ) in the terminal nuclei of the accessory optic tract system. Similarly, some of the secondary visual pathways have been elucidated by silver-staining techniques. This is particularly true of the ascending efferent projections of the superior colliculus to certain posterior thalamic nuclei which either receive primary optic fibers

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(lateral geniculate nucleus; pretectum ) or which project corticopetal pathways probably involved in visual function ( pulvinar; lateralis posterior nucleus). Further studies using silver-staining procedures may contribute new information to many additional problems and controversies in this field, Thus, a precise map of the retinogeniculate projection in the cat is needed to supplement the older Marchi studies. Such studies, following retinal lesions, might also aid in resolving questions concerning the presence of a retinohypothalamic tract in the cat. Although repeatedly studied, there is no agreement on the histological spectrum of optic nerve fibers; the complete origins of the supraoptic commissures are still largely unknown; the thalamic and midbrain connections of the two parts of the lateral geniculate nucleus are beginning to emerge, but these observations need confirmation and elaboration; the connections of the reticular formation with the visual system and the presence of centrifugal fibers to the retina in the cat remains equivocal; and the efferent paths from the terminal nuclei of the accessory optic tract system are unknown. A beginning has been made with silver-staining methods to supplement and extend the few excellent Marchi studies of the efferent projection, associational, and commissural pathways from particular cortical areas, but much remains to be accomplished before the intricacies of the interconnections of cortical and subcortical visual centers emerge. Finally, it should be re-emphasized that this paper has reviewed the experimental evidence relating to the visual pathways in the cat and has indicated certain specific anatomical properties of the cat’s visual system, as compared with other species. Extension of many of the specific features of the cat’s visual system to a general plan of the visual system of other mammals, particularly primates, must presently await further investigations. In summary, the authors have presented their attempt to synthesize the available neuroanatomical and neurophysiological data into a schema of the visual system of the cat. This schema is obviously not complete nor final, and it will hopefully become obsolete rapidly. The authors will consider this review successful only if it will have contributed to hastening this process by stimulating additional work in this field.

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ACICSOWIXDCMEEITS

The authors are grateful to hlrs. Marian Fischman and Miss Mary Perez for their assistance in preparing the manuscript, and to Miss Elizabeth Brodel and htrs. Dorothy hiiles for their execution of the illustrations. REFERENCE5

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PROPERTIES OF AFFERENT SYNAPSES AND SENSORY NEURONS IN THE LATERAL GENICULATE NUCLEUS

. .

By P 0 Bishop Department of Physiology. University of Sydney. Sydney. Australia

I. Introduction . . . . . . . . . . . . . . . 192 I1. The Lateral Geniculate Nucleus . . . . . . . . . . 194

I11. Waveforms of Geniculate Responses . . . . . . A. Types of Electrodes and their Effect on Waveforms . B. Multineuron Responses . . . . . . . . . C. Waveforms of Unit-Responses . . . . . . . IV. Spontaneous Activity . . . . . . . . . . A. Single-Unit Discharges . . . . . . . . . B., Spontaneous Slow Waves . . . . . . . . V Repetitive Firing . . . . . . . . . . . Mechanism of Repetitive Discharge . . . . . . VI . Fractionation of Unit-Waveforms . . . . . . . A. The Single-Unit S Potential . . . . . . . B. Multineuron S Potentials . . . . . . . . C. A and B Potentials . . . . . . . . . . D . Generation of Postsynaptic Spikes . . . . . . E Properties of Dendrites of Geniculate Neurons . . F. After-Potentials of Geniculate Neurons . . . . VII . Refractory Period of Geniculate Neurons . . . . . A. Orthodromic-Orthodromic Responses . . . . . B . Antidromic-Orthodromic Responses . . . . . C Antidromic-Antidromic Responses . . . . . . D. Antidromic Intracelldar Stimulation . . . . . VIII. Recovery of Responsiveness . . . . . . . . A . Single Orthodromic Volleys . . . . . . . B . Repetitive Orthodromic Volleys . . . . . . IX. Pharmacology of Geniculate Neurons . . . . . . A. Intra-Arterial Drug Application . . . . . . B. Electrophoretic Drug Application . . . . . . X . Concluding Remarks . . . . . . . . . . References . . . . . . . . . . . . . 191

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1. Introduction

In view of the extensive information available about the synaptic mechanisms of spinal motoneurons (see Eccles, 1957) the tendency to regard the motoneuron as the prototype of the central neuron is perhaps not surprising. It has, however, long been known that the synapses which interrupt the main afferent pathways to the brain have properties distinguishing them from the motoneuron synaptic system (G. H. Bishop and O’Leary, 1940; Marshall, 1941; Therman, 1941; G. H. Bishop and O’Leary, 1942; Grundfest and Campbell, 1942; Marshall, 1949; Lloyd and McIntyre, 1950; Amassian, 1953; I?. 0. Bishop, 1953; Rose and Mountcastle, 1954). Because of the technical difficulties associated with the study of afferent nuclei, particularly in the brain, our knowledge about their synaptic mechanisms has grown relatively slowly. The nuclei are for the most part not as accessible as the spinal motor centers; the available input is usually complex and the output is not readily available for measurement and experimental manipulation, such as by antidromic activation. Our knowledge of motoneuron synapses was greatly advanced by the introduction of the technique of intracellular recording ( Brock et al., 1952). Unfortunately, a comparable intracellular technique has yet to be developed for the small sensory neurons. Most of the early investigations into the properties of sensory neurons involved the interpretation of complex waveforms extracellularly recorded from cell populations. Although this approach necessarily lacks much of the precision frequently possible with single-cell analyses, it still retains an important place among the techniques available for studying sensory systems. For many years, the extracellular recording of unit-responses has been the established method for studying neural organization in the brain, but it is only in the last few years that interest has developed in the interpretation of the waveform of these responses. No doubt, this interest has been heightened by lack of success in obtaining satisfactory intracellular records from the smaller sensory-type neurons, but it is now clear that extracellular recording has its own distinctive contribution to make to the biophysics of the neuron and of junctional transmission. A satisfactory interpretation of the extracellularly recorded unit-responses is now beginning to emerge. In many respects this interpretation is more complex than for the cor-

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responding intracellular record but extracellular recording offers the possibility of studying localized parts of the neuron in a way that is not possible with an intracellular electrode. With intracellular recorcling, it may be difficult to differentiate active and inactive regions of the membrane, particularly in the vicinity of the cell body. This follows from the fact that the impedance of the neuronal membrane is extremely high compared with the resistance of the cytoplasm so that potentials arising in contiguous zones tend to merge into one another as a result of electrotonic spread. The use of external microelectrodes largely avoids this averaging process. The dorsal nucleus of the lateral geniculate body (LGN) can be taken as a typical sensory synaptic system. The main purpose of this review is to discuss the contribution that single-unit studies have made in recent years to our understanding of the properties of sensory neurons as exemplified by the principal cells in the LGN. Much information has already accrued as a result of the older technique of multineuron recording but, in order to keep this review within bounds, an arbitrary decision has been made to exclude these data from detailed consideration. Furthermore, the present review concentrates on certain aspects of the properties of geniculate neurons that have been the particular concern of the author and his colleagues. An adequate review of all the information that is now available about the properties of sensory neurons must, therefore, wait for another occasion. All the single-unit studies on the LGN have used the technique of extracellular recording and the waveforms that have been obtained are critically dependent upon factors such as the type of electrode used and the distance separating it from the cell body and its processes. A brief discussion of these factors will be included since their importance is not generally well understood. In addition to the new data about the properties of geniculate neurons, these single-unit studies have also provided important confirmation of the interpretations made using the older technique of multineuron recording. An inquiry will, therefore, be made concerning the aspects of single-unit activity which may be expected to be reflected in the field potentials obtained from neuron populations and the manner in which the latter responses are built up from single-unit potentials.

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II. The lateral Geniculate Nucleus

Since this review will be primarily concerned with the principal cells in the LGN in the cat a brief account of the morphology of this region is desirable. The LGN is a cell-station on the central visual pathway, the principal neurons in the nucleus providing a monosynaptic relay from the eye to the cerebral cortex. The LGN in the cat can be briefly described as consisting of three cell laminae [labeled A, A,, and B by Thuma (1928)l placed one above the other to give a flattened S-shaped structure in parasagittal sections (Fig. 1 ) . Layers of .4 and .4,are almost identical in histological

N. Intertarn.Cent.

f ic.

N.

FIG.1 A. A semi-schematic drawing of a parasagittal section through the central region of the lateral geniculate nucleus (pars dorsalis) of the cat showing the 3 cellular layers, A, A,, and B. (From Hayhow, 1958.)

structure and are separated from one another by a conspicuous fiber plexus. Layers A, and B, each with a characteristic cellular structure, are separated from one another not by a fiber lamina like the one separating A and A, but rather by a cellular transitional zone containing scattered large deeply-staining cells. The optic tract fibers approach mainly from below and pass up into the substance of the nucleus approximately at right angles to the cell layers, The optic fibers from the contralateral eye terminate in layers A and B, while fibers from the ipsilateral eye terminate in layer A,. The s o n s of the principal celIs issue from the upper surface of the nucleus

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FIG. 1 B. Semi-diagrammatic composite drawing of the principal features presented by the dendrites, cell bodies, and axons in the LGN of the cat as revealed by the Golgi method. Details only shown of cell layers A and B. Insert (top): Diagram of the parasagittal section through the LGN showing the general direction taken by fibers in optic tract (ot) and optic radiation (or). (From P. 0. Bishop, 1953.)

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to form the optic radiation and, curling round the fimbria of the hippocampus and lateral ventricle, they pass upwards to the cerebral cortex. For a more detailed account of the morphology of the LGN in the cat see particularly Thuma (1928), Hayhow (1958), and P. 0. Bishop et al. (1962d). I l l . Waveforms of Geniculate Responses

In this review, two categories of response will be considered: synchronous multineuron discharges [Fig. 2 and Fig. 4 ( d, e ) ] produced by electrical stimulation of the optic nerve, and single-unit responses [Fig. 4 (a, b, c); Fig. 5 and Fig. 81, which may also be conveniently produced by electrical stimulation in the same way. For details of the interpretation of the waveform of the multineuronal responses in the LGN see particularly G. H. Bishop and O’Leary ( 1 9 4 q P. 0. Bishop ( 1953), P. 0. Bishop and McLeod ( 1954), Vastola (1957,1959a), and P. 0.Bishop and Davis (1960b). The interpretation of the waveform of single-unit responses in the LGN has been the particular concern of Tasaki et al. (1954), Freygang (1958), P. 0. Bishop et aE. (1958a), Griisser-Cornehls and Griisser ( 1960) and P. 0. Bishop et al. ( 1962a, b, c ) . .4. TYPESOF ELEIXRODES AND

THEIR

EFFECTON WAVEFORMS

One of the most important factors which determine the form of the response is the type of microelectrode used for recording. Both because the amplitude of the multineuron responses are rarely more than about 1 mv and the fact that their recording is not critically dependent upon location with respect to individual neurons, they are best recorded with low resistance (and hence low noise) metal microelectrodes (Bishop and McLeod, 1954), These multineuron potentials cannot be satisfactorily recorded with high-resistance glass capillary microelectrodes because of the relatively poor signalnoise ratio occasioned both by the inherent noise level of electrodes of this type and the signal loss due to capacitative blunting. It is only when the glass capillary microelectrode becomes relatively large (DC resistance less than 1 Mn and tip diameter more than about 1p ) that the responses obtained resemble those recorded with low-resistance metal microelectrodes. The extracellular field about individual neurons is best explored with capillary electrodes having

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a resistance of 2-5 M a (P. 0. Bishop et al., 1962a, b). If the electrode resistance becomes greater than 10 M a the increased noise level requires the microelectrode to approach much more closely to the single neuron, and nearly always the electrode will be in contact with the cell membrane by the time the recorded potentials rise quite clear of the noise level. While stable extracellular potentials can be recorded under these circumstances, the danger of impalement of the cell is always imminent particularly with the sharp-edged glass capillary microelectrode. So far, no satisfactory intracellular records of geniculate neurons have been obtained. Recently, Naka et al. (1960) have succeeded in obtaining intracellular responses from the small ganglion cells in the retina using micropipettcs having resistances around 200 Mn. Presumably, micropipette electrodes of comparable resistance will be required when recording intracellularly to avoid serious damage to the geniculate neurons. In our experience, the most satisfactory microelectrodes for recording multineuron responses are electrolytically-pointed fine steel needles (Bishop and Collin, 1951; Grundfest et al., 1950). The typical low-resistance steel microelectrode averages the potential over quite a large distance, e.g., 1OOp or more. This is equivalent to recording at a relatively large distance from cells by means of a fine capillary microelectrode. Hence, the unit-responses obtained by the glass micropipette electrode at a distance from the cell resemble those obtained with the steel microelectrode (type “c,” Fig. 4).For exploring the electrical field about single neurons, relatively lowresistance (=M a ) glass capillary microelectrodes have the advantage that the location of the tip (i.e., tip size) is known, a fact which can be of considerable theoretical importance in the interpretation of the waveforms. For the routine extracellular recording of single geniculate neurons, the tungsten microelectrode ( Hubel, 1957) is to be preferred. The smooth tip of the electrolyticallypointed tungsten can apparently touch or indent the cell membrane without the same danger of impalement as would occur with the ultra-sharp edges of the glass capillaries. Stable extracellular records from the same single-unit have been recorded continuously for periods up to 14 hours using tungsten microelectrodes (Levick and Williams, 1962). Effective tip size (i.e., the portion bare of insula-

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tion) is presumably also of critical importance in relation to the metal microelectrode although no satisfactory information is available in this respect. Another important technical consideration concerns the location of the second electrode. Unless otherwise indicated, the potentials described in this paper h a w been obtained with an "indifferent" electrode located in tissue well nwav from the region of the LGN. The use of differential recording with the second electrode in or close to the LGN increases the complexity of the waveforms though it may have advantages in special circumstances (e.g., Vastola, 1957; Width and Ajmone Marsan, 1960).

R. MULTIXEUROS RESPOXSES The interpretation of the multineuron responses is greatly simplified if the afferent tract volley is restricted to the large-diameter fiber group ( t l ) (see G. H. Bishop and OLeary, 1942; P. 0. Bishop and McLeod, 1954). Unfortunately, the contribution of the smallfiber ( t z ) volley to the geniculate response following electrical stimulation of the optic nerve is not always as clear cut as the records of Fig. 2 might suggest. The latter, obtained with a steel microelectrode, are the multineuron responses recorded in the LGN with low resistance metal microelectrodes following electrical stimulation of the contralateral optic nerve. The records in the left-hand column show the development of the response with increasing shocks applied to the contralateral optic nerve. The postsynaptic propagated spikes ( rl and r z ) in the corresponding records in the right-hand column have been eliminated by a brief high-frequency train of conditioning shocks applied to the optic nerve (P. 0. Bishop and McLeod, 1954). The latter records show only the two tractvolleys, tl and t2, respectively, and the corresponding synaptic potentials, s1 and sz. The effect of the location of the recording site in the LGN on the form of the multineuron geniculate response particularly in relation to the optic tract fiber-groups has been discussed by P. 0. Bishop and Evans ( 1956). A typical multineuron geniculate response to a contralateral afferent volley restricted to tl fibers is shown in Fig. 4, at d, corresponding to recording site d in Fig. 3. The first positive-negative diphasic spike represents the arrival of the tl volley and this is followed after a synaptic delay of 0.30.4msec (P. 0. Bishop and

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FIG.2. Developmcnt of the multineuron response recorded in the LGX with a low-resistance steel microelectrode following increasing electrical stimulation of the contralateral optic nerve. Left-hand column: unconditioned responses.

Right-hand column: responses conditioned by a brief high-frequency train of shocks applied to the optic nerve. Insert: Outline tracing of a Nissl-stained parasagittal section through the LGN showing the recording site for the multineuron responses. The recording site, indicated by the filled circle, was labeled by the iron-deposition method. Arrows indicate the Horsley-Clarke vertical and horizontal planes. For details see text. (From P. 0. Bishop and R. Davis, unpublished. )

Evans, 1956) by a postsynaptic negative spike ( rl ) representing the discharge of geniculate neurons. The rl spike is commonly followed by a low positivity though this may be obscured by the negative after-potential and occasionally also by the repetitive firing of the

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geniculate neurons (P. 0. Bishop and Davis, 1960b). As will be shown below, the rl spike is mainly due to the discharge of the cell body of the geniculate neurons though the initial segment of the axon probably contributes to some extent. The optic tract terminals are distributed across the cellular layers of the LGN in a preciseIy ordered paraIIeI array (Telio, 1904; see P. 0. Bishop et al., 1962d) and even the fine fibers within a lamina

FIG.3. Diagram of the recording sites in the LGN for responses of unittypes a, b, and c, and for multineuron types d and e. Arrows indicate 00ws of current during the active phase of the excitatory postsynaptic potential. The lines of current flow are purely diagrammatic and do not represent the actual paths taken by the ions. OTT, optic tract terminals; OR, optic radiation; A, initial segment of axon; B, cell body of geniculate neuron.

of termination retain much of this parallel arrangement within the so-called arborization spindles. It is this open-field type of arrangement (Lorente de N6, 1939, 1947) which makes the tl spike such a striking feature of the multineuron geniculate response. By contrast, the singfe-unit responses of the geniculate neurons do not have any corresponding presynaptic component apart from the multifiber tl spike which becomes relatively small or entirely insignificant by comparison ( see, however, Grusser-Cornehls and Griisser, 1960). The elecbical field about single nerve fibers attenuates extremely rapidly with distance from the fiber (Tasaki and Tasaki, 1950;

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Tasaki et al., 1954) and this attenuation would be greatly accentuated in the case of the very fine presynaptic fiber terminals. The fact that these terminals are very weak electric generators and are spread widely over dendrites and cell body, means that the conven-

FIG. 4. Types of responses recorded extracellularly in the LGN. ( a ) unitresponse type a from axon; ( b ) unit-response type b from cell body; ( c ) unitresponses type c from vicinity of cell body; ( d ) multineuron response recorded with a steel microelectrode in the LGN following submaximal stimulation of contralateral optic nerve; ( e ) multineuron response corresponding to ( d ) but recorded in optic radiation immediately above LGN. Traces to right of ( d ) and ( e ) show the corresponding responses after the synapses have been blocked by brief high-frequency stimulation. Unless otherwise indicated all the responses dustrating this review were obtained with glass micropipette electrodes. (a, b and c, from P. 0. Bishop et al., 1962a; d and e, from P. 0. Bishop and McLeod, 1954).

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FIG. 5. a to j: extracellular responses in LGN. a, background multineuron response following an ipsilateral optic nerve shock just subthreshold for the unit-response shown in traces b to j; b to j: successive transformations of the unit-waveform from type c to type b as the microelectrode approaches the cell

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tional capillary microelectrodes would not reveal any new component within the presynaptically generated electric field about single neurons. There is no localized presynaptic generator in any way comparable in size to the cell body and initial segment of the postsynaptic neuron. It is not surprising, therefore, that the very fine capillary microelectrode markedly attenuates the tl spike without revealing any new and localized presynaptic potential. To what extent the fine presynaptic terminals contribute to the multifiber tl spike, it is not, as yet, possible to say. Griisser-Cornehls and Griisser (1960) have described a presynaptic component (a component) in their records of geniculate single-unit responses although in some respects this component seems to resemble the A potential of P. 0. Bishop et al. (1962~).Rose and Mountcastle (1954) also described a presynaptic complex in their records from single thalamic neurons. There is as yet no evidence of any prolonged electric action in the presynaptic terminals in the LGN such as that described in the spinal cord (e.g., Lloyd and McIntyre, 1949; Eccles et al., 1962). These potentials should be looked for, however, because the morphology of the optic tract would favor their detection. If the terminal few microns of the tract fibers are randomly orientated and the flows of current are restricted to these portions, multifiber summated potentials large enough to be recorded may not be established, and the individual electric generators may remain undetected by even the very finest micropipette electrode. The way in which the multineuronal rl spike of the type d response (Fig. 4 ) is built up from unit responses (type c, in Fig. 4) having a similar waveform, is illustrated in Fig. 5, k to n. These records were obtained with a 4 Ma glass capillary microelectrode. Trace k shows the geniculate response to electrical stimulation of the contralateral optic nerve at threshold for the unit-spike which body. Time calibration constant throughout series. Stimulus parameters and amplifier gain settings were varied to emphasize different features of the waveform. Voltage calibrations, 0,p, and q refer to traces h, i, and j, respectively. For details see text. k to n: simultaneously recorded multineuron and unit-responses in the L,GN illustrating the formation of the multineuron r , spike from unit responses of type c. Time calibration: lo00 c/s refers to k and 1; 5ooo c/s refers to m and n. Voltage calibration: 1 mV refers to k; 2 mV refers to 1, m, and n. For further details see text. (From P. 0. Bishop, W. Burke, and R. Davis, unpublished.)

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is represented here by a train of 4 negative-positive spikes towards the end of the sweep. The potential complex immediately after the stimulus artifact is the multineuronal tl, rl response and this is followed by a low negative wave (r3) representing a synchronous multineuronal repetitive discharge. In 1 the stimulus strength has been increased to approximately 3.5 times threshold for the unitspike. The unit repetitive-discharge moves earlier and is reduced in number from 4 to 3. The first unit-spike is now superimposed on the peak of the rl spike and the second unit coincides with the early part of the multineuron repetitive wave, r3. These details are shown more clearly in traces m and n taken with a faster sweep-speed. The shock strength used to produce trace n was somewhat higher than that used for m. As will be described below, this alteration in the latency and number of repetitive spikes is a characteristic reaction of the geniculate neurones to increasing shock strength applied to the optic nerve (Bishop, 1960). The multineuronal rl spike is, therefore, compounded of type c unit-responses, the waveform in each case being initially negative without any evidence of a preceding positivity. In Fig. 3, the recording site for these two responses are diagrammatically represented by ( d ) and ( c ) , respectively. As will be described below, movement of the microelectrode close to the cell body [position ( b ) ] leads to the appearance of an initial positivity in the unit-response but, despite this, the time of onset of the negativity remains almost unchanged, the positive-going deflections appearing at an earlier time. This indicates that the type c unit-response and, hence, the multineuronal rl spike are due mainly to the discharge of the cell body. It is only as the electrode nears the cell body that the distinctive contributions made to the waveform by the synaptic depolarizations and the discharge of the initial segment become evident. When the electrode recording the multineuronal geniculate response is moved up among the optic radiation fibers the waveform undergoes a characteristic sequence of changes (P. 0. Bishop and McLeod, 1954; P. 0. Bishop and Davis, 1960b). In the present account the multineuron waveform recorded just above the nucleus is labeled “response type e” (Figs. 3 and 4). In Fig. 4, the two tracings on the right of d and e show the geniculate and radiation responses, respectively, after synaptic bansmission in the nucleus has been blocked by brief high-frequency stimulation. The slow

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negative and positive waves following the tract spike are the multineuron equivalents of the unit synaptic potentials (P. 0. Bishop et al., 1962c (see below).

C . WAVEFORMS OF UNIT-RESPONSES As pointed out above, the type c unit-spike (Fig. 4,c) is the one that is commonly recorded using electrodes of large size (e.g., the steel rnicroelectrodes used for the recording of multineuron responses) and are, in fact, the only type of unit activity that may be recorded with these larger-tip electrodes. Responses of type c are rarely of large amplitude, being usually only a few times the noise level when high-resistance electrodes are used. They are obtained when rhe electrode is some distance from the cell, possibly up to 50 p or more away. ( Mountcastle et aZ., 1957). Further consideration of these responses will be deferred till later. The sequence of changes of waveform of the unit-responses as a microelectrode (5 MQ glass micropipette) moves closer to the cell, is shown in Fig. 5, a to j, the response being changed from type c to type b. (Bishop et al., 1962a; see Granit and Phillips, 1956; Phillips, 1959). The particular cell was activated by electrical stimulation of the ipsilateral optic nerve, the stimulus strength being well above threshold for the unit. Trace a shows the background multineuron response. The unit first appears as a small, entirely negative spike on the crest of the multineuron response. As the microelectrode is advanced closer to the cell, the negative spike grows in amplitude and a very brief initial positivity makes its appearance. With further advance, both the initial positivity and particularly the negative spike continue to grow in amplitude. At the particular “gain” setting, the negative spike moves off the screen of the cathode ray tube in records e to i. Trace h (reduced gain) clearly shows evidence of two inflections on the initial phase of increasing positivity of the unit response. Both inflections are shown more clearly in records i and j in which the safety factor for synaptic transmission has been reduced by a high-frequency train of 5 conditioning shocks, applied some 20 msec before the test shock. The first inflection (first arrow, trace j ) is shown more clearly by increasing the amplifier gain in trace i. It represents the onset of a propagated A spike rising out of a synaptic potential (S-A step, Fig. 8A; Bishop et al., 1962~;see also Bishop et al., 1958a; Freygang, 1958). This

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undoubtedly corresponds to the generation of the A (or IS) spike in the spinal motoneuron by the excitatory postsynaptic potential (EPSP) as described Fuortes d aE. (1957) and Coombs et aE. (1957a, b ) . The second inflection (second arrow, trace j in Fig. 5) is shown somewhat more clearly by reducing the gain (trace j ) so that now the whole response appears on the screen of the cathode ray tube. The second inflection ( A-B step, Fig. 8A) represents the onset of the B spike. This again is the counterpart of the situation in the spinal motoneuron. Trace j is a typical b-type unit-response which is recorded only when the microelectrode is very close to the cell body (recording site b, Fig. 3). Striking confirmation of the interpretation of these extracellularly recorded waveforms has been provided by studies on the giant neuron of ApZysia (Tauc, 1962a, b ) . The second negative spike of variable amplitude that occasionally appears after the main negativity in these records, is probably an abortive B spike. In this case, it was almost certainly due to the direct initation of the membrane by the electrode tip since the negativity arose without any obvious preceding positivity, indicating that generation occurred in the region of the tip (see Tomita et al., 1961).The irritation was, however, without apparent harm to the membrane in this instance since the repetitive firing was readily stopped by withdrawing the electrode. In fact, the sequence of changes of potential illustrated in Fig. 5 was observed several times by withdrawing and readvancing the microelectrode without apparent harm to the cell. The peak-to-peak amplitude of the type b responses is usually between 2 and 10 mv with very occasional giant spikes up to 30 mv or more (Fig. 4, b; see the “giant spikes” of Granit and Phillips, 1956). In order to obtain these larger potentials the recording resistance, whatever its nature and location, must also be large. This resistance is usually regarded as the result of electrical isolation of the membrane under the microelectrode either by indentation of the surface membrane or by some process of “sealing of the electrode to the membrane (Hild and Tasaki, 1962; Gillespie, 1962). The membrane is regarded as being locally inactivated due to the depolarization consequent on mechanical deformation. This idea has been strengthened by the close similarity that is frequently observed between the extracellular record and the first derivative of the intracellular record (Freygang, 1958; Freygang and Frank,

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1959). Such a state of affairs probably obtains when the negative phase of the extracellular response has been largely lost, but type b responses cannot be explained in this way (Tauc, 1962b). However, declining negativity and increasing positivity probably indicates that the electrode is touching or indenting the cell membrane, leading to a local loss of responsiveness. That the above responses may be recorded extracellularly and without electrode sealing by membrane indentation is, nevertheless, clearly indicated by the following observations, namely: (1) the absence of a resting potential; ( 2 ) the negative phase of the spike is often much larger than the initial positivity; ( 3 ) two and, very occasionally, three separate unit-responses have been recorded at the same time with the one electrode (Fig. 6 see also Hubel and Wiesel, 1961; Levick and Williams, 1962); (4) the response may occasionally be lost as the electrode is moved on and fully recovered on withdrawal of the electrode. The records in Fig. 6 were taken from immediately adjacent parts of a moving film record of responses obtained in the LGN with the one tungsten microelectrode; three separate single-unit spike responses can be seen. Whereas the individual spikes usually occur separately, they may superimpose, and the pattern of the firing suggests some loose coupling between them. 'The largest response is clearly of type b with well-marked inflections on the initial phase of increasing positivity. The small slow positive waves in these traces are extracellularly recorded excitatory synaptic potentials (Bishop et al., 1962c) which are only obtained when the electrode is very close to the cell-body. The amplitude of the other two triphasic responses in Fig. 6 is too small for proper identification but both appear to have at least one inflection on the initial phase of increasing positivity. The high recording resistance under these circumstances may possibly be due to the presence of glial membranes investing or associated with the neurons. In order to complete the catalog of unit-waveforms a brief mention must be made of axon spikes (response type a, Fig. 3 ) . Singleunit potentials attributable to axons are either entirely positive or initially positive with a very small after-negativity (Bishop et al., 1962a; Levick and Williams, 1962). The very rapid initial downstroke (duration 0.25 msec or less) is usually devoid of inflections, though notching may occur as the unit deteriorates. These responses

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FIG.6 . Three separate single-unit responses simultaneously recorded in the LGN with the one tungsten microelectrode during intermittent ( 80/second) photic stimu1,ition of the retina. The records were taken from imniediately adjacent parts of a moving film record. (From T. 0. Ogawa, unpublished.)

are usually suddenly encountered, indicating that the current density declines very rapidly in the extracellular medium about the axon. The spike amplitude is usually from 1 to 10 mv and with glass micropipette electrodes it is commonly associated with a low resting potential. These responses are usually much less stable than those attributable to the cell body. Markedly diphasic or triphasic waveforms attributable to axons were not encountered in our records obtained in the vicinity of the LGN, although these have been described in other parts of the nervous system. The retina is a particularly favorable location for differentiating between extracellular1y-recorded cell body and axon potentials ( Kuffler, 1953; Barlow, 1953; W. Kozak, P. 0. Bishop, and W. R. Levick, unpublished). In the unopened eye, when the tip of the microelectrode is optically projected onto a tangent screen (P. 0. Bishop et al., 1962e), polyphasic spikes can be attributed to axons with some certainty if the locus of the tip is found to lie at a distance from the position of the corresponding receptive area of the unit and situated in such a position between the receptive area and the blind spot to be expected from the known distribution of the ganglion

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cell axons over the surface of the retina. Our failure to record markedly polyphasic axon spikes in the LGN is possibly due to inadequate interpretation on our part and also the fact that the axons are largely myelinated as opposed to the unmyelinated fibers in the retina. IV. Spontaneous Activity

A. SIXGLE-UNIT DISCHARGES

With the exception of those taking part in postural activity, motoneurons differ from other central neurons in that they are normally silent when the body is at rest. Under the usual experimental conditions of anesthesia and spinal transection, motoneurons only discharge when stimulated transsynaptically or antidromically. By contrast, sensory neurons are for the most part continuously active even in the apparent absence of sense organ stimulation. Granit (1955) has reviewed the earlier literature on the latter topic. It is obviously extremely difficult to provide conclusive evidence that neurons in l;iz;o are, by their own intrinsic mechanisms, capable of self-generated spontaneous activity. Isolated neurons in zjitro may, however, be spontaneously active (Hild and Tasaki, 1962). Almost without exception, investigators who have recorded unit activity in the visual system have commented upon the spontaneous activity (see Levick and Williams, 1962, for references) but there have been only a few systematic attempts to study it. Kuffler et al. (1957) have examined the statistical properties of the retinal ganglion-cell discharges under a variety of conditions (see also Bornschein, 1958a, b ) and a systematic study of the resting discharge in various parts of the visual system in the cat has been carried out in this laboratory, particular attention being given to the discharge that occurs in the complete darkness. Apart from preliminary reports (Levick et al., 1961), the first detailed account of our work is that of Levick and Williams (1962) (see also Arden and Soderberg, 1961; Jung, 1958). An understanding of the nature of spontaneous firing is of considerable importance because it is against this background that information is coded and transferred in the visual system. The problem becomes of particular importance in the consideration of various visual thresholds ( Barlow, 1957). As observed under pentobarbital anesthesia in the cat progres-

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sive changes occur in the spontaneous geniculate discharge during dark adaptation but these changes have been difficult to analyze and no clear-cut pattern of dark-adaptation has emerged from these studies. In the complete darkness, many geniculate units show a stable firing rate for short periods (5-10 minutes) but it is uncommon for these stable periods to be longer than about 30 minutes. The rnajoritv of these stable mean rates were below 10 per sec, the commonest observation being 4-6 per sec. This is in contrast to the retinal ganglion cells under similar conditions since these cells fire at 10-100 per sec (average mean rate about 30 per sec). These values must be treated with some reserve, however, since the darkfiring patterns showed wide variations, not only from unit to unit, h i t also in tile one unit at different times. Geniculate firing patterns classified as unstable were frequently quite irregularly so, but occasionally they displayed a remarkably regular cyclic behavior having periods ranging from a fraction of a second up to 50 minutes. Regular cycles of frequency modulation recurring at rates of 0.510 per sec have also been observed in the resting discharge of spinal interneurons (Frank and Fuortes, 1956). Another type of cyclic spontaneous discharge was the occurrence of short bursts fairly regularly repeated at intervals of 0.1-1 sec (P. 0. Bishop and Davis, 196Ob; Hubel, 1960; P. 0. Bishop et al., 1962a). These short highfrequency bursts will be discussed later on. There is little doubt that both types of cvclic behavior are of physiological origin. Pressure block of the retinal discharge showed that it was the principal factor causing the discharge of the geniculate neurons since the block completely stops (61%) or markedly suppresses the geniculate dark discharge (Levick and Williams, 1962). The proportion of cells (39%) that remain active under these circumstances is very nearlv the same as the proportion of geniculate cells (37%)and optic radijtion axons ( 3 6 a ) found by P. 0. Bishop et al. (1962a) to be spontaneously active after the excision of the two eyes. Gross lesions which destroyed the midbrain at the intercollicular level in conjunction with continued barbiturate anesthesia had no obvious influence on the geniculate dark discharge (Levick and Williams, 1962). That the brainstem does, however, influence the geniculate discharge is indicated by the fact that 85%of the units are now silenced by retina1 pressure block. In the rabbit, the brainstem has a greater influence than it does in the cat since, in the rabbit “cerveau

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isol6‘ preparation, Arden and Soderberg (19f3l) found many genicd a t e units to be no longer active, though those that remained active were all silenced by retinal pressure block. These findings argue against true “spontaneous” geniculate activity. In the rabbit “enckphale isole” preparation (Arden and Soderberg, 1961) the resting geniculate spike-discharge is practically unaffected by the loss of retinal activity. Levick and Williams (1962) have made important observations on the many experimental variables such as body temperature, depth of anesthesia, degree of hyperventilation or hypercapnia, adjuvant drugs, etc., which might be thought to have a significant influence on the rate and pattern of the spontaneous geniculate discharge. Fluctuations in these variables such as might occur under the usually-maintained experimental conditions were shown to be without effect on the rate and pattern of the spontaneous discharge. Powerful new techniques involving the use of special purpose digital computers have recently become available for studying unitfiring patterns (Gerstein and Kiang, 1960; Levick, 1962). Much more elaborate analyses of the spontaneous discharge are now possible such as the construction of interval histograms, joint interval distributions, conditional probability distributions, etc. Only brief preliminary accounts of this work have so far been published (Levick et al., 1961; Levick and Williams, 1962) but detailed analyses have now been carried out and will be published elsewhere. One of the features of the geniculate firing which sharply distinguishes it from the retinal discharge is the tendency to fire repetitively (see below). No satisfactory evidence is as yet available about the intrinsic mechanisms responsible for the spontaneous geniculate discharge or whether, indeed, the geniculate neurons are capable of a true spontaneous activity. U. SPONTANEOUS SLOWWAVES

Using microelectrodes suitable for recording the mu1tineuron evoked response (see above), the spontaneous slow waves occurring in the LGN have been studied by Bishop and Davis ( 1960b). While consideration of these potentials falls outside the scope of this review, they call for brief mention here since they appear to depend upon processes largely or even wholly independent of the spike generating mechanisms. Thus, the geniculate synaptic mechanisms

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can be blocked by the action of lysergic acid diethylamide without materially affecting the spontaneous slow waves. This finding recalls Li and Jasper’s (1953) observation that temporary arrest of artificial respiration or careful administration of barbiturates caused the loss of spontaneous unit-firing in the cerebral cortex without necessarily reducing the ‘‘dike” waves. The available evidence indicates that the geniculate slow waves arise from the dendrites and possibly the cell bodies of the principal cells (P. 0. Bishop and Davis, 1960b).

V. Repetitive Firing One of the striking and characteristic features of geniculate neurons is the tendency to fire repetitively in short high-frequency bursts (Fig. 7). This phenomenon was first described in the multineuronal geniculate response following a singIe electrical shock applied to the optic nerve (P. 0.Bishop ef al., 1953b) but it has since

FIG. 7 A. Esnmplcs of repetitive firing by single geniculate neurnns following s i n g l ~ ~ h n cstimulation k of the optic nerve. The pause between the first and second response is a common but not constant feature. Voltage calibration, 2 mV. (From P. 0. Bishop et al., 1962a.) B. Superimposed S potentials and repetitive full spikes obtained from the vicinity of the cell body of a single geniculate neurone in response to variable shocks applied to the optic ncxve. With stimuli subthreshold for the full spike the S potentials appear to fall into 4 or 5 amplitude steps. Voltage calibration, 5 mV. (From P. 0. Bishop et ul., 19F2c.) C. Single unit (axon) records obtained from LGN following stimulation of contralateral optic nervc. a, effect of repetitive stimulation a t about 300/sec. b to e, effect of increasing strength of stimulation on the pattern of the repetitive discharge. (From P. 0.Bishop, 2960. )

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been repeatedly observed by most investigators who have made single-unit studies on the LGN (Tasaki et al., 1954; P. 0. Bishop et al., 1958b; Freygang, 1958; Erulkar and Fillenz, 1960; Hubel, 1960; Widen and Ajmone Marsan, 1960; P. 0. Bishop et d.,1958a, 1959c, 1962a, b,c; Hubel and Wiesel, 1961; Ajmone Marsan and Morillo, 1961; Negishi et al., 1962). As yet, no systematic study of repetitive firing in the LGN has been published; such a study is in progress in this laboratory. Apart from possible mechanisms, the only detailed study of the phenomenon itself as it occurs in other parts of the central nervous system is that of Rose and Mountcastle (1954). These authors studied the repetitive responses of single thalamic neurons to transient peripheral stimuli. Similar repetitive firing has been described in afferent systems elsewhere (e.g., Amassian, 1953; McIntyre et al., 1956; Wall, 1959; McIntyre and Mark, 1960; Darian Smith, 1960) and in spinal internuncials, cortical, reticular, and hippocampal neurons (e.g., Amassian, 1953; Amassian and de Vito, 1954; Frank and Fuortes, 1956; Hunt and Kuno, 1959a, b; Kandel and Spencer, 1961). From the viewpoint of the analysi:s of the phenomenon of repetitive firing, Rose and Mountcastle’s (1954) study suffers the disadvantage that the stimulus was applied to the skin or peripheral nerve trunks relatively remote from the thalamus. Repetitive firing in the form of a short high-frequency burst may occur spontaneously or in response to single afferent volleys, The repetitive firing is usually associated with spontaneous activity. Cells which do not fire spontaneously usually show no evidence of repetitive firing although marked spontaneous activity does not necessarily produce burst discharges. The repetitively-firing neurons are in a marked contrast to motoneurons which scarcely, if ever, discharge more than once to single-shock presynaptic stimulation applied to the monosynaptically connected afferent fibers of the stretch-reflex arc. Repetitive firing in motoneurons can, however, be produced if the membrane is depolarized in the absence of orthodromic stimulation by direct currents. In these cases, frequency of firing is a simple function of current intensity (Fuortes, 1959). Although much of the “spontaneous” activity of geniculate neurons is a consequence of retinal activity, the repetitive firing is a characteristically geniculate activity. The spontaneous discharge of retinal ganglion cells rarely occurs in the form of short high-fre-

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quency bursts (Kuffler, 1953; W. Kozak, W. R. Levick, and P. 0. Bishop, unpublished). After destruction of the two retinas, burst activity still characterizes both the spontaneous unit-firing in the LGN and that evoked by a brief single-shock stimulation of the optic nerve. Under these conditions P. 0. Bishop et uZ. (1962a) found that 33%of the geniculate neurons fired repetitively in response to single-shocks applied to the optic nerve whereas in the case with intact eyes, Negishi et al. (1962) found burst activity in only 15.6%of the spontaneously discharging geniculate neurons. It is interesting that the response of optic radiation axons to singleshock optic nerve stimulation showed a much higher incidence of repetitive firing (79%) than did the parent geniculate neurons (33%) (P. 0. Bishop ct al., 1962a). This suggests that the cell body may not always be involved in the postsynaptic discharge. As mentioned ‘tbove, however, optic radiation axons show no more evidence of spontaneous activity than do the geniculate cells. Repetitive firing of geniculate neurons has also been observed following cortical stimulation (Bishop and Davis, 1960b; \Tiid& and Ajmone Marsan, 1960). Some, at least, of these discharges are probably due to true antidromic activation of the geniculate neuron. A similar antidromic repetitive discharge occurs in the ventral roots of the spinal cord (Renshaw, 1941). The repetitive firing of geniculate neurons is a characteristic feature of the anesthetized or sleeping brain since EIubel (1960), working with the unrestrained animal, seldom if ever saw repetitive firing of geniculate units when the animal was alert. During sleep, the clusters (bursts) were accentuated by stimuli that activated the unit, From data at present available in the literature, the features of the repetitive discharge in the LGN appears to be much the same in the various circumstances in which it has been observed. The burst may contain 3-10 or more spikes, most commonly 3 or 4, firing at frequencies up to 500 per sec or higher. Intervals between spikes are usually least initially and increase during the course of the burst but occasionally the initial intervaI may be the longest in the burst (Fig. 7 ) . The latter pattern is also found in the repetitive responses of spinal interneurons (Hunt and Kuno, 195913) although the former is more usual in the sensory relay neurons (Rose and Mountcastle, 1954). The spontaneous geniculate bursts may recur at intervals

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between 0.1 and 1 second, most commonly about 0.2 second. This fast cyclic behavior has been briefly described by Hubel (1960), P. 0. Bishop d al. (1962a) and Levick and Williams (1962). Fairly regularly-recurring bursts of repetitive firing may also be triggered by singleshock stimulation of the optic nerve (P. 0. Bishop et al., 1958b). Repetitive responses to shocks applied to the optic nerve do not survive rapid rates of stimulation (P. 0. Bishop et ul., 1953b). With progressive increase in the frequency of stimulation, Wid& and Ajmone Marsan (1960) found that the spike bursts are usually cut off “from behind leaving only the first two spikes and eventually only the first one responding to every stimulus. Bursts of 3-5 spikes could, however, follow stimulation frequencies up to 10 per sec. In most situations in the nervous system where repetitive firing has been observed the latency of the first spike in the burst (minimal latency value) progressively decreases with increasing afferent stimulation. This is found to be the case in the LGN, also. Thus, the decrease in the minimal latency values for the repetitive genicd a t e discharges, k and 1 of Fig. 5, is about 3.5 msec and even greater reductions have been observed. In addition, at threshold there are considerable spontaneous variations in the minimal latency -the longer the minimal latency value the greater the variability ( Ajmoiie Marsan and Morillo, 1961). With a minimal latency value of about a millisecond or slightly longer, Ajmone Marsan and Morillo found the range of variability, spontaneously or in relation to changes in optic tract stimulus strength or to conditioning influences, to be as great as 6 msec or more. These latencies are hardly compatible with a monosynaptic pathway and complex neuronal pathways are almost certainly involved. Associated with the decrease in minimal latency, increasing stimulus strength also leads to a progressive increase in the number of spikes in the burst in most situations in the nervous system (Rose and Mountcastle, 1954; McIntyre and Mark, 1960). In striking contrast, it is very frequently the case in the LGN that an increase in the stimulus strength applied to the optic nerve leads to a decrease in the number of spikes in the burst (Bishop, 1960). With strong stimulation, only a single spike may remain (Fig. 7). An important finding is the range of stimulus strengths usually required to reduce the burst to a single spike. Thus, a low threshold geniculate unit,

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firing repetitively, may require a shock close to maximal for the whole optic nerve to produce a single spike. Whatever the inhibitory mechanism, this indicates that a large number of optic nerve fibers, scattered throughout the range of fiber diameters, can influence a single geniculate cell. There is considerable convergence, largely of an inhibitory kind, on geniculate cells. These findings account for the earlier observation (P. 0. Bishop et al., 1953b) that repetitive firing is particularly associated with large-fiber activity in the optic nerve. In contrast to the above descriptions, Rose and Mountcastle (1954) reported that only a small minority of thalamic units responded with shorter trains of spikes with increasing stimulus strength, and the reverse of the usual pattern described above may also be seen in the LGN. The increasing tendency of geniculate neurons to fire repetitively as the afferent stimulation is reduced is evidenced also in the action of various drugs such as lysergic acid diethylamide, bufotenine, and 5-hydroxytryptamine, which block transmission through the geniculate synapses (P. 0. Bishop et al., 1960). The effect of the intracarotid injection of these drugs on the repetitive firing in the geniculate response evoked by electrical stimulation of the optic nerve, has been repeatedly observed. More recently, Curtis and Davis (196%) have confirmed these findings by more direct iontophoretic techniques. During the onset of the action of these drugs and before synaptic block is established the repetitive firing may appear or be accentuated in the evoked response. The repetitive firing may then reappear as recovery ensues. Even after full recovery the repetitive firing may remain an established feature of the evoked response although little in evidence before the injection of the drug. This increase in the repetitive firing was always associated with an increase in the amplitude of the negative after-potential of the multineuron response (P. 0. Bishop et al., 1960). Several features of the unit-waveform of the repetitive discharge have been described which have an important bearing on the origin of the repetitive discharge. Usually, the first spike in the burst is preceded by a synaptic potential which is usually absent from the remaining spikes in the series [Fig. 6 (upper trace), Fig. 7A, a and b] (Hubel and Wiesel, 1961; P. 0. Bishop et al., 1962a). This indicates that a single optic-tract synaptic potential can probably initiate

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a whole burst. However, it is not known whether or not an extracellular recording can be so localized that a synaptic potential in one part of the cell can be picked up but not another in a more remote part, even though both synaptic potentials may be of sufficient amplitude to discharge the cell (P. 0. Bishop et al., 1958a). Such a possibility is suggested by the fact that in occasional bursts, even ithe first spike is not preceded by a recognizable synaptic potentiall. This appearance may, however, be due to the fact that the first geniculate spike follows on the rising phase of the synaptic potential so rapidly that an inflection is not produced in the record. An alternative explanation is that the burst discharge can arise spontaneously as a result of mechanisms intrinsic to the cell. Some form of maintained depolarization is generally associated with the repetitive firing wherever it has been studied (e.g., Eyzaguirre and Kuffler, 1955). In multineuron geniculate responses, Bishop and Davis (1960b) have shown that the repetitive discharge is nearly always sharply limited to the duration of the negative afterpotential which immediately follows the principal geniculate spike discharge. It is obviously dBcult to decide how much of this depolarization is a true after potential process and how much is summed asynchronous repetitive firing, as suggested by McIntyre and "ark (1960) for the dorsolateral tract response in the spinal cord. The presence of a maintained depolarizing process is, however, indicated by the study of single-unit records from the LGN (Width and Ajmone Marsan, 1960; P. 0. Bishop et al., 1962~).In some records (Fig. 7B) the spikes in the burst appear to be superimposed upon a slow positive potential which is presumably due to a longlasting depolarization of the membrane elsewhere in the soma or dendrites. The later spikes in the burst rise out of this slow potential without any intervening synaptic potential. Slow negative waves may also occur (Rose and Mountcastle, 1954). The repetitive discharge shown in the upper trace of Fig. 6 rides up on a slow negative wave (see Rose and Mountcastle, 1954).

MECHANISM OF REPETITIVE DISCHARGE It is beyond the scope of this review to discuss in detail the various mechanisms which have been proposed to account for repetitive firing (see particularly Rose and Mountcastle, 1954; Wall,

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1959; McIntyre and Mark, 1960; Kandel and Spencer, 1961). A brief inquiry will, however, be made to see to what extent these mechanisms suffice to explain the particular features of the geniculate repetitive discharge. In this respect, it will be convenient to consider the various factors as separable into two categories (see Kandel and Spencer, 1961), namely: ( a ) sustaining factors, and ( b ) limiting factors. a. Sustaining Factors.

As far as central neurons are concerned, the sustaining factors apparently always lead to a prolonged or maintained depolarization which can be of two types: ( 1 ) those due either to multiple synaptic potentials produced by dispersed synaptic volleys (Hunt and Kuno, 1959b; Wall, 1959) or to prolonged transmitter action (Eccles d QI., 1956; Curtis et al., 1958; Eccles et al., 1961d) and (2) those due to the electrical properties of the neuronal membrane, i.e., a nonsynaptically maintained excitatory state such as the depolarizing after-potential in hippocampal neurons ( Kandel and Spencer, 1961) or the rhythmically recurring spontaneous fluctuations in membrane potential in cortical cells (Li et al., 1961). There is still a sharp difference of opinion regarding the origin of the residual transmitter action that survives the spike process in many neurons such as the cells of the dorsal spinocerebellar tract (Eccles ef al., 1961d). Rall (1960) has suggested that, with monosynaptic activation of motoneurons, no transmitter substance survives for more than 2 msec and that the rebuilding of the EPSP after the spike is due to electrotonic spread from regions of the motoneuron not invaded by the spike. Eccles (1961) discounts this effect and has put forward the case for continued action by the transmitter substance. Long continued action by the transmitter substance seems to be the most likely explanation for the repetitive firing of Renshaw cells (Eccles et al., 1954; Eccles et al., 1961a). It is probable, however, that the above factors can occur in various combinations. Thus, the synaptic potentials can trigger bursts and may contribute to the maintained depolarization produced by the depolarizing after-potential as suggested by Bishop and Davis (196Ob). Since the depolarizing afterpotential in hippocampal neurons occurs with directIy initiated spikes, Kandel and Spencer (1961) regard it as an endogenous process intrinsic to the neuronal membrane.

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b. Liiniting Factors. These factors include the normal excitation processes and the changes in membrane properties occurring with time such as refractoriness and accommodation (Fuortes and Mantegazzini, 1962) and spike inactivation by depolarization (Granit and Phillips, 1956; von Ehler and Green, 1W) and the depression of excitability due to hyperpolarizing after-potentials (Brock et al., 1952; Bishop and Davis, 1960b). The data at present available are inadequate to provide a satisfactory model of the repetitive firing of lateral geniculate neurons. While the progressive reduction in the complexity of the presynaptic pathways seems the only adequate explanation for the considerable reduction in latency that may occur with increasing afferent stimulation, neither presynaptic nor postsynaptic reverberating activity in chains of interneurons provides a satisfactory explanation for the repetitive discharge itself. The relatively stereotyped pattern of the actual burst under widely diverse conditions suggests either that there is an origin intrinsic to the geniculate neuron or that any external influences serve only to maintain and possibly modify the activity of these intrinsic cellular mechanisms. Like optic tract axons (P. 0. Bishop et al., 1962a) the initial segment of lateral geniculate neurons probably fire repetitively to maintained depolarization (see Fuortes, 1959, for motoneurons) . The initial segment may possibly respond repetitively when the cell body discharges only singly. This is suggested by the finding mentioned above that a much greater proportion of optic radiation axons show repetitive activity than do their parent cell bodies (P. 0. Bishop et aZ., 1962a). A further consequence of this suggestion is that the repetitive firing observed in the LGN should frequently consist of a single cell-body response followled by a further one or more responses having only A spikes. Unfortunately, this has only rarely been observed (P. 0. Bishop et al., 1 9 6 2 ~ )The . second and subsequent members of repetitive discharge may be reduced in amplitude with respect to the first response and show a more pronounced A-B step but they seldom fractionate at this level. A somewhat analogous situation occurs in the double discharge found in nerve cells of the crustacean stretch receptor (Eyzaguirre and Kuffler, 1955; Edwards and Ottoson, 1958) and in the giant

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neuron of Aplys-iu (Tauc, 1962a, b). In the latter neuron, Tauc has shown that the site of origin of the orthodromic spike is situated some distance along the axon from the cell body in the same region as the axon-axonic synaptic scale. This region also acts as the pacemaker of spontaneous activity, The important finding in the present context, however, is that the axonal region can be reactivated by the current sink produced by the discharge of the cell body giving a double axonal spike but only a single soma spike. The repetitive firing in the LGN would clearly require a more prolonged current sink than that produced by the cell body spike since the first spike of the burst is normally always completed well before the onset of the second. On the other hand, a maintained depolarization could be produced by the prolonged action of the transmitter substance or the persistence of synaptic depolarization in dendrites which are not invaded by the propagated postsynaptic spike. Again a somewhat analogous situation may occur in the antidromically activated giant neuron of Aplysia if the somatic membrane is not immediately invaded by a propagated response (Tauc, 1962a ) . Under these circumstances, a prolonged after-depolarization occurs due to the recharging of the uninvaded somatic membrane. However, somatic local responses developed by and summating with this after-depolarization may then lead to the discharge of the cell body. The latter in turn leads not only to the possible reactivation of the axon but also to the abolition of the after-depolarization. When the discharge of the cell body is much delayed the afterdepolarization may itself directly reactivate the axon after the latter has recovered from refractoriness. In the LGN, it is possible also that spontaneous fluctuations of the membrane potential may reach threshold for spike discharge. Once the geniculate cell has discharged the negative after-potential of the cell body (P. 0. Bishop and Davis, 196Ob) might provide a maintained depolarizing action on the initial segment. This would explain the spontaneous burst discharges which may occur in the absence of any evidence of repeated synaptic activation. Kandel and Spencer (1961) have postulated a similar basis for the repetitive discharge of hippocampal neurons. Once appropriately triggered the hippocampal pyramidal cell appears to be capable of sustaining a brief period of repetitive &ing by the summation of depolarizin g after-poten tials. Despite continuing depolariza tion ,

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however, Kandel and Spencer suggest that the hippocampal burst is self-limiting because the depolarization itself leads to spike inactivation. The depressant effects of a maintained depolarization appear to limit not only the frequency of firing but also, by persistent and increasing inactivation of the inward Na' current, the duration of the burst. Such an interpretation is supported by the progressive decline in spike height and change in spike configuration which occurs during the hippocampal burst. By contrast, no such spike changes appear to occur during the geniculate burst, making it unlikely that spike inactivation is an important limiting process. In the LGN, the repetitive firing only occurs during the negative afterpotential, and it appears to be limited by the relatively abrupt onset of a long-lasting positive after-potential (P. 0. Bishop and Davis, 1960b). In a similar way, the hyperpolarizing after-potential limits the frequency of firing of motoneurons (Eccles et al., 1958). The discharge of hippocampal pyramidal neurons is not associated with a hyperpolarizing after-potential. The feature of the geniculate repetitive discharge that is most difficult to explain in terms of the ideas already put forward is the fact that the number of spikes in the burst usually decreases with increasing optic nerve stimulation. It is possible that many of the high-threshold, more slowly conducting fibers in the optic nerve are inhibitory to geniculate neurons, progressively eliminating the last members of the burst as their spikes enter the optic tract volley. AS yet, no potentials have been described as occurring in the LGN that might be regarded as analogous to the inhibitory postsynaptic potentials of spinal motoneurons. The absence of electrical evidence of synaptic inhibition in the LGN would, however, be explained if the inhibition is presynaptic as proposed by Frank and Fuortes ( 1957) and Eccles et al. (1961b) in the case of the primary afferents in the spinal cord. On the assumption that the discharge is due to the persistence of synaptic depolarization in the dendrites, an alternative explanation for the progressive reduction in the duration of the burst is that, with increasing synaptic depolarization of the dendrites, the spike process penetrates more deeply into them from the cell body, thus tending to cut short the long-lasting passive repolarization phase of the synaptic potential. This alternative explanation emphasizes the increasing synaptic drive to the geniculate neurons rather than any special effect of impulses in higher thresh-

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FIG.8. A. Diagram to illustrate the nomenclature used in discussing LGN cell responses. The solid line indicates the full response; it has two steps in its positive phase; the S-A and A-B steps. The dotted line indicates the time course of the S potential ( S ) , and the interrupted Iine the time course of the A potentiaI ( A ) superimposed on the S potential. The A potential is shown as monophasic but it may be diphasic. For details see text. (From P. 0.Bishop et al., 1962c.) B. Single-unit type b responses recorded from the vicinity of

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old fibers. It gains support from observations on the effects of drugs electrophoretically applied to geniculate neurons ( see Section IX, l3, 3 ) . VI. Fractionation of Unit-Waveforms

The single-unit orthodromic responses recorded extracellularly from the vicinity of the geniculate neuron cell-body have been described above as consisting of three components-S, A, and B. The corresponding antidromic responses have only the A and B components. A number of different methods have been employed to accentuate the features of the waveform that indicate the transition from one component to another or to fractionate the waveforms into their separate components (P. 0. Bishop et al., 1962a, b, c). These methods include: 1. Threshold stimulation. 2. Double and repetitive stimulation. 3. The combination of orthodromic and antidromic stimulation. 4. Conditioning by brief high-frequency stimulation. 5. The use of drugs such as lysergic acid diethylamide (LSD ) . The various components and the contribution they make to the single-unit discharge of the geniculate neuron are shown diagrammatically in Fig. 8A. The full response of the geniculate neuron to orthodromic stimulation is shown by the solid line. There are two inflections on the downslope of the initial positivity, indicating the the cell body of a geniculate neuron. a, superimposed antidromic responses showing fractionation at the A-B step (indicated by arrow); b, antidromic conditioning and orthodromic testing responses from same unit as in trace a. Shock artifacts indicated by filled circles. Antidromic response consists of an A potential only. Orthodromic response has all 3 components, S, A, and B, respectively. Arrows indicate S-A and A-B steps. Voltage calibration, 5 mV, for both traces. (From P. 0. Bishop, W. Burke and R. Davis, unpublished). C. Extracellular records from 2 geniculate neurons; a, b, and c, three examples of multiple superimposed orthodromic responses from the one geniculate neuron following constant threshold shocks to the ipsilateral optic nerve. Note the constant amplitude of the S potential, the play of latency of the A spike, and the prominent A-B step when the B spike is delayed. Arrows indicate S-A and A-B steps, respectively. f and g, extracellular responses from the one geniculate neuron; f, antidromic response following cortical stimulation; g, orthodromic response following contralateral optic nerve stimulation. e, baseline for antidromic response ( f ) obtained by using a cortical stimulus just subthreshold for the unit response in f.

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S-A and A-B steps. The dotted line indicates the time course of the S potential and the broken line the time course of the A potential. The A potential may be either monophasic as shown or diaphasic (see below). The responses shown in Fig. 8B were obtained from the same neuron using different methods of stimulation. Two antidromic responses have been superimposed in trace a; one response consists of an A potential only and the other has both an A and B potential. In the case of trace c, a conditioning antidromic stimulus has been followed by a testing orthodromic shock, the two shock artifacts being indicated by the filled circles. The antidromic response consists of an A potential only while the orthodromic response contains all three components, S, A, and B. The S-A step has been accentuated here because the onset of the orthodromic A potential is delayed by refractoriness since it occurs only about 1.5 msec after the antidromic A response. The slight broadening of the positive phase of the conditioning antidromic A-potential, due to a deforniation soon after the peak, is probably the result of a local response on the part of the membrane responsible for the B potential (see below). The S-A and A-B steps are also clearly evident in the traces shown in Fig. 8C. Traces a, b, and c were all selected from a series of responses by the one geniculate neuron to a constant threshold stimulus applied to the optic nerve. In a, using a constant threshold stimulus, five traces have been superimposed, two stimuli failed to evoke any response, two produced S potentials having identical amplitude and time course, and the remaining trace shows the full response. The traces in b show the play in latency of the onset of the A-B complex with the orthodromic stimulus held constant. The orthodromic response can rarely be made to fractionate at the A-B step, though the step becomes more clearly evident if the onset of the A-B complex is delayed (Fig. SC, c). In Fig. 8C traces e, f, and g were obtained from the one recording site in the vicinity of another geniculate neuron. Trace f shows the antidromic response with an obvious A-B step, the trace itself being superimposed on a long-lasting stimulus artifact. The baseline produced by the stimulus artifact is shown at e using a just subthreshold stimulus. The orthodromic response by the same neuron is shown at g. The S-A step is clearly evident but the A-B step is much less obvious than in the case of the antidromic response.

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A. THESINGLE-UNIT S POTENTIAL The S potential recorded extracellularly from geniculate neurons undoubtedly corresponds to the excitatory postsynaptic potential (EPSP) as recorded intracellularly from the motoneuron. The extracellularly-recorded single-unit S potential is a slow positive monophasic wave. The S-A step or the S potential is commonly about one-fifth the amplitude of the positive phase of the full cell response (P. 0. Bishop et al., 1 9 6 2 ~ ).The rise time ranged from 0.37 to 0.75 msec (mean, 0.48 msec) and time of half decay from peak ranged from 0.36 to 1.40 msec (mean, 0.83 msec). Measurements of total duration were unreliable but varied from about 2 to 10 msec. While the S potential can occasionally be graded, in the great majority of cases it behaves in an all-or-nothing manner with a constant amplitude (Fig. 8C, a ). Even when the S potential can be graded, usually only a few relatively discrete amplitude steps can be obtained. From this it is concluded that an S potential sufficient to excite an A potential can be generated by a single optic tract axon and that normally each geniculate neuron is innervated by a relatively small number of tract axons. It is probable that a single optic tract axon can be effective in this way only because the terminals arborize extensively, thereby applying a large number of endings to each of the cells that it innervates. The most unusual feature of the S potential is its positive polarity. This indicates that, in addition to the initial segment, the cell body is also a source of current throughout the duration of the potential. The most likely explanation for this finding is that the surface of the dendrites has a much greater density of presynaptic terminals than does the cell body. Hence, the dendrites would suffer a relatively greater synaptic depolarization than the cell body so that the latter would act as a source of current to the dendritic sinks. Glees ( 1941) thought that axon-dendritic contacts predominated over axon-somatic contacts in the LGN, and recently J. SzentAgothai (personal communication, 1962) has confirmed this impression. Undoubtedly, artificial deaff erentation by the electrode and damage to the cell body would tend to accentuate the positivity by rendering the membrane in the vicinity of the electrode partially or completely unresponsive. That the usual positive S potentials are not produced

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by damage to the membrane is, however, indicated by the fact that they are regularly observed in the presence of a B potential having a large negative component. A consideration of the morphology of the lateral geniculate neurons provides a satisfactory interpretation for the polarity of the S potential. The dendrites of the geniculate neurons extend out more or less radially in all directions from the cell body and occupy a very much larger volume in the LGN than do the cell body and initial segment. The density of current entering the widely scattered dendritic sinks would, therefore, be very much less than that leaving the concentrated cell body source. It is not surprising, therefore, that a positive S potential can be readily obtained in the vicinity of the cell body, whereas the corresponding single-unit negative dendritic potential is very small and difficult to record. In addition, the latter tends to be swamped by, and difficult to differentiate from the multineuron negative S potential (discussed below). By activating only a single motoneuron, Fatt (1957) was able to plot the external potential field much more widely than would have been possible had other cells also been active in the vicinity. This possibly explains why his potentials are mainly negatively directed.

B. MULTINEURON S POTENTIALS In the absence of a postsynaptic spike an afferent tl volley produces a negative multineuron S potential as recorded by a lowresistance electrode from among the LGN cells and a positive S potential from among the optic radiation axons above the LGN (Fig. 4, d and e ) (P. 0. Bishop and McLeod, 1954). As far as synaptic currents flowing between cell body and dendrites are concerned, the radial arrangement of the latter leads to a closed field of current distribution. .4n electrode located on the outskirts or beyond the spread of the dendrites would fail to record any potentials generated by these currents. It is only as the axon approaches and leaves the limits of the field of distribution of the dendrites that an open type of current distribution is produced. The essentially parallel arrangement of the optic radiation axons means that, during the course of the EPSP, relatively large potentials can be recorded by a low resistance electrode, due to current flowing from sources located along the axon into sinks on the cell body and dendrites. Except for its reversed polarity, the wavefonn and time course of

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the multineuron negative synaptic potential closely resemble those of the positive single-unit S potential. Single-unit waveforms of type c occasionally have a step on the rising phase of the initial negativity. Such a step could be due either to the S potential of the single cell in question recorded at a distance or more probably the multineuronal S potential from the cells in the vicinity. Multineuron synaptic potentials in response to optic nerve volleys have been recorded while the geniculate synapses were blocked by lysergic acid diethylamide and as a result of posttetanic delayed depression, the level of block being monitored by recording from the cerebral cortex (P. 0. Bishop and Davis, 1960b). The time course of the synaptic potentials was the same in either case. Measured from the onset of postsynaptic activity, the initial negativity has a duration of about 12 msec, the succeeding positivity reaches its maximum at about 25 msec, the baseline is recrossed soon after 60 msec and the late negative wave has a peak at about 100 msec. The subsequent oscillations of potential are indistinguishable from the spontaneous slow rhythms which also occur in the LGN during synaptic block. Since the later phases of the synaptic potential have yet to be studied at unit level, they will not be further discussed here.

C. A AND B POTENTIALS While evidence of an A-B inflection is almost invariably present on the downstroke of the initial positivity in both the orthodromic and antidromic responses of geniculate neurons, the step is always much more prominent in the antidromic records. The orthodromic responses rarely fractionate at this level but can be made to do so by stimulation during the refractory period of a B potential of anti. such a fractionadromic origin (P. 0. Bishop et al., 1 9 6 2 ~ )When tion occurs in response to a single orthodromic stimulus the cell is probably damaged to some extent. Even with repetitive orthodromic stimulation the geniculate response rarely fractionates at the A-B step. By contrast, the appearance of an A potential in isolation is comparatively common with antidromic stimulation. Bishop et al. (1962b) found that half the units activated by a single cortical stimulus responded occasionally with only an A potential. Those of the remaining units that were investigated all were readily reduced to an A potential by repetitive cortical stimulation.

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For each neuron, the level of the A-B step is the same whether the stimulus is orthodromic or antidromic. Whenever an A potential in the absence of a B potential was obtained from the one neuron as a result of both orthodromic and antidromic stimulation, the response was similar, both being either monophasic or diphasic. The A potential is frequently diphasic (positive-negative ) and this occurs only when the B potential is also diphasic (positive-negative). A monophasic A potential can, however, occur in the presence of a diphasic B potential. The reduction in amplitude or absence of the negative phase of the B potential is regarded an indicating damage to the cell body membrane (P. 0. Bishop et aZ., 1962b, c ) . When the A potential is diphasic, the amplitude of the negative phase averages slightly less than half that of the positive phase. The ratio of the amplitude of the negative phase of the A potential to that of the negative phase of the B potential averages 0.16. Some further features of the A and B potentials noted by P. 0. Bishop et al. ( 1 9 6 2 ~ )are of importance in relation to the steps in the production of the postsynaptic spikes. The negative phase of the A potential in isolation commences at a time when the positive phase of the B potential in the full response is largely over. Also, the positive phase of the B potential increases in amplitude more or less p r i passu with that of the negative phase during recovery from refractoriness. Griisser-Cornehls and Griisser ( 1960) described two components ( a and p ) in the unit-responses they obtained in the LGN. They decided against the interpretation that they were the extracellular equivalent of the IS and SD components described by Brock et aZ. (1953) and considered the CY component to be presynaptic. It is not and components of Griisser-Cornehls easy to homologize the and Griisser with the A and B potentials described above particularly since these authors did not describe any component as being equivalent to the EPSP’s in the motoneuron. Furthermore, they found that the a and p components could usually he separated by two orthodromic shocks separated by a critical interval. In the present writer’s experience, the fractionation takes place under these circumstances at the S-A step and rarely if ever at the A-B step, at least in a normal cell. It is possible that the a component corresponds to the S potential and the component to the A-B complex. (Y

a

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Again, many of the units may have been axons, a and /3 being nodal components. Phillips (1959) has pointed out that the behavior of Betz cells to antidromic activation differs considerably from that characteristic of spinal motoneurons (Brock et al., 1953). While many spinal motoneurons respond to a single stimulus with only an A potential, A-B transmission is in any case easily prevented by close spacing of impulses. In Betz cells, on the other hand, it is exceptional to prevent the generation of a B spike even with antidromic shocks separated by only 0.5 msec although the A-B step becomes more prominent under these conditions. P. 0. Bishop et aZ. (1962b) have drawn attention to the fact that there is a gradation of properties from Betz cells through LGN cells to spinal motoneurons, the ease with which the antidromic impulse invades the cell body decreasing in that order. These differences are probably related to the rate of expansion of the membrane at the axon hillock region (Phillips, 1959). The time course of the action potential also varies considerably in different neurons. The time course of the intracellularly recorded Betz-cell action potential (Phillips, 1959) is shorter (duration, 0.5 msec or less) than that of the similarly recorded spinal motoneuron (duration 1.0 msec or more). The total duration of the intracellularly recorded A-B potential of the spinal motoneuron has been variously reported as having a mean value of 1.0 msec (Brock et al., 1952) and 1.57 msec (Frank and Fuortes, 1955). Both values are, however, shorter than the 1.84 msec found by Kandel et al. (1961) for hippocampal neurons, Considering extracellularly recorded responses, the total duration of the Betz-cell action potential is no more than about 1msec (Phillips, 1959) whereas the duration of the geniculate neuron response is never less than 1.5msec and is usually 2-5 msec. The difference lies mainly in the negative phase since the positive phase of the geniculate response is only slightly longer than that of the Betz cell (both 0.25-0.5 msec). In general, the negative phase of the geniculate response is up to 5 times or more as long as the positive phase, whereas in the Betz cells it appears to be about the same duration. These differences are due to variations in the duration of the negative phase of the B spike, presumably reflecting differences in the way that the impulse invades the cell body.

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The extracellularly recwrded responses of geniculate neurons (described above) differ considerably from those obtained from spinal motoneurons by Fatt ( 1957) and Terzuolo and Araki ( 1961). No adequate explanation for these differences can, as yet, be provided.

D. GENERATIOX OF POSTSYNAPTIC SPIKES The original suggestions of Forbes (1934, 1939), Gesell (1940) and P. 0. Bishop (1953) that the spikes take origin in the initial segment of the axon have been confirmed by the studies of Araki and Otani (1955), Fuortes et ul. (1957), Coombs et uZ. (1957a, b ) on the spinal motoneurons and it is now clear that the sensory neurons in the LGN react in the same general way. In the case of spinal motoneurons (Eccles, 1957), crustacean stretch receptors (Edwards and Ottoson, 1958) and Aplysiu giant neurons (Tauc, 1962a, b ) the membrane in the initial segment of the axon is more excitable than is any other part of the soma-dendritic membrane. Whatever the form of the stimulation, whether it be an electrical stimulus, an EPSP, an antidromic spike, or an intracellularly applied depolarization, the first excitation occurs in the initial segment membrane. The rest of the electrically excitable soma-dendritic membrane is then fired by local circuit current from the initial segment. The steps in the production of geniculate neuron spikes by orthodromic and antidromic stimulation have been described in detail by P. 0. Bishop et al. (1962a, b, c ) . Since the sequence of events closely parallels the now familiar situation in the spinal motoneuron it will only be necessary in this context to discuss features which may be either peculiar to geniculate neurons or specifically revealed by the extracellular method of recording. When slowly advancing the microelectrode through neural tissue, the tip is likeIy to be preferentially moved into the immediate vicinity of the cell body since the latter generates much larger potentials than any other portion of the neuron. In interpreting responses recorded extracellularly from single units it is important to take this bias into consideration since activity in parts of the neuron other than the cell body will nearly always make only an indirect (and therefore positive-going ) contribution to any waveforms that are large enough for detailed study. The synaptic depolarization of the dendrites and cell body leads to the generation

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of the A potential in the initial segment of the axon at some little distance from the cell body. As recorded from the cell body, the A spike has an initial positive phase. Preferential recording from the vicinity of the cell body would explain why A spikes with an initial negative phase have not been recorded. The area of membrane responsible for the A potential is small and the portion with the lowest threshold is situated at some distance from the cell body. When recorded in isolation from the B potential, the A spike positivity is not infrequently followed by a negative phase indicating that conduction occurs up to the vicinity of the electrode tip. Presumably this A spike conduction still occurs even when a B spike is produced, although electrical evidence for this conduction would be obscured by the altered pattern of extracellular current flow. Bennett et al. (1959), working on the supramedullary neuron of the puffer (Spheroides maculatus) recorded an A spike with a shallow and prolonged terminal negativity. They did not regard this as representing activity under the recording electrode but rather that the membrane was inactive and that part of the current flow induced in it by the electronically spread potential was capacitative. Regardless of whether the geniculate A spike is diphasic or not, the B spike usually commences with an initial positivity which indicates that the latter spike also arises at a distance from the recording site, presumably in the direction of the initial segment of the axon. Subsequent propagation into the main portion of the cell body is signaled by the prolonged negativity of the B spike. As mentioned above, the positive phase of the B potential is largely over before the time at which the negative phase of the A potential commences when the latter is recorded in isolation (see Fig. 9B). In explanation of these findings, P. 0. Bishop et al. (1962~)suggest that the boundary between initial segment and cell body is highly irregular, strands of more excitable A-type membrane extending for considerable distances into the cell body. This situation has been diagrammatically represented in Fig. 3 by the finger-like prolongations extending from the axon hillock region into the body of the cell. Such a possibility has also been considered by Coombs et al. (1957a). When a second B spike is elicited by orthodromic stimulation immediately after an antidromically produced B spike, the second B spike is much smaller than normal but still diphasic . indicates that positive-negative (P. 0. Bishop et al., 1 9 6 2 ~ )This

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only a relatively few strands of B-type membrane are becoming active and the response is abortive as far as the main B-area is concerned. As recovery from refractoriness proceeds more strands respond; the positive-negative wave increases in amplitude until the full B spike is produced. As P. 0. Bishop et al. ( 1 9 6 2 ~ )point out these prolongations of A-type membrane would have the effect of grading transmission from the initial segment into the cell body, thereby increasing the safety factor for invasion of the cell body. Both Tauc (1962a, b) and Fuortes et al. (1957) envisage between initial segment and soma a transitional zone of gradually changing properties, whereas the model of Coombs et al. (1957a) postulates a sharp transition of thresholds. While our findings are compatible with the idea of a transitional zone, they seem to require in addition fairly extensive interdigitating of A- and B-type membranes. There is still a difference of opinion concerning the nature of the invasion of the cell body by the B spike. Since the impedance of the neuronal membrane is high compared to the resistance of the cytoplasm in the cell body, it is generally held that the depolarization of the soma membrane is virtually simultaneous over its entire surface (Eccles, 1957; Svaetichin, 1958; Freygang and Frank, 1959). By placing an external electrode on different parts of the lobster stretch-receptor cell, Edwards and Ottoson ( 1958) were able to show that the impulse does propagate over the surface of that cell. Conduction of the B spike also takes place at least in the axon hillock region of both the supramedullary puffer neuron (Bennett et al., 1959) and Aplysia giant neuron (Tauc, 1962b) since in both cases the B spike, as recorded from the surface of the cell body, commences with a brief phase of positivity. Presumably, conduction over the main surface of the cell body would be very much faster and. by comparison with the axon hillock region, virtually instantaneous. Much the same variation in conduction velocity on the part of the B spike also occurs in the geniculate neurons. There is a portion of the B-type membrane, presumably in the region of the axon hillock, over which conduction occurs at a slower rate than over the main surface of the cell body. When the B spike loses its negative phase, the electrode is probably indenting the cell membrane, rendering it locally inactive. Under these circumstances, the cell body is still invaded since the positive phase now becomes very large, indicating that the locally

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inactivated region is acting as a source of current not only to the axon hillock region, as before, but also now to the remainder of the cell body, No doubt, part of this increase is due to more effective electrode sealing, increasing the recording resistance. It is frequently assumed that the intracellular glass capillary microelectrodes do not cause any significant alteration in the behavior of the neuron. Experience with extracellular recording indicates that the region of puncture is almost certainly rendered inactive (see Freygang and Frank, 1959; Murakami et a?.1961 , ) . Thus, extracellular recording, under conditions in which the B spike has a prominent negative phase, is likely to reflect the normal behavior of the neuron rather more faithfully than would be the case with intracellular recording. The latter has the added hazard of electrolyte leakage from the tip of the microelectrode. In our experience, however, loss of B-spike negativity in the extracellular record may leave the behavior of the cell unchanged as a whole, insofar as we have been able to judge this by its discharge pattern. Certainly the most stable extracellular recording conditions are generally obtained when the B spike has only a relatively small negative phase and the discharge may be recorded for very many hours without apparent change.

E. PROPERTIES OF DENDRITES OF GENICULATE NEURONS Relatively little precise information is available about the properties of dendrites. Whether or not dendrites are electrically excitable and can sustain a propagated action potential is only one of the many controversial issues awaiting a satisfactory solution (Eccles, 1960). Our interpretation of both unit and multineuron responses from geniculate neurons has required that the principal sites for synaptic depolarization should be located on the dendrites. During the course of the EPSP it is likely that the dendrites act as a sink for current drawn from the cell body and axon. In addition the dendrites are probably largely responsible for the slow oscillations of potential that occur in the LGN not only spontaneously but also as a result of both orthodromic and antidromic activation (Bishop and Davis, 1960b). However, the interpretation of our records has not required conduction along dendrites nor have we been able to assign any portion of our unit responses to propagated activity in dendrites. If the negative phase of the B spike represents invasion of the cell body, one would expect a terminal positivity as

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the impulse propagates away from the cell body up the dendrites (see Svaetichin, 1958). A shallow terminal positive phase is occasionally seen in orthodromic records but this apparently docs not occur in antidromic records. The shallow terminal positivity in the orthodromic records is more satisfactorily explained as residual synaptic depolarization in dendrites uninvaded by a propagated impulse. This synaptic partial depolarization of the cell body and dendrites accounts for the other differences in the waveforms of the orthodromic and antidromic geniculate responses noted by P. 0. Bishop et al. (1962b). The slightly greater amplitude of the 13 spike in orthodromic records is probably due to the momentarily greater external current density achieved by the very rapid invasion of the synaptically depolarized cell body and the shorter duration of the orthodromic B spike is probably due to the effect of the residual synaptic depolarization of the invaded dendrites reducing the external current density and occasionally reversing its direction to give the B spike the shallow terminal positivity mentioned above. Our

FIG. 9. A. The regions of the giant neuron of Aplysia corresponding to different components of the antidromic spike recorded simultaneously from the soma with intracellular ( I n ) and extracellular (Ex) electrodes. I, soma; 11, axon hillock; 111, transitional zone; IV, normal site of origin of spike; V, distant axon. (From Tauc, 1962b.) B. Extracellular antidromic waveforms of a geniculate neuron (negative downwards for comparison with Aplysia neuron response in Fig. 9 A ) . Three superimposed responses to a constant cortical stimulus of threshold strength (note blank sweep). Partial response is a diphasic A potential. Full response has a well-marked A-B step. (From P. 0. Bishop d at., 1962b.)

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extracellular records of antidromically invaded geniculate neurons are remarkably similar to those obtained by Tauc (1962a, b ) near the soma of the Aplysia giant neuron (Fig. 9A). The A-B step in the geniculate record in Fig. 9B is rather more prominent than usual, the B spike occasionally failing altogether. The invasion of the cell body in Aplysia is equally uncertain. More important in the present context, however, is the fact that the extracellular records from the Aplysia neuron have no terminal positive phase in keeping with the fact that the soma is devoid of dendrites. It is interesting that the extracellular potentials from antidromically invaded Betz cells (Phillips, 1959) show no evidence of a terminal positive phase despite the fact these cells have a large apical dendritic system. Terzuolo and Araki (1961) have, however, recorded a late positive wave with a microelectrode located outside the soma membrane of the spinal motoneuron. They interpret this as due to propagation along some portion, at least, of the dendrites, as pointed out above. Tasaki et al. (1954) have obtained slow, diphasic, all-or-none responses from geniculate neurons which they have interpreted as due to slow propagation along dendrites. We have obtained similar records (e.g., P. 0. Bishop et al., 1962a) but have interpreted their slow time course as the result of injury to the cell body. Even in the case of the very elaborate dendritic system of the Purkinje cell in the cerebellar cortex Granit and Phillips (1956) were unable to assign any potential to the dendrites with the possible exception of the slow D response. The slow D potential has, however, properties akin to a synaptic potential rather than that of a propagated impulse. Castillo and Katz (1954) have obtained evidence which indicates that the chemoreceptor areas at the end-plate of the muscle membrane are probably electrically inexcitable. It is possible that, if dendrites become sufficiently encrusted with synaptic scale, the mosaic of chemically and electrically excitable patches on the membrane has an insufficiency of the latter electrically excitable patches to sustain a propagated impulse, This may well be the situation with respect to the principal neurons in the lateral geniculate nucleus. In other situations in the nervous system, it is possible that the dendrites may be capable of conduction (e.g., Fatt, 1957; Terzuolo and Araki, 1961) or at least have graded response characteristics ( G . H. Bishop, 1958).

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F. AFTER-POTENTIALS OF GENICULATE NEURONS The after-potentials which follow the postsynaptic spike have SO far only been studied in multineuronal responses (Vastola, 1957, 1959a, 1960a; Bishop and Davis, 1960b) so that only a brief reference will be made to them here. Measured from the onset of postsynaptic activity following optic nerve stimulation, the initial negative after-potential of the orthodromic response has a mean duration of 7.3 msec; the subsequent positive after-potential has a sharp decline to a minimum at about 92 msec and returns to the baseline at about 80 msec. There is a late negative peak soon after 100 msec. The subsequent oscillations of potential, which have a mean frequency of 9 cps in the lightly barbitalized animal, cannot be distinguished from the spontaneous slow rhythms. Except for the initial after-negativity, the antidromic after-potentials are the same as those evoked at the same site by orthodromic volleys. VII. Refractory Period of Geniculate Neurons

Neurons have as many different refractory periods as there are ways of activating them, whether by orthodromic volleys, antidromic volleys, or by direct extracellular or intracellular stimulation. The refractory period will further depend upon the particular stimulus combination used for conditioning and testing. In addition, the several components of the cell response have different refractory periods. From the point of view of neuronal organization, the most significant refractory period will be determined by conditioning and testing volleys both of which are orthodromic. The data obtained by other methods of conditioning and testing may, however, be used to analyze the properties of the various components of the neuron response. P. 0. Bishop and Evans (1956) have made a detailed study of the recovery of the geniculate neurons from the refractory state using orthodromic conditioning and testing volleys and recording multineuron responses in the LGN. Recovery from refractoriness in the LGN is principally determined by the rate at which normal conducting ability returns in the presynaptic fibers. The testing volley, traveling in the wake of the conditioning volley, conducts more slowly in the relatively refractory axons. As separation between the two volleys increases, however, the second progressively

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gathers more speed, so that the interval between the two volleys asymptotically approaches a constant value which is independent of the initial interval at which they started (Tasaki, 1959). For this reason, the refractory period is best stated as a “least-response-interval” rather than as a “least-stimulus-interval.”The least-responseinterval is the least interval between two responses whereas the least-stimulus-interval is the least interval between two stimuli both of which provoke a response. The absolutely refractory period of a synaptic system is greater, the longer the effective presynaptic pathway. Using the multineuron response, Bishop and Evans (1956) set an upper value of 0.85 msec for the least-response-interval with conditioning and testing stimuli applied to the optic tract about 10 mm from the LGN. With stimulating electrodes applied to the optic nerve in the orbit, the conduction distance to the LGN is 3035 mm and, under these conditions, the least-response-interval of the geniculate synaptic system is about 1.1msec. The geniculate neurons are always capable of responding as soon as conduction becomes possible in the optic tract fibers. Using the multineuron spike height as an index, the relatively refractory periods of the presynaptic ( tl) and postsynaptic ( r l ) geniculate responses were 1.7 and 1.6 msec, respectively. Supernormal responsiveness on the part of the geniculate neurons commences while the optic nerve fibers are still relatively refractory. For further studies using the multineuron response see Vastola ( 1957). The only systematic single-unit study of the refractory period of geniculate neurons is that of Griisser-Cornehls and Griisser (1960) who restricted their attention to orthodromic stimulation. Using various methods of stimulation, P. 0. Bishop et al. (1962b, c) have also studied the refractory period of a few geniculate neurons. Comparison between the respective results is complicated by the differing interpretations that are placed upon the unit-waveforms (see above).

A. ORTHODROMIC-ORTHODROMIC RESPONSES It is not normally possible to fractionate the geniculate response at the A-B step using doubIe orthodromic volIeys and the same is true of the spinal motoneuron. Under these circumstances, both neurons fractionate to an EPSP, not to an A potential. Bishop et al. (1962b) found, for one geniculate neuron, that the least-response-

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interval for the full response using orthodromic stimulation was 1.7 msec. Of the 60 geniculate units studied by Griisser-Cornehls and Griisser (1960), nineteen had discernible LY and /3 components regarded by them as characteristic of the cell body response. Considering only these 19 units, the least-stimulus-interval for the 01 component was 0.66 msec (0.35-1.05 msec) while that for the 3./ component was 0.1-0.5 msec longer. The corresponding least-response-interval for the a,@ spike was L1.5 msec (0.71-1.6 msec). The least-response-interval in the latter case was presumably measured with respect to the component. These values correlate well with those obtained in the multineuron studies of Bishop and Evans (1956). The spinal motoneurons are much less readily stimulated orthodromically than are geniculate neurons and many require the summation of EPSP’s in order to generate an impulse. Even when an impulse has been generated by a single orthodromic volley, the generation of a second impulse by the motoneuron is improbable until there has been some recovery from the after-hyperpolarization (Eccles, 19%). When the orthodromic volley has a powerful stimulating action, a second motoneuron spike may, however, be generated as early as 4.8 msec after the first (Eccles, 1953). In the case of orthodromic volleys which fail to generate a motoneuron spike, a second EPSP may follow the first with a delay that is determined only by recovery in the presynaptic pathway (Curtis and Eccles, 1960). Subnormality limits the response frequency of motoneurons to about 50/second but many spinal interneurons can follow rates of stimulation in excess of 500/second and in some cases over 1000/ second (Hunt and Kuno, 1959b; Eccles et al., 1960). In order to respond at such frequencies refractoriness following an impulse must be nearly as brief as in A fibers. Interneurons usually respond repetitively to a single afferent volley (Frank and Fuortes, 1956) but with repetitive stimulation above about 2-!5/second the repetitive discharges begin to fail and at high rates of stimulation only the earliest spike continues to be evoked. Lateral geniculate neurons are also capable of responding to high rates of stimulation, up to 1000/second or more, but usually fail after 3 or 4 impulses (Bishop and Evans, 1956; Bishop et al., 1962a). By contrast, spinal interneurons may follow frequencies in afferent volleys up to 700/second for as many as 30 responses ( Eccles et al., 1960). (Y

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B. ANTIDROMIC-ORTHODROMIC RESPONSES The use of an antidromic conditioning volley avoids the limitation imposed by the refractoriness of the presynaptic pathway although the response to the orthodromic testing volley will now depend upon the extent to which the antidromic impulse invaded the cell, In one geniculate neuron with antidromic conditioning (full response) and orthodromic testing the least-response-interval of the A potential was 1.12 msec and that of the B potential, 1.25 msec (Bishop d. al., 1 9 6 2 ~ ).These values are in good agreement with the earlier multineuron studies with double orthodromic stimulation indicating once again that the limiting factor, as far as the principal cells in the LGN are concerned, is refractoriness in the presynaptic pathway rather than in the synaptic mechanisms of the postsynaptic cell. Following an antidromic A-B spike in spinal motoneurons, an orthodromic volley cannot begin to generate an EPSP until the end of the B potential, i.e., about 1 msec or slightly longer after the onset of the antidromic spike (Coombs et al., 1955; Eccles, 1957). Thereafter, full recovery of the EPSP is prolonged being about 70% of normal at about 5 msec from the start of the spike. C. A~~;TIDROMIC-A~TIDROMIC RESPONSES Using double antidromic stimulation, the least-response-interval has been determined for a number of geniculate neurons (P. 0. Bishop et al., 196213). The A potential first appeared at a response interval of about 1.0 msec and the B potential at an interval of 1.5-4.0 msec. In all instances the conditioning volley produced a full A-B potential. The corresponding least-response-intervals for spinal motoneurons are rather longer, being 1.25-1.45 msec for the A potential and 2.5-50 msec for the B potential (Brock et al., 1953). If only an A potential is generated in the motoneuron by the conditioning volley the least-response-interval for a second A potential is rather less, being 09-12 msec. Thus, if the cell body discharges, it lengthens the refractory period of the initial segment, presumably as a result of the catelectrotonic effect of the currents generated by the initial B spike (Brock et al., 1953; Fuortes et at., 1957). The coupling between the initial segment and the cell body is even closer in the case of cells of the dorsal and ventral spinocerebellar tracts (DSCT and VSCT, respectively) (Curtis et al., 1958;

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Eccles ct al., 1961c), than it is with geniculate neurons. The leastresponsc-interval for VSCT cells using double antidromic stimulation was 1-2 msec. In these circumstances, the A-B inflection was accentuated but there was never a transmission block, i.e., an A spike in isolation, such as invariably occurs in motoneurons under such conditions. The A-B coupling is also very close in the case of Betz cells since full invasion of the cell by a second antidromic volley occurs with a least-stimulus-interval of 0.5 msec (Phillips, 1959). A stimulus-interval of 0.5 msec probably corresponds to a response interval of about 1.0 msec under these circumstances (see Phillips, 1959, Fig. 8). Again, the A-B inflection was exaggerated but, at a sufficiently brief stimulus-interval, the Betz cell response failed as a whole without fractionation.

I>. AXTIDROAIIC ISTRACELLULAR STIMULATION The most direct method of measuring the recovery of the excitability of the components of a neuron response is to test the excitability by brief depolarizing pulses intracellularly applied at various intervals after a conditioning response. Such a technique has been developed for motoneurons but not as yet for geniculate neurons. Following a full spike of antidromic origin, the absolutely refractory period of the A spike in the motoneuron is as brief at 1.3 msec and that of the B spike, 2.0 msec (Fuortes et al., 1957, Fig. 8 ) . Using the same technique, Coombs et al. (19574 observed similar intervals for the two refractory periods. The recovery of excitability following a simple A spike of antidromic origin is rather more complex since a B spike can be generated before the end of the refractory period of the A spike (Coombs et al., 1957a). It is instructive to note that the absolutely refractory periods of the A and B components of the motoneuron response, as measured by the technique just described, are still rather longer than those described earlier for the geniculate neuron using double antidromic stimulation, namely: A, 1.0 msec; B, 1.5 msec. It must be remembered that the latter values include the increased conduction time of the testing volley traveling in the recovering optic radiation axons. This effect, tending to increase the least-response-interval of geniculate neurons, was not present in the case of the directly stimulated motoneurons. Since, in every way that it is tested, the geniculate neuron recovers its excitability more readily than does

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the motoneuron, the two cells probably differ in respect to their membrane properties rather than simply in respect to morphology. The graded amplitudes of the A and B components of the geniculate response when elicited at critical intervals after a previous response are similar to the gradations found in the corresponding components of the motoneuron response under similar circumstances (Brock et al., 1953; Frank and Fuortes, 1955). These gradations may be ascribed to inactivation of the sodium carrier mechanism and increased potassium permeability occurring during the period of relative refractoriness. VIII. Recovery of Responsiveness

The time-course of the recovery of responsiveness of optic tract fibers following single and repetitive antidromic shocks (P. 0. Bishop et al., 1953a) and of lateral geniculate neurons following corresponding orthodromic volleys (P. 0. Bishop and Davis, 1960a) have been reported but since multineuronal responses were used they will only be briefly referred to here (see also Vastola, 1957, 1959a, b, 1960a, b) . A. SINGLEORTHODROMIC VOLLEYS

During recovery from refractoriness following a single orthodromic volley, the geniculate neurons pass through a brief phase of relative supernormality (114%).When the optic tract axons have regained normal responsiveness (shock interval, 1.7 msec) the geniculate neurons may be slightly subnormal. The early phase of relative supernormality is followed by a late phase of true supernormality with a peak (113%)at a mean shock interval of 4.7 msec and ending at 6.7 msec. The succeeding phase of depression is maximal at a mean shock interval of 19 msec (32%).At this time synaptic transmission may be largely blocked. Thereafter, recovery proceeds relatively rapidly until about 200 msec and then much more slowly until normality is achieved at 2 sec. The above sequence of events occurs in the cat under barbiturate anesthesia and it may not be characteristic of the normal unanesthetized animal. A similar early phase of increased excitability following discharge has recently been reported as occurring in the spinal trigeminal nucleus of the rat (Erickson et al., 1961).

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B. REPETITIVE ORTHODROMIC VOLLEYS The study of the responses of optic tract axons and of lateral geniculate neurons during and after repetitive stimulation (Hughes et aZ., 1956; Evarts and Hughes, 1957a, b; Bishop et al., 1959a; Vastola 1959a, 1960a) has brought to light the curious phenomenon of posttetanic delayed depression. If the repetitive orthodromic volleys are of sufficient frequency and duration the geniculate synapses may be blocked for many hours. Evarts and Hughes (1957b) and Morlock et al. (1962) found that this prolonged depression only occurs in the anesthetized preparation. Contrary to this experience Bishop et (11. (1959a) still found a delayed depression in the cerveau is014 preparation which was quite similar to that occurring during anesthesia. IX. Pharmacology of Geniculate Neurons

While the nature of the transmitter substance at geniculate synapses is still unknown much detailed information has nevertheless become available in recent years concerning the action of pharmacological agents on geniculate neurons. In the majority of these studies the drugs were injected either intra-arterially (intra-carotid) or intravenously and their action assessed by recording the multineuronal geniculate response to optic nerve stimulation. These early studies have however been recently confirmed and extended by Curtis and Davis (1961, lWh, b ) using single-unit recording and local electrophoretic application of the drugs. A. INTHA-ARTERIAL DRUG APPLICATIOK

The earlier studies may be briefly reviewed. Evarts et aE. (1955) first reported that the intracarotid injection of D-lysergic acid diethylamide (LSD) or bufotenine reduced the postsynaptic responses of geniculate neurons to optic nerve volleys. Using both intracarotid and intravenous injections of LSD, Bishop et uZ. ( 1 9 5 8 ~ )confirmed these findings. The observed failure of LSD to affect the latency or amplitude of the presynaptic spike, the increase it produced in the synaptic delay, and the fact that repetitive stimu1959b), lation of the optic nerve overcame the block (Bishop et d., indicated that the compound was probably interfering with the release of the natural excitatory transmitter or with the attachment of

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the latter to its appropriate subsynaptic receptors. Evarts (1958) examined a series of tryptamine derivatives for their effect on synaptic transmission through the LGN. Tryptamine and dimethyltryptamine had actions similar to LSD but 5-hydroxytryptamine was ineffective. More recently, Bishop et al. (1960) reported the relative potencies of a number of substances that they had found to have a blocking action on geniculate synapses when administered by intracarotid injection. The order of potency was (from high to low) : LSD, bufotenine, 2-bromolysergic acid diethylamide ( BOL148), psilocybine, mescaline, and Ei-hydroxytryptamineat the bottom of the list with a very weak and variable action.

B.

DRUGAPPLICATION The early studies described above suffered the serious disadvantage that drugs administered intravenously or intra-arterially may not reach the neurons because of the presence of diffusional barriers such as the blood-brain barrier, Thus, the order of potency reported by Bishop et al. (1960) probably reflect the ability of the various drugs to penetrate the diffusional barriers as much as their effectiveness with respect to geniculate neurons. This difficulty is avoided by the electrophoretic application of the drugs in the immediate extracellular environment of the neuron (Curtis and Eccles, 1958; Curtis and Watkins, 19-60), This technique, combined with both multineuronal and single-unit recording, has now been applied to geniculate neurons by Curtis and Davis (1961, 1962a, b ) . Their results are summarized in Table I. Curtis and Davis tested the drugs on the extracellularly recorded single-neuron spikes produced not only by orthodromic and antidromic stimulation but also by chemical excitation using electrophoretically applied L-glutamic acid. The glutamate ion had the same excitant-depressant action on geniculate neurons as they had found previously on various spinal neurons (Curtis et al., 1960) and neurons in the brain stem (Curtis and Koizumi, 1961). Without exception, glutamate ions excited every neuron to which they were applied in the LGN. It probably acts by membrane depolarization which is nonspecific and unrelated to the naturally occurring excitatory synaptic transmitter (Curtis et al., 1960). In the LGN Curtis and Davis used the negative-positive type c unit-spikes (Fig. 4, c ) of 0.2-1 mv in amplitude. When the spikes were larger than this, ELECTROPHORErIC

Naturct of action - . -

Depression Depression Depression Depression

PoBnc y" -~. .-

~

24-36

18-24 12 12-18 9-12

1

6-10 7-1 I

Lysergic :wid t1eriv:ttives

Phenylettiylaminc* cterivativw (including noradrenalin) Quaternary ammoniiim compoiinda Choline and esters

Betainc esters Other quaternary ammonium compounds Miscellaneous compounds (including enzyme inhibitors arid drug antagonists)

Ergometrine MethyIergomc.tiiric, All of low pott~llry

Depression Depression Depression

.\wtylrholint~Imnnide (:arbamoylcholine chloride No action -411 of low potency

Excitatioii Exei tati on

Low potency

9-12 6-9 Mostly < L

20-30 Prolonged 1-4

++ ++++

Excitation

Gener:tlly

Depression Excitation

0-1 0-i-

+

~

After Curtis and Davis, l962a,b. * Potency: Depression, relative t o that of 5-hgdroxytryptamine = 12. Excitation, relative to that of acetylcholine = c Duration of depressant activity: relative to that of 5-hydroxytryptamine = 1. 0

+ +.

cd '

P

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and particularly when a spike was initially positive, the cell responses were altered by current flowing from the drug barrels (see Curtis and Koizumi, 1961) . The latter were each about 2 p in diameter. As judged by the action of drugs, the negative-positive spikes apparently belonged to a homogeneous group, confirming our impression that, in the LGN at least, type c responses are always recorded from cell bodies and rarely, if ever from axons. With respect to drug action Curtis and Davis found no differences between the synapses operated by either low- or high-threshold afferent fibers in the optic nerve. They did not attempt intracellular recording. 1. Depressants The local electrophoretic application of certain indoles, particularly 4-,5-, and 7-hydroxytryptamine, and also some lysergic acid derivatives, prevents the geniculate neurons from responding to orthodromic volleys. These substances do not, however, affect the excitability of the cells as tested by antidromic volleys or by chemical activation by L-glutamic acid. Impulse conduction in presynaptic fibers is probably unaffected. The above observations render unlikely at least three possible modes of depressive action: 1. The depression is unlikely to be due to an intracellular action following drug-penetration into the cell. 2. The failure to affect antidromic potentials and chemical excitability suggests that the drugs do not alter the membrane conductance of the geniculate cells. In this respect, the action of these drugs differs from that expected of inhibitory transmitters or that characteristic of depressant amino-acids. 3. The electrically excitable component of the postsynaptic membrane is not stabilized by an interference with spike-generating mechanisms, such as occurs with procaine (Curtis and Phillis, 1960). The above conclusions coupled with the observation that LSD first fractionates the unit-responses of geniculate neurons at the S-A step and subsequently reduces the amplitude of the S potential (Bishop et al., 1962c), directs attention to the presynaptic terminals and the subsynaptic receptors specialized for combination with the excitatory transmitter released from these terminals. One possibility is that the drugs are affecting transmitter release by interfering with the presynaptic membrane sites through which the transmitter is

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discharged. The rapidity with which the drugs af€ected synaptic transmission in the LGN suggests that they do not interfere with the synthesis and storage of excitatory transmitter. The other possibility is that the drugs have a curare-like action, combining with the postsynaptic receptors and so denying access of the natural transmitter to them. If curare-like, such an action would cause no change in the conductance of the postsynaptic membrane. Until the natural transmitter substance becomes available, it will be difficult to decide between these alternatives. The structural requirements for the combination of these substances with receptors at lateral geniculate synapses are of some importance since, apart from cholinergic synapses, this is the only central synapse for which a specific blocking agent is known (Curtis and Davis, 196%). It is possible that the natural transmitter may bear some structural similarity to these active depressants, particularly since the concentrations of substances, such as 4-hydroxytryptamine, which were used, were within what might be considered a physiological range. Neither Shydroxytryptamine nor ergometrine were active when applied to spinal neurons (Curtis and Davis, 1962a). It is probable that the structure of the tryptamine derivatives require the presence of a phenolic hydroxyl group and of a terminal unsubstituted amino group upon the tryptamine side-chain, for interaction with the receptor. The phenylethylamine derivatives were relatively ineffective depressants so that neither adrenalin, noradrenalin, or dopamine are transmitters acting upon lateral geniculate synapses. The active depressants derived from lysergic acid are of importance since they penetrate the blood-brain barrier and are potent when administered systemically. The high potencies of ergometrine and rnethylergometrine suggested to Curtis and Davis the possibility that these compounds interact with the membrane receptors by means of the terminal hydroxyl group of the side-chain and the nitrogen atom at position 6. 2. Excitants

Of all the substances tested by Curtis and Davis (1962a, b ) only the quaternary ammonium derivatives (mainly the choline esters) produced excitation of the geniculate neurons. None of these choline esters were, however, of high potency when compared with

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their actions upon cholinoceptive Renshaw cells (see Curtis and Eccles, 1958). Apart from carbachol, the agents usually failed to activate cells unless a background discharge was provided either by the application of L-glutamic acid or by synaptic action. The excitant action of acetylcholine and the other active agents was revealed by the increased frequency of firing above the background discharge. The potencies, relative to acetylcholine, were assessed by comparing the electrophoretic currents which were necessary to provide approximately equal degrees of potentiation of the neuronal responses. The electrophoretic application of acetylcholine to Renshaw cells, using currents of 10-100 nA produced h i n g at rates of 50-120 spikes per second (Curtis and Eccles, 1958) (1 nA = amp). However, with a similar application, quiescent geniculate neurons were not excited even though currents of 100-150 nA were used. The activity of acetylcholine on geniculate neurons was exceeded only by that of carbamoylcholine. In several instances, application of this choline ester fired quiescent geniculate neurons though the frequency did not exceed 20 spikes per second. The features of the excitant action of acetylcholine, however, make it unlikely that the transmitter released from optic nerve terminals is closely related structurally to this choline ester. Thus, 5-hydroxytryptamine suppressed the synaptic excitation of lateral geniculate neurons by optic nerve volleys but did not affect the excitatory action of acetylcholine. Conversely, dihydro-p-erythroidine did not affect the synaptic responses but prevented the action of the choline ester. Furthermore, there is very little choline acetylase in the optic nerve (see Hebb, 1957). Since the electrophoretic currents used to apply these excitant quaternary ammonium compounds to the geniculate neurons were of the same order as those necessary to excite Renshaw cells, Curtis and Davis (19621,) suggest that the receptor sites for the drugs in the LGN might lie beneath excitatory synaptic terminals of fibers other than those arriving in the optic tracts. The features of its action, however, still make it unlikely that acetylcholine is the transmitter even at these synapses, Nevertheless, the excitatory effect of acetylcholine on geniculate neurons is of considerable interest since neither this substance or the other choline esters have any excitatory activity with respect to spinal interneurons (Curtis and Davis, 1962b). Furthermore, these substance do not potentiate the firing of spinal inter-

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neurons by excitant amino acids. Thus, the action of acetylcholine on the geniculate neurons is not a nonspecific one.

3. Repetitive Firing The effects produced by drugs are somewhat complicated by the tendency of geniculate neurons to fire repetitively in response to threshold optic nerve volleys whereas maximal volleys evoke a single spike (see above). Potentiation of geniculate firing by drug action may, therefore, convert the 2 or 3 spikes that are evoked by a submaximal synaptic stimulus to a single response, Thus, the repetitive firing evoked by a just-threshold optic nerve volley was abolished by acetylcholine though the first spike remained and fired with a decreased latency (Curtis and Davis, 1962b). The effect was reversible. Conversely, when Shydroxytryptamine was applied to neurons excited by a maximal optic nerve volley the effect was identical with those observed when the strength of the optic nerve stimulus was reduced, During the first few seconds of application the nerve volley evoked 2 or 3 spikes in each discharge and this phase of repetitive firing was followed by a complete suppression of the postsynaptic response. With very weak depressants, such as morphine, complete suppression of spike response occasionally was not obtained. The production of this poststimulus repetitive firing could mistakenly lead to the classification of the drug as an excitant. The above observations indicate that the suppression of repetitive firing by maximal optic nerve volleys is due to an increase in synaptic drive to the cell, rather than any special effect of impulses in higher threshold nerve fibers. It was suggested above that the increased synaptic depolarization of the dendrites consequent upon the increased synaptic drive allows the propagation of the B spike to proceed further out along the dendrites than would otherwise be the case. The membrane conductance change associated with the B spike produces a rapid repolarization of the dendrites after the brief action of the transmitter substance. The repetitive firing does not occur under these circumstances because the initial segment of the axon is no longer subjected to a prolonged depolarizing action by the dendrites. X. Concluding Remarks

Axonal conduction has changed very little in the long course of evolutionary history. Much the same seems to be true of the be-

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havior of the cell body also, since the generation of the postsynaptic response has many similarities in molluscs, crustacea, and mammals. It is no surprise therefore that, within the mammalian nervous system, we should find sensory and motor neurons having the same general pattern of behavior. These two neuron types and the synapses associated with them do, however, have some striking differences, though the latter are still perhaps more of degree than of kind. Thus, if we take the sensory neurons in the LGN as typical of sensory neurons in general, they are much more excitable than are motoneurons. The former are spontaneously active and commonly fire repetitively in short high frequency bursts. Sensory synapses have a much greater safety margin for transmission than is the case with motoneuron synapses and seem to require much less afferent summation. Sensory neurons respond more readily to afferent volleys, recover their excitability more promptly and can follow, for short bursts at least, afferent stimulation at frequencies perhaps as high as 1000 per second in a way that is beyond the range of the motoneuron. To what extent these differences can be explained in terms of the morphology of the cells rather than differences in respect to membrane properties is a matter for the future. Undoubtedly, the large size of the motoneuron determines many of its properties making it more sluggish than the smaller sensory neurons but nevertheless fitting it for the role of a “finalcommon-pathway” neuron. It would be an enormous advantage to have available accurate models of representative neuron types, complete with cell processes and associated synaptic endings, as could be reconstructed from serial electronmicroscopic sections. Most of the properties of the motoneuron synapses appear to have their counterpart in the synapses in the lateral geniculate nucleus. One striking exception is the lack of any direct electrical or other guide to the actual mechanism of synaptic inhibition in the LGN. This is certainly surprising since inhibition is such a dominant feature of the visual process. Working with single-unit recording and natural stimulation of the retina, one is continually struck by the observation that inhibition assumes a character as positive in its way as excitation itself. This is true at the geniculate level quite as much as it is at the level of the retina, Lack of direct electrical evidence of an inhibitory process comparable to the inhibitory postsynaptic potential in the motoneuron would perhaps be explained if inhibition in the LGN is largely presynaptic in origin. Recent el-

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tronmicroscope studies of the s,ynaptic endings in the nucleus suggest that this may well prove to be the case. There are clearly wide differences between neurons in the central nervous system in respect to their reactions to pharmacological agents. These differences have been confirmed by the local electrophoretic method of drug application which obviously avoids many of the limitations associated with systemic administration. There are many possibilities to account for these differences: considering only modes of action related to the surface of the cell, there are differing mechanisms for transmitter release, differing transmitter substances, differing synaptic chemoreceptors, differing membrane properties not directly connected with the synaptic process, diffusion barriers associated with the glial cells and still more intimate juxtasynaptic barriers, and many others. It is, however, just this variety of modes and sites of action that holds out such promise for the future in respect to the differential control of parts of the nervous system. There are still many problems to be solved before a fully adequate interpretation of the extracellularly recorded response is available. The most intriguing problem is the nature of the contribution made by the dendrites. Another deficiency is that we still do not have a satisfactory appreciation of the mechanoelectric effects of the presence of the electrodewhat happens when dendrites are tugged or squeezed or when synaptic knobs are pulled off the surface of the neuron. Just as the glial cells with their complex enveloping processes probably play an important role in determining the nature of drug action, so too perhaps are they important in relation to the waveforms extracellularly recorded from single neurons. Perhaps the large type ( b ) responses are recorded when the electrode tip is fixed or jammed between glial cell processes in close proximity to the cell. This may possibly provide mechanically stable recording conditions and also the high recording resistance necessary for the large amplitude of the response. AcxxowmDcm E x T s

The experimental work reported from this laboratory was carried out in collaboration; the author is particularly grateful to his colleagues, Dr. W. Burke and Dr. R. Davis, both for their collaboration and for allowing him to use their unpublished material. I t is a pleasure to thank Miss Johnson for considerable secretarial assistance, particularly in respect to the bibliography. The

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work has been aided by grants from the National Health and Medical Research Council of Australia, the Ophthalmic Research Institute of Australia and the Post-graduate Medical Foundation of the University of Sydney. REFERENCES Ajmone Marsan, C., and Morillo, A. (1961). Electroencephabg. and Clin. Neurophysiol. 13, 553-563. Amassian, V. E. (1953). Electroencephabg. and Clin. Neurqphysiol. 5, 415438. Amassian, V. E., and de Vito, R. V. (1954). J. Neurophysiol. 17, 575-603. Araki, T., and Otani, T. ( 1955). J. Neurophyswl. 18,472-485. Arden, G. B., and Soderberg, U. ( 1961). In “Sensory Communication” (W. A. Rosenblith, ed.), pp. 521-544. Wiley, New York. Barlow, H. B. (1953). J. Physiol. (London) 119, 58-68. Barlow, H. B. ( 1957). J. Physiol. (London) 136,469488. Bennett, M. G. L., Crain, S. M., and Grundfest, H. (1959). 1. Gen. Physiol. 43, 189-219. Bishop, G. H. (1958). Electroencephabg. and Clin. Neurophysiol. Suppl. 10, 12-21. Bishop, G. H., and O’Leary, J. L. (1940). J. Neurophysiol. 3, 308-322. Bishop, G. H., and O’Leary, J. L. (1942). J. Cellular Comp. Physiol. 19, 315331. Bishop, P. 0. (1953). PTOC. Roy. SOC.B141,362-392. Bishop, P. 0. (1960). In “Mechanism of Colour Discrimination” (Y.Galifret, ed.), pp. 129-133. Pergamon, New York. Bishop, P. O., and Collin, R. (1951). J. Physiol. (London) 112, &lop. Bishop, P. O., and Davis, R. (1960a). J. Physiol. (London) 150, 214-238. Bishop, P. O., and Davis, R. ( 1960b). J. Physiol. (London) 154, 514-546. Bishop, P. O., and Evans, W. A. (1956). J. Physiol. (London) 134,538-557. Bishop, P. O., and McLeod, J. G. (1954). J. Neurophysiol. 17, 387414. Bishop, P. O., Jeremy, D., and Lance, J. W. (1953a). J. Physiol. (London) 121,415-432. Bishop, P. O., Jeremy, D., and McLeod, J. G. (1953b). J. Neurophysiol. 16, 437447. Bishop, P. O., Burke, W., and Davis, R. (1958a). Nature 182, 72&730. Bishop, P. O., Burke, W., Davis, R., and Hayhow, W. R. (1958b). Trans. Ophthalmol. SOC. Australia I S , lS35. Bishop, P. O., Field, G., Hennessy, B. L., and Smith, J. R. ( 1 9 5 8 ~ )J.. Neurophysiol. 21,529-549. Bishop, P. O., Burke, W., and Hayhow, W. R. (1959a). Ezptl. N E U T O1, ~ . 534-

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REGENERATION IN THE VERTEBRATE CENTRAL NERVOUS SYSTEM' By Carmine D. Clemente Department of Anatomy, School of Medicine and the Brain Research Institute, University of California, 10s Angeles, California and the Veterans Administration Hospital, Sepulveda, California

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I. Introduction . . . . . . . . . . . . . . 11. Developmental Considerations . . . . . . . . . 111. Theories of Nerve Growth and Orientation . . . . . . IV. Regeneration in the Central Nervous System of Primitive , . . . Vertebrates V. Regeneration in the Central Nervous System of Fishes . . A. Spinal Cord Regeneration in Teleost Fishes . . . . B. Optic Nerve Regeneration in Teleost Fishes . . . . VI. Regeneration in the Amphibian Central Nervous System . . A. Studies on Embryonic Urodeles . . . . . B. Studies on Embryonic Anurans . . . . . C. Central Nervous System Regeneration in Amphibian Larvae D. Regeneration in the Central Nervous System in Adult , . . . . . Amphibians . VII. Regeneration in the Reptilian Central Nervous System . VIII. Regeneration in the Central Nervous System of Birds . . . A. Regeneration in the Embryonic Chick Central Nervous System B. Regeneration in the Central Nervous System of Adult Birds . IX. Regeneration in the Mammalian Central Nervous System . . A. Emergence of the Concept of Abortive Growth . . . . B. Theories Offered to Explain Limited Regeneration in the . . . . . . . . Central Nervous System C . More Recent Studies on Central Nervous System Regeneration . . . . . . . in Mammals . . D. Peripheral Nerve Implantation Studies and Cortical Grafts . E. The Biochemical Search . . . . . . . References . . . . . . . .

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The author's research on this subject has been supported by United States Public Health Service Grant number B987. 257

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I. introduction

That severed neurons in the brain and spinal cord of adult mammals do not possess functionally significant regenerative power, has been firmly established in the literature for decades. During the last 25 years, however, experiments involving different approaches to this same problem have indicated the possibility that a limited growth capacity exists following injury to adult mammalian central neurons. Other studies indicate little or no significant growth of CNS fibers in adult mammals. Certainly it must be admitted that an injury in the human central nervous system which destroys or severs large numbers of nerve fibers is not followed by functional repair to the extent observed following a similar injury in a peripheral nerve. This statement does not hold for all submammalian vertebrates as will be seen further in this review. The interesting phenomena encountered during the restorative events following injury in the central nervous system of lower vertebrates must form a preamble to a consideration of problems in the mammal. Since the proximal end of the severed nerve fiber encounters many of the features in its regrowth which are characteristic of initial fiber outgrowth during embryogenesis, additional factors related to developmental mechanisms should likewise be considered. Thus, a phylogenetic analysis of the problem of regeneration in the vertebrate central nervous system might well be introduced by pointing out the factors considered important in leading or guiding a developing nerve fiber to its destination during ontogenesis. II. Developmental Considerations

Experimentalists in the past have forwarded certain hypotheses in attempts to explain the basic nerve patterns that are established in the developing nervous system. The outgrowth theory of neuron development originally proposed by H. F. Bidder and C. von Kupffer and upheld by W. His, Sr. and S. Ram6n y Cajal was most firmly established by the now classical experiments of R. G. Harrison (1907, 1910) which demonstrated that nerve fibers were formed by neuroplasm anabolized in the cell body and distributed to nerve sprouts. The work which Harrison accomplished, by successfully initiating the method of tissue culture, virtually put to an end the century-old problem of nerve fiber origin and disproved such pro-

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posed ideas as the cell chain theory offered in 1839 by Th. Schwann and the protoplasmic bridge theory of Hensen (18f34, 1868), which, although valuable in their day, were shown to be scientifically inaccurate. That the periphery assumes some role in central nervous morphogenesis has long been recognized. Shorey (1909) showed that following the ablation of peripheral fields there resulted alterations in the corresponding segmental spinal ganglia and that the changes observed were dependent on the amount of skin and musculature destroyed and on the stage of development of the embryos at the time when the larvae were preserved. Such experimental conditions resulted in a reduction in numbers of spinal ganglion cells due to a failure in development, since she was unable to demonstrate degenerating nerve cells. Numerous investigators have agreed that the developing nervous system responds in some manner to alterations in the periphery (Braus, 1906; Hamburger, 1934; Detwiler, 1920, 1924a, 1926, 1927, 1936; Bueker, 1943, 1944, 1945; Hamburger and Keefe, 1944; Piatt, 1946). Detwiler (1919) transplanted limbs of AmbZystomu embryos a short distance either cranial or caudal to their normal positions. When this was done, the respective limbs were supplied by spinal nerves which normally would have innervated them. If, however, he placed the limb several segments away, the innervation was derived from segments of the spinal cord other than those which would normally innervate the limb. Histologically, sections of the spinal cord revealed hyperplasias of sensory areas in those segments from which the transplanted limbs were innervated. He noted by making cell counts that motor areas in the spinal cord were uneffected by limb extirpation and concluded that although the periphery exerted an influence on sensory neuron proliferation, the development of motor neurons remained independent of peripheral factors. Hamburger (1934), Hamburger and Keefe (1944) and Bueker (1943), using the chick embryo did not completely agree with Detwiler concerning the reactions of motor cells after wing bud extirpation or after embryos had been overloaded peripherally by implantations of supernumerary limbs. In either type of experiment these latter investigations revealed either decreased or increased numbers of cells in the lateral motor cell areas, i.e., in those areas which specifically innervated limb musculature. When, however, total cell counts,

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including the nonmotor cells, were made in the ventral horn there was little difference in the numbers of neurons in experimentals and controls. In this point, their results agreed with Detwiler’s, however their reasoning differed. It was felt that although the mitotic activity of embryonic motor neuroblasts was not influenced by changes in the periphery, the processes of motor neuroblastic differentiation were affected, and that the ratio of mature motor neurons and of potential motor neurons was a factor of peripheral regulation similar to the observations made in sensory areas of the cord. Not only has it been postulated that the outlying peripheral areas exert some influence on central neurogenesis, but additional experimental data have been presented by various investigators which either uphold or tend to disprove the thesis that intrinsic factors of an intramedullary nature also influence neurocellular proliferation and differentiation. Thus, Detwiler ( 192313) substituted spinal cord segments 3-4-5 with the normally smaller segments 7-8-9 in Amblystoma embryos. Cell counts in the transplanted segments revealed a hyperplasia comparable to the numbers of cells expected in the normal brachial segments. The same type of experimentation in various cord regions led Detwiler to the conclusion that the more rostra1 spinal-cord levels possess a greater inherent capacity for self development than the more caudal segments. Neural proliferation of the more caudal areas was dependent in large extent, therefore, on the developing longitudinal fiber tracts descending from above. That such a dependence existed in A m b l y s t m was also expressed by Nicholas (1930) and experiments by R. G. Williams (1931) tended to confirm the work in chick embroys. Williams removed segments 19-23 and inserted a mechanical block so that the lumbar cord could not receive any descending tracts. The tail was also severed caudal to the twenty-ninth segment, eliminating the ascending fibers. He found a 40% motor hypoplasia in the chick embryos and concluded that cellular proliferation was considerably regulated by stimuli from other regions of the central nervous system. Other research on this problem, however, revealed that descending tracts exerted little influence on neural proliferation and differentiation. Work by Levi-Montalcini (1945) and Hamburger (1946) which involved either extirpation or isolation of cord segments in the chick embryo (but in any event, removal of incoming fiber tracts) re-

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vealed normal motor cell counts in the spinal areas of the experimental animals. The conclusion that the periphery exerts a much greater influence on differentiation and proliferation than intracentral factors has been found in an extensive series of experiments by Bueker (1943, 1944, 1945). He has shown that after radical lower limb extirpation in the chick embryo, neurocellular hypoplasias resulted of up to 90%.Because of such extensive reductions in cell numbers, he concluded that central conduction pathways played little role in the development of lumbosacral motor elements in the cord. For much more complete reviews of the factors concerned in neurogenesis, the reader is referred to the excellent articles by Piatt (1948) and Straus ( 1946). Ill. Theories of Nerve Growth and Orientation

Through observations on the developing nervous system by the methods of experimental embryology, and through observations on nerve regeneration in the adult nervous system, there have evolved three hypotheses regarding the factors involved in eventual guidance of a growing nerve fiber toward its goal. These have been termed, respectively, the chemical, electrical, and mechanical theories of nerve outgrowth orientation. The chemical theory was advanced by Ram6n y Cajal late in the nineteenth century and states that particular specific chemical substances secreted by localized centers attracts the growing nerve fiber. Other neurologists, Marinesco and Lugaro, at the turn of the twentieth century, shared in this opinion (Ram6n y Cajal, 1928). It was postulated that from degenerating myelin and from Schwann cells in the degenerating nerve stump, there emanated chemical agents capable of attracting nerve sprouts which emerge from the proximal stump. Forssman (1900) referred to this theory as neurotropism and Cajal, who had previously discussed this relationship in the embryonic development of a nerve fiber, interpreted it as being chemotactic in nature. It was even suggested that different types of nerve fibers might be guided selectively by different chemical discharges. The electrical theory proposed that differences in electrical potentials have an orienting effect on nerve fibers. Kappers’ ( 1917,

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1921) elaboration of Strasser's (1892) ideas led to the formulation of the theory of neurobiotaxis as an explanation of how the various brainstem nuclei become arranged ontogenetically, and of their differences in anatomical location phylogenetically. In essence, Kappers states that the growth of the chief dendrite of a neuron proceeds in the direction from which the largest number of stimuli are discharging, and that there is an eventual shifting of the neuron cell bodies toward the direction of stimulus. Ingvar (1920) and more recently Marsh and Beams (1946) observed modifications in the growth of nerve processes in tissue culture by subjecting them to galvanic currents. Much direct and indirect evidence contradicts these results. Weiss (1934) and S. C. Williams (1936) did not find instances in which electrical currents resulted in an orientation of growing nerve fibers in tissue culture. Speidel (1933), working on the tail of living tadpoles, at times found nerve fibers growing in the same pathway but proceeding in opposite directions. The third theory, which to date appears to have very convincing experimental data emphasizes the importance of mechanical factors in the development of nerve patterns. The importance of mechanical influences in directing and determining the pathways of growing nerve processes was advocated by His (1887), Hamson (1910, 1914), and Dustin (1910). This theory stresses the role of solid mechanical structures as being largely responsible for nerve orientation. Unquestionably, the most outstanding work in more recent years in support of this theory has come from the experiments of Paul Weiss and his collaborators. In 1934 Weiss showed that the growth of tissue cultures composed of brain fragments or spinal ganglia of chick embryos remained unoriented when subjected to chemical or electrical stimuli. However, when the medium was stroked gently with a brush in a certain direction, the nerve processes developed in parallel paths in the same direction in which the culture medium had been stroked. According to Weiss, the stroking produced an orientation of the fibrillar or ultrafibrillar particles (micellae), and the growing nerve fibers followed the paths of the oriented medium. This phenomenon has been called contact guidance and supposedly acts indirectly on the nerve fiber through the ground substance. In another experimental procedure in which two spinal ganglia

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cultures grew in the same medium, it was observed that the processes of the explants grew in parallel lines toward each other. In this experiment, Weiss reasoned that the proliferation of the cells caused a dehydration in the surrounding medium, resulting in contraction and the eventual formation of tension forces between the two centers of growth. This tension was reflected in the ultrastructure, and the growing nerve fiber followed the tension lines. In order to disprove the chemical attraction of degenerating nerve material for regenerating in vivo peripheral nerve, Weiss and Taylor (1944) severed the sciatic nerve, extirpated the distal stump, and allowed the proximal stump to regenerate into a forked artery. These experiments were performed in the rat, and a piece of abdominal aorta including its bifurcation into the iliac arteries was used as the regenerating site. The proximal stump of regenerating sciatic nerve was placed in the aorta, and when the regenerating fibers confronted the two iliac channels at the bifurcation, the pathway of choice remained to the individual fibers. Into one iliac vessel was placed a “bait” consisting of degenerating nerve fragments. The other channel was left with no “lure” and only a blood clot filled its path. In all cases the regenerating nerve fibers divided themselves about evenly between the two routes and, the evidence did not indicate that the degenerating tissue inserted into the one iliac vessel attracted the regenerating processes. The assumption that mechanical effects are the only influencing factors cannot be established, for, indeed, even though much evidence has been presented, a good deal is indirect or negative evidence. It is conceivable that if chemical and electrical activity exert an effect on the guidance of growing nerve fibers, such influences may not be acting directly on the nerve process but indirectly through an orientation of the growing medium. Thus, the problem is not closed. Factors influencing regeneration in the central nervous system may in some respects be similar to those in the peripheral nervous system, but there are also many differences. It must first be established that regeneration is possible in the central nervous system. The literature is extremely voluminous and contradictory. An evaluation must also be made as to the type of restoration observed. Certain lower forms have a greater innate plasticity within the cen-

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tral nervous system than is evident in many of the higher animals; thus, what is often called regeneration in lower forms is in reality a differentiation of more embryonic cells into neuron types. IV. Regeneration in the Central Nervous System of Primitive Vertebrates

No information is available to my knowledge on the regenerative capacity of the central nervous system in elasmobranch or ganoid fishes, although two interesting recent studies were reported on cyclostomes. Mar6n (1959), from S. Skowron’s active group in Krakow, found that severance of the spinal cord in larvae of the European river lamprey ( Lumpetra fluoiatilis, 3-18-cm long), resulted in reconnection of the severed stumps of the cord by nerve fibers as early as 10 days after severance. Following removal of 2-3-mm segments of the spinal cord in other specimens, there was re-establishment of the cord stumps by neural tissue after 20 days. Mar6n comments on the abundant mitotic reactivity of ependymal elements characterizing the cord lesion sites and on the fact that Muller’s fibers were also restored. Similar findings were reported by Hibbard (1963) using larvae of Petromyzon marinus. V. Regeneration in the Central Nervous System of Fishes

A. SPISAL CORDREGEKERATION IN TELEOST FISHES Experimentation on the regenerative capacity of the central nervous system in fishes has been limited exclusively to work done on the teleost spinal cord, optic nerve, and retina. Forty years ago Koppanyi and Weiss (1922) transected the vertebrae and spinal cord of an adult fish ( Carussiris vulgaris) and demonstrated a functional return in the paralyzed region 6-8 weeks following section. The coordinated swimming movements were correlated with histological evidence that bundles of nerve fibers could be traced across the original site of transection (see Pearcy and Koppanyi, 1924). These latter authors continued the original work by making behavioral observations in large goldfish ( Carussius auratus, 8-14-inch specimens ) that had sustained spinal lesions indicative that “the spinal cord must have been sectioned.” Functional return was described as swimming movements which were rhythmic and coordinate behind the transection site and which required two-and-a-

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half months for maximum recovery. Although no histological studies were done on these fish, Pearcy and Koppanyi entertained the suggestion by A. J. Carlson that undeveloped nerve cells in the spinal cord above the section had retained their embryonic potentialities and had been stimulated to grow as a result of the transection. Because of the older age of the animals the authors preferred, however, to consider that “morphological regeneration” of the sectioned fibers had actually occurred (see also Koppanyi, 1926). Experiments by Nicholas on the regenerative efforts of transected spinal cord neurons in Fundulus embryos reportedly failed to indicate even a trace of morphological reconstitution ( Nicholas, 1927; Hooker and Nicholas, 1930; Hooker, 1930, 1932). This failure was explained by the high degree of determination in FunduZus tissues at an early period of development, even though Morgan (1900) had shown fin regeneration was able to occur in adult Fundulus specimens. These seemingly contradictory results were the background for the more complete studies of Hooker (1932), Tuge and Hanzawa (1937) and Kirsche ( 1950). Each of these three excellent papers established that anatomical and physiological restitution of the transected teleost spinal cord could occur, and that coordinated function returned after varying periods of time in the different species. Hooker (1932) using the young rainbow fish, Lebistes ~ e ticulatus, found that 4 days after spinal transection there was already an anatomical fusion of the severed stumps by masses of fibers issuing from both cut ends. At the transition site there were few cellular mitoses but a large number of “indifferent neural cells that had wandered out from the cord ends.” Complete physiological and anatomical regeneration of the cord usually occurred in 6 days. These studies were repeated in Hooker’s laboratory on adult rainbow fish by Keil (1940) who described functional return 2 weeks after complete spinal cord transection. Tuge and Hanzawa (1937) reported that a somewhat longer period was necessary for both morphological and physiological regeneration to occur in the transected spinal cord of adult Japanese rice minnows (Oryzias latipes). Anatomically, it was found that after 2-3 weeks a connective tissue scar had formed, filling the gap between the cord stumps. By 4 4 weeks there was a gradual infiltration and bridging of scar tissue by increasing numbers of regenerating fiber bundles. Functional recovery was first noted by the

re-establishment of complicated and coordinated movements in the caudal fin. There was then an abolition of spinal reflexes, and finally a restoration of muscle tone and the return of normal behavior. The attainment of maximum recovery was observed 3 months after the operation. Kirsche (1950) described experiments carried out on 150 adult teleost specimens ( Lebistes reticulatus) , The spinal cord was completely transected and physiological and morphological evidence of regeneration was obtained. The first stage of regeneration (4 days after the operation) was characterized by the growth of the severed spinal cord fibers in an aimless fashion. There were cones of growth at the tips of the growing fibers. These were considered truly regenerating elements and not cells restored by mitosis, which was thought to occur later. The second phase ( 7 days after the operation) was characterized by mitotic reproduction of ependymal cells which developed into neuroblasts and glioblasts. This was observed in both stumps of the cord simultaneously; the newly developed neurons sent parallel bundles of fibers across the transection site by the twelfth day, although at this time functional connections had not yet been established. Kirsche electrically stimulated above the lesion and used caudal fin movements below the lesion induced by the stimulation as evidence of established functional connections. By the fifteenth day such functional connections were observed.

B.

OPTIC

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ZnT TELEOST FISHES

From the fascinating studies of Sperry (1948), functional optic nerve regeneration following its severance was shown to result in 5 different species from 3 families of marine teleost fish and in 2 fresh-water species ( Sperry, 1955)- Microscopic examination revealed copious regeneration through the site of severance in the optic nerve, which led to the re-establishment of anatomical connections between the retina and the brain. Further, he described other experiments in which optic nerve severance was combined with 180" rotation of the eye. Functional return in these fish indicated reversed vision, and 18 days after the operation, the fish exhibited optokinetic responses in the direction opposite from normal. Thus, not only was restoration of vision accomplished in these specimens, but the re-establishment of functional connections of the regenerated optic nerve fibers was orderly and systematic and seemed

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destined along some predetermined scheme. Good visual recovery in still another series of marine teleosts (Bathygobius soporator) was also reported by Sperry (1949, 1955). Furthermore, Sperry (1955) reported success in tests of color perception following regeneration of visual neurons in fishes that had been trained in a color discrimination task prior to optic nerve section. Other evidence that optic nerve fibers bypass many neurons in order to make functionally appropriate connections has come from this same laboratory ( Attardi and Sperry, 1963; Sperry, 1963). Some of the earliest experiments on the regenerative capacity of teleost optic nerves were carried out by Przibram ( 1923), Koppanyi (1923a, b, c ) , and Kolmer (1923), and these were discussed more recently by Koppanyi ( 1955). Although these early experiments leave something to be desired from the standpoint of control observations and detailed histological analysis, nevertheless, they indicated long ago the possibility that regeneration of teleost optic nerve could occur. Blatt ( 1924), impressed with Koppanyi’s “sensational communications,” reported on 340 eye transplantation experiments and 60 eye re-implantation experiments done on 3 species of fish, the carp (Cyprinus carpio), Barbus fluviatilk, and Scardinius eythrophthulmus. From the 400 fish, there were 19 cases of anatomical healing of transplanted eyes and 7 cases of healing of reimplanted eyes. In no case did Blatt feel that he had observed functional restitution and he stated that all of the transplanted eyes in his fish were blind, On the other hand, F. Ask (1926), F. Ask and Anderson (1927) and Anderson and 0. Ask (1933) described unquestionable evidence of a copious and forceful regenerative capacity of optic nerve fibers following reimplantation experiments in goldfish and in a European fresh-water cyprinoid tench (Tinca uulgaris).

Meanwhile Matthews (1933) in Philadelphia offered a very plausible answer to the contradictory results of the various authors cited above. Using Fundulus heteroclitus (4-6 cm in length) he showed that when the optic nerve was cut in such a way that the blood supply to the eyeball was left intact, no degeneration of the retina was observed. This was followed by an extensive “neuromalike” growth of optic nerve fibers that had grown from the stumps of the severed nerve. On the other hand, when the optic nerve and the blood vessels to the eyeball were both cut, the pars optica

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retinae degenerated completely, obviating the possibility of regrowth of the original ganglion cell processes. The pars caeca of the retina in the ischemic eyes, however, did not degenerate (results similar to those of Fugita, 1913, in Triton) and within this rudiment, mitotic figures appeared and proliferated into variable amounts of new retinal tissue. Rasquin (1949) also stressed that vascularization must take place prior to degeneration of the retina and lens. She also considered it necessary for the severed stumps to be very close €or regeneration of severed optic nerves to occur in the characin, Astyanux mexicanus. Thus, she reported good functional recovery of visual feeding reactions in 5 of 12 fish with severed optic nerves. Less successful, however, were those with transplanted eyes in which the vascularization was maximally interrupted. VI. Regeneration in the Amphibian Central Nervous System

Taxonomically the amphibia are subdivided into three orders: ( a ) the Apoda ( Gymnophiona) comprised of small headed, virtually tailless members which are also limbless and almost eyeless and which are found in tropical parts of the Old and New World; ( b )the Urodela (Caudata), whose species have long tails which are retained throughout life and long bodies with short weak limbs (represented by the salamanders and newts); and ( c ) the Anura (Salientia), which are distinguished by the complete absence of a tail in the adult stage and which possess long strong hind limbs (the frogs, toads, and tree toads). Since no information could be found on the regenerative capacity of central neurons in any specie of Apoda, this discussion will be limited to findings in urodeles and anurans during their embryonic, larval, and sexually mature adult periods. A. Smm ox EMBRYONIC URODELE~ Information is plentiful on the central nervous system’s restoration potential following lesions in the brain and spinal cord of embryonic salamanders and newts. During the early phases of development there is quick and orderly return of function following spinal transecting lesions. Resumption of normal swimming movements occurred as early as the eighth postoperative day when the spinal cords of developmental stage-40 A m b l y s t m punctatum were com-

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pletely cut. Severance at the earlier stage 32 (the time of first neuromuscular reflex response to light touch) did not result in an alteration of the normal developing behavioral pattern, and the spinal-sectioned embryos began swimming at the same time as unoperated specimens (Piatt, 1955). For information on the restorative character of the embryonic urodele central nervous system at even earlier stages, one can refer to the studies of Lewis (1910), Hooker (1922, 1930), Wieman (1922, 1925a), and Detwiler (1923a, b, 1924a, 1925, 1929). Although the primary objective of these experiments for the most part dealt with the further understanding of the underlying mechanisms involved in amphibian neurogenesis, the techniques utilized and the results obtained pointed clearly to the conclusion that complete restitution can be expected following CNS lesions in embryonic Amblystoma from stage 21 (completely closed neural folds) to the stage 32 animal used by Piatt (1955). Not only could certain segments of developing spinal cord from a donor embryo be successfully transplanted into different regions of the host neuraxis, but developing donor brainstem could also be successfully transplanted to host spinal levels. Additionally, even heteroplastic transplantation of spinal segments from Amblystm punctatum to corresponding regions in the cord of Amblystoma tigrinum and vice versa were shown to be successful at these early stages ( Wieman, 1925b, 1926; Detwiler, 1931). A more recent group of papers by Detwiler (1944a,b, 1945, 1946a, b, 1947), Harrison ( 1947), Piatt (1949, 1951), Holzer (1951, 1952), Hollinshead (1952), Sibbing (1953), and others have indicated to some extent the limits and the qualitative features of this restorative process in embryonic urodeles. Thus, maximal recovery could be expected when unilateral portions of the medulla, mesencephalon, or spinal cord had been removed, whereas less complete restitution occurred following bilateral removal of the same regions. With respect to higher centers, Detwiler (1945) stated that “when the right half of the forebrain (including the optic and olfactory rudiments) is removed from embryos in stage 21, there is no regeneration.” These findings essentially confirm those of Burr (1916) that the cerebral hemispheres of Amblystoma will not regenerate in the absence of the developing nasal placode. Thus, factors other than those inherent within the developing urodele brain are also of

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significance in the restoration process observed following CNS lesions. The exact nature and the extent of these influences are still to be determined. B. STUDIESox EMBRYONIC ANURANS Similar to the process described in embryonic urodeles, a very favorable degree of restoration is observed in the central nervous system of anurans following the production of lesions during the embryonic period. From experiments as early as those of Harrison (1898) on three species of American frogs, it can be said that successful restoration of the spinal cord occurs following complete amputation of the tail bud in anuran embryos of 4 mm. Harrison, utilizing the methods of G. Born (1896, 1897), essentially confirmed and extended earlier studies of Vulpian (1859) and showed that growth of the spinal cord from one species of frog would take place into a grafted tail bud of another species. During the early part of this century, Bell (1906, 1907), utilizing 2.5-4-mm embryos of RUM fusca showed that an entire half of the brain of young frog embryos regenerates readily if the remaining half is intact. Removing the brain entirely, however, in 33-mm specimens did not result in restitution (Schaper, 1898; Rubin, 1903a, b ) . Bell ( 1907) argued that these somewhat varying findings might be explained on the basis that the central nervous system in these embryos is better capable of regenerating laterally from intact cerebral masses but its regrowth craniocaudally is more limited. Lewis (1910) removed the anterior end of the neural plate in frog embryos and noted that even after “large pieces” were removed, regeneration was practically complete and that all of the cranial nerves were present. Spirit0 (1929, 1930) called attention to the fact that increased mitotic activity over relatively long periods was an important feature of the restoration process. More recently Terry (1956) has studied the regenerative capacity of the midbrain in R a m pipiens embryos. He found that reorganization and restitution occurred in all cases of partial optic lobe excision. Animals of a slightly older developmental stage regenerated incompletely. Ferguson ( 1957) unilaterally excised the medulla in frog embryos ( R u m pipiens and R a m cutesbianu) at the neural-fold stage and earlier, and obtained complete morphological restitution of the missing half. Migration from the intact side

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and increased mitotic activity on the defective side were characteristics of the restitution. Somewhat similar results were obtained by Stevens (1959) in the brachial spinal cord. From the foregoing studies and those of Hooker (1915, 1916, 1917) and Spemann (1912), it can be concluded that the central nervous system of the embryonic anuran is at least equal to that of the embryonic urodele in its restorative capacity. Following simple severance, healing occurs per primam. If the stumps of the severed cord are intentionally not placed in direct apposition, fibers grow from each end of the cord and from the epithelioid cells of the central canal in order to establish anatomical continuity of the severed stumps. Furthermore, the elements which enter into this restorative process are derived from the cord itself and not from surrounding connective tissue. The development of behavior in such operated embryonic anurans appears generally to keep pace with that of normal animals. C. CENTRAL NERVOUS SYSTEMREGENERATION IN AMPHIBIAN LARVAE Successful regeneration following lesions in the central nervous system of urodele larvae has been described by many authors. Piatt (1955) observed both morphological and functional recovery following spinal cord transection in Amblystoma punctatum larvae of 35 and 4550 millimeters. In the younger larvae, nerve fibers had already bridged the transection site on the fifth postoperative day, and normal swimming movements occurred by the twentieth day. The older larvae (45-50mm) required a somewhat longer interval for the return of coordinated hind-limb function and of normal swimming movements but, “structurally,” Piatt states that “no essential difference in the regenerative capacity of the spinal cord was observed between the two larval series.” The source of the regenerating fibers at these stages appeared to be from already differentiated neurons, since mitotic activity was not observed at the transection sites. Interestingly, the Mauthner fiber did not regenerate in these larvae, whereas Baffoni (1952) and Stefanelli (1952) did describe Mauthner fiber regeneration in larval newts following tail amputation. On the other hand, Stefanelli (1951, 1952) did not find this fiber capable of regeneration in the caudal spinal cord of adult newts. Regeneration in the larval urodele central nervous system was

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also studied by Koppanyi and Weiss (1922) and by Weissfeiler (1924, 1925). The former reported functional recovery in larval newts 5-7 weeks following spinal cord transection while the latter quite thoroughly described the regeneration of olfactory lobes and portions of the cerebral hemispheres in the Axolot2. Actually, as early as 1864 research studies have described the regenerative capacity of urodele larval spinal cords. Muller (1864-1865) noted regeneration of the spinal column and spinal cord in triton larvae (Triton taeniatzcs and T . cristatus) and in 1885 Fraisse described regeneration in the lower spinal cord of various larval urodeles (both salamanders and newts) following tail amputation. Even more reports are available on the regenerative capacity of larval anurans. Lorente de N6 (1921) described an extensive regeneration capacity of spinal cord fibers in frog larvae (Rana, 2035 mm) and, furthermore, claimed that regenerated dorsal root fibers were capable of penetrating the spinal cord and of growing in both directions within its substance. A few years later Hooker (1925) utilized tadpoles of R a m syluatica and Rana catesbianu ranging in length from 6.75 mm (tail-bud stage) to 25 mm (hindlimb stage; in process of metamorphosis ) and carried out a complete transection of the spinal cord in the cervical region. Animals in the hind-limb series demonstrated the return of completely normal behavior 20 days to 3 months after the operation and neural transmission through the severed segment of the cord was “always accompanied by at least a fairly complete restitution of the form of the cord.” Hooker describes the outgrowth of neuraxes from both ends of the cord, the establishment of a central canal, and the proliferation and migration of indifferent cells in the original cord stumps. Recent studies have been reported from a group of Polish investigators utilizing tadpoles of the African tongueless frog, X m q u s laeois. Jordan (195!5, 1958) and Srebro (1957) reported a remarkable regeneration of the extirpated telencephalon of tadpoles and metamorphosed forms. Srebro (1957) states that “after four days the cut end of the brain is covered with a layer of ependymal cells.” Large numbers of mitoses can be observed as long as 20 days after the operation, and by this time the telencephalon is almost completely redeveloped. Jordan (1958) felt that 8 weeks was required to accomplish the same process. If, however, the olfactory organs are also removed bilaterally, regeneration of the telencephalon is

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abortive (Kosciuszko, 1958). The studies from this group have reported a lack of regeneration in diencephalic areas ( Srebro, 1959). “After total removal of the between brain regeneration of this organ is insignificant,” Srebro states, “and the regenerating optic nerves unite with the midbrain.” Kwaitkowski (1959) studied the regenerative capacity following transections in various areas of the neuraxis in Xenopzcs tadpoles and concluded that distinct connections could be found as early as the fourteenth day after section in the more rostra1 parts of the brain, whereas it required a longer interval (up to 6 weeks) for this to occur in the lower brainstem. Regeneration of the caudal spinal cord and other tail tissues following operations on and amputations of the tail in larvae of the true toad (Hyla urbmea) has been described ( Stefanelli, 1950b; Stefanelli et al, 1950a; Stefanelli et al., 1950b; Themes, 1950a, b; Santa, 1951). In the studies of Stefanelli, incipient hind-limb budstage specimens were subjected to removal of 24-mm segments of the caudal spinal cord. As early as 8 hours after the operation, undifferentiated ependymal elements created ampullae at both stumps and by the fourth day, these outgrowths came into anatomical continuity. D. REGENERATIONIN AMPHIBIANS

THE

CENTRAL NERVOUS SYSTEMOF ADULT

1. Urodeles Although many experiments have been reported on the regeneration capacity of the adult urodele tail and caudal spinal cord, only a few studies have dealt with other regions in the adult urodele neuraxis. Piatt (1955) using the Japanese water newt (Triturus Pywhogastm) carefully and completely severed the spinal cord in the middle trunk region. These animals lost their ability to swim and there was no functional use of the hind limbs 24 hours after the operation. In 8-10 days, first placement movements on land were noted in the hind limbs and, thereafter, walking improved rapidly. Coordinated fore-limb and hind-limb motion was noted 70 days postoperatively. Between 90 and 120 days, rhythmic swimming coordination was achieved and “the behavior of the oldest animals (175 days) was normal in all respects.” Piatt retransected above the level of original severance in one regenerated cord and

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noted an immediate resulting paralysis, stressing that true functional recovery had, indeed, occurred. There was no obvious evidence of cellular proliferation or increased mitotic activity. Successful regeneration had, therefore, occurred from the severed fibers and not by neocellular replacement. Another very complete study on 6-14-cm specimens of a different species of urodele ( Arnblystma m a a n u r n ) was published by Kirsche (1956). This investigator severed the spinal cord in the lower trunk region and, in addition to studying his animals histologically, he used electrical stimulation techniques. Kirsche reported that 14 days postoperatively he obtained the first unequivocal evidence of restored function. Out of 112 animals, 85 gave a clear picture of restored motor function below the site of severance, between 12 and 95 days after the operation. Anatomically, nerve fibers were observed to have regenerated across the transection site 2 weeks after the operation. The findings of Piatt and Kirsche, although thorough and important, were not surprising in the light of many previous studies on the tail and caudal spinal cord of adult urodeles. Stefanelli and Cervi (1946) extirpated 5-6-mm segments of the spinal cord near the base of the tail in adult tritons and noted that after 2 months the newly formed spinal cord segment appeared normal. Earlier Stefanelli (1944) and Stefanelli and Capriata ( 1944) had amputated the tails in the adult newts, Triton cristatus and Triton taeniatus. Three processes were thought successful in accounting for the attainment of a regenerated caudal cord: first a process of degeneration, then a process of cellular migration of ependymal elements from the remaining cord and finally a process of proliferation and multiplication of the cellular elements. Many investigators, before the turn of the twentieth century, were interested in the process of tissue regeneration. The urodele tail served well as an experimental model and, although many observations were made on the consequent regenerative capacity of the spinal cord in these studies, the primary objectives were often broader and extended to tissues other than the nervous system. Amongst the earlier workers that described regenerated urodele tail spinal cord were Fraisse (1885), Colucci (1884), Barfurth (1888, 1891), Caporaso ( 1889), Sgobbo ( 1890), Goldfarb ( 1909), S’imoes-

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Raposo (1922, 1923, 1925), Duesberg (1922, 1924a, 1924b) and McCreight ( 1924, 1931). Others ( SchottB, 1926; Locatelli, 1924, 1925, 1929) were interested in the influential role of the nervous system within the general question of tail regeneration. 2. Anwuns Studies on regeneration in the postmetamorphic anuran central nervous system have been limited in number and those that have been published deal primarily with spinal cord or optic nerve. Masius and Vanlair (1869, 1870a, b) reported partial regeneration of the severed spinal cord in adult frogs following the excision of 2-mm segments. Physiological regeneration resulting in a return to “normal function as well as anatomical restitution of neurons which successfully developed neural processes” were reported by these authors. Certain subsequent studies were unable to confirm these results (Sgobbo, 18%; Marinesco, 1894) setting the background for Hooker’s (1925) finding that there was a gradual reduction in the regenerative capacity of the frog spinal cord as the animal approached metamorphosis. More recent studies (Piatt and Piatt, 1958; Jordan, 1958; Roguski, 1959) have shown clearly that although significant intraspinal regeneration can occur in the early postmetamorphic and adult frog (see Figs. 8 and 13in Piatt and Piatt, 1958),it is more the exception than the rule. In this respect perhaps adult X m w p u s Zumi.~ responded somewhat better than Runa pi@em. Why does significant regeneration occur only in a few adult frogs? What factors endow the CNS fibers of some animals with a higher capacity for growth while in others of the same species and age there results a virtually complete failure? Are these factors genetic, biochemically metabolic, or environmental? What combination of factors leads to success? No one really knows the answers to these questions although extensive regeneration of the optic nerve usually does occur in adult urodeles (Matthey, 1927; Stone and Chance, 1941) and anurans (Sperry, 1944), and functional return to a remarkable degree of performance following optic nerve severance has been observed. Since an excellent review stressing the ontogenetic implications of the visual system problem has been published in this journal recently (Gaze, 1960), the author will not comment further on this subject.

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VII. Regeneration in the Reptilian Central Nervous System

The works published by Stefanelli and his collaborators have stressed the events coincident with regeneration of the tail in lizards following the periodic tail amputation that occurs naturally in these animals. These studies followed their observations on amphibia and have been limited to animals from two suborders of the order Sauria, the Geckones, and the Lacertae. Marrotta (1946) and Stefanelli (1951) in Lacerta mudis and Zannone ( 1953) in the gecko, Tarantola muuritunica, concluded that regeneration of the caudal spinal cord following amputation commences by an invasion of the blood clot with connective tissue elements, and an ameboid migration of ependymal elements which effectively obstructs the lumen of the central canal. This is followed by a proliferation of the ependymal cells and a regrowth of nerve fibers from above to form a regenerated caudal spinal cord. This grows into the center of the cartilaginous spinal column, which forms the skeleton of the lizard's new tail. Since both the motor and sensory innervation of the regenerated tail is derived from ganglia rostra1 to the site of amputation ( Temi, 1920; Stefanelli, 1950, 1951; Zannone, 1953), the spinal regenerate begins to involute, probably because of a lack of peripheral connections. The once regenerated caudal spinal cord regresses to a thin-walled filamentous ependymal tube by the third month after tail amputation. Marrotta (1946) concluded that regeneration of the caudal spinal cord in Lacertae occurs and that the mechanism of ependymal proliferation, differentiation and regrowth are similar to that seen in the amphibian urodele. To this author's knowledge a systematic study of the regenerative capacity of the adult reptilian spinal cord following transection at various levels and utilizing acceptable physiological and neurohistological techniques does not exist. A short description of spinal transection experiments again in the tail of Lacertn niuralis by Themes ( 1950), however, provides reason to believe that care must be taken in such experiments to insure an adequate blood supply in both spinal stumps. If this is done, there is reason to predict that successful regeneration might occur in the adult reptilian spinal cord (Rossi, 1910a, b). Effective experiments in these species, however, await investigation. It has been recognized since the studies of Gegenbaiir ( 1862), MiiIIer (1863, 1864-

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1865) and Fraisse (1885) that the spinal cord in the lizard tail is in continuity with the more rostra1 spinal cord, but it is composed simply of ependymal epithelium with some nerve fibers and neuroglia (Hughes and New, 1959). It seems that higher spinal transections or cerebral lesioning experiments would reveal more conclusive information on the capacity of adult reptilian central neural elements to regenerate. VIII. Regeneration in the Central Nervous System of Birds

A. REGENERATIONIN

THE

EMBRYONIC CHICKCENTRAL NERVOUS

SYSTEM

Although a number of studies have been carried out on the restorative capacity of the central nervous system during the early developmental stages in the chick, very little work has been done in older embryos ( Hamburger, 1955). During the early developmental stages, experimental evidence warrants the statement that some degree of regeneration occurs. Waddington and Cohen (1936) removed one lateral half of the forebrain in embryos of 5-25 somites and showed that the organ could remodel itself into a complete forebrain. The repaired forebrain even induced the formation of a nasal placode from the overlying nonpresumptive nasal epidermis, but the formation of the optic evagination did not occur if the optic vesicle had been entirely removed. These findings were confhned and extended by Spratt (1940) who also pointed out that the restorative powers diminished in older specimens. Less success was obtained in operations involving complete removal of the forebrain, following which there was no replacement from the midbrain, but simply a nonneural healing anteriorly. Regeneration within the chick brain during early embryonic development, thus, mimics the situation observed in other areas of the body. If a complete presumptive region is removed, there is no regenerative replacement, but if a portion is left, it may be capable of replacing the entire region (Weiss, 1939; Waddington, 1952). Bearing this premise in mind, it is not surprising that when extensive bilateral ablations in developing chick spinal cord or brainstem have been performed, regeneration has been rarely described. On the other hand, when Kallen (1955) extirpated one or two hindbrain neuromeres on one side in embryos and allowed them to survive to

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an age of 4.5-7.5 days, almost half of the cases showed a regenerating cell mass developing in a normal way. The regeneration was correlated with an increase in mitotic activity on the intact side, but bilateral extirpation was wt followed by regeneration. The positive results obtained following hemiablation in the forebrain (Waddington and Cohen, 1936) and in the spinal cord (Watterson and Fowler, 1953) were confirmed by Kallen (1955), and were contrary to the contention of Wenger (1950) who claimed that lateral halves of the chick neural tube were incapable of regulation or regeneration. Other pertinent studies on the regenerative capacity of the developing chick central nervous system include the earlier findings of Hoadley (1925) who transplanted the midbrain of 48-hour chick embryos to the choriallantoic membrane. Growing central tract fibers emerged from the transplant and large fascicles innervated the host tissue in the vicinity of the implantation. R. G. Williams (1931) found that the 56-hour chick embryo spinal cord “showed remarkable powers of reconstitution.” In attempts to produce isolated areas in the cord, he resorted to the use of mechanical blockades such as packed egg shelling instead of merely transecting the spinal cord. Rhines (1943) and Rhines and Windle (1944), producing lesions in the midbrain and hindbrain of chick embryos of 30-40 hours incubation, also reported a case in which regeneration had occurred in a descending bundle of the medial longitudinal fasciculus and the individual fibers mingled freely with the elements in the posterior stump. In over half of their experimental specimens central nervous system regeneration was evidenced by emergence of nerve fibers from the brain into the surrounding mesenchyme. In an extensive series of experiments, Clearwaters (1946, 1954) produced transections in spinal cord areas of embryonic and newborn chicks. Her operations were performed behind the wing-bud region at the levels of the twenty-first and twenty-third somite and in some cases one or two segments of the cord were removed. Embryos operated on after 48 hours incubation showed essentially a complete repair through the lesion site 6 days after transection. When operations were performed on incubated embryos of 72 hours, longitudinal spinal cord sections taken on the fifth day revealed bundles of nerve fibers crossing the gap to join the two stumps of the cord. An animal operated on after 96 hours of incubation and

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sacrificed after hatching showed “no impairment of voluntary function of its legs and appeared to be perfectly normal in its reactions.” Histological examination of the cord revealed a scar in the dorsal cord region and many regenerating fibers in the ventral areas. Interestingly enough, the dorsal scar area was composed of neuroglial tissue, and very few fibers succeeded in penetrating this portion of the scar, Other animals operated on after hatching and sacrificed up to 5 weeks later revealed that the ends of the cord were completely rounded off by neuroglial tissue. Regenerating nerve fibers turned laterally or medially when this barrier was reached and the physical resistance offered to regeneration by the neuroglial proliferation was evident, When certain fibers did succeed in breaking through this barrier, they could be traced into the inter-stump region which consisted of a wedge-shaped connective tissue scar continuous with the dura. Thus, it was shown in these prenatal birds that a forceful regeneration process commenced, and that the majority of the regenerative vigor was spent through the blocking property of the neuroglial tissue. Glial proliferation to injury appeared only in animals studied after the sixth day of incubation and embryos studied in later stages showed a marked decrease in success of spinal cord fibers bridging the site of transection even though the forceful innate regenerative or reparative properties of the neurons were still present.

B. REGENERATION IN THE CENTRAL NERVOUS SYSTEMOF ADULTBIRDS Among his many studies dealing with the central nervous system, Brown-Sequard (1848, 1850, 1851) also studied the regenerative capabilities of the adult pigeon spinal cord. He reported that if the cord was severed “immediately behind the wings” the animals appeared to recover and would show voluntary movements between the third and sixth postoperative month. He claimed that by the fifteenth month the gradual restoration of function resulted in a motor and sensory return that was almost normal. Voit (1868) ablated a portion of the cerebral hemispheres in a pigeon and after 5 months, he found within the area of ablation a cystic mass whose walls consisted entirely of nerve cells and fibers. His observation might be questioned since the cystic mass described may have become larger than the original lesion site and may have invaded areas of undamaged tissue. Grunert (l899), after hearing of Voit’s often

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quoted findings, made lesions in the cerebral hemispheres of 14 pigeons and was unable to detect any regeneration of central elements in his animals. He described neuroglial scars which did not contain nerve fibers or neuron cell bodies. A few years earlier Sgobbo (1890) was unable to confirm the older findings of BrownSequard and found neither anatomical nor physiological evidence of regeneration in the adult pigeon spinal cord. A description of the results of Sgobbo also appeared two years later in Archives de Physiologic (Gley, 1892). To this Brown-Sequard ( 1892) responded that the reasons for the success of his experiments were the meticulous postoperative care that he rendered his animals and the fact that he was patient enough to await the long period of time that functional return required. Furthermore, Brown-Sequard claimed that 4 anatomists had assisted him and each had agreed that regeneration must have occurred in the adult pigeons. It should be remembered, however, that very limited histological procedures were in vogue in the midnineteenth century, when Brown-Sequard carried out his experiments, methods not precise enough by today's standards to be considered critical. During the twentieth century, very few significant studies have been carried out on the regenerative capacity in the central nervous system of adult birds, although Foster (1911) in Ram6n y Cajal's laboratory described degenerative and regenerative events following spinal cord lesions in newly hatched chicks and pigeons. Since her animals were allowed only to survive for about a week following the production of lesions, her conclusions were more relevant to degenerative events than regenerative phenomena. A more thorough study was reported by Cattaneo (19.23) on optic nerve regeneration in birds and rabbits. He reported experiments on 18 chickens, 4 pigeons, and 1 falcon, and in some animals he not only severed the optic nerve but also inserted between the cut stumps excised pieces of the trigeminal nerve or the peripheral end of a severed branch of the trigeminal to act as a guiding path for the newly regenerated fibers. It is interesting that the greatest success was achieved in animals in which a peripheral nerve graft had been employed. Regenerated optic nerve fibers were described emerging from both the retinal and central stumps of the severed optic nerve in chickens, 23,35, and 40 days after operation. Instances of complete traversing of the lesion site by regenerating optic fibers were observed with

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the Cajal silver-nitrate impregnation methods. Cattaneo appeared to be influenced toward a neurotropism interpretation of his successful experiments. A detailed analysis of spinal cord regeneration in adult birds still remains to be done. IX. Regeneration in the Mammalian Central Nervous System

A. EMERGENCE OF THE CONCEPT OF ABORTIVE GROWTH

Regeneration in the central nervous system of mammals has interested some of the greatest experimental neurologists that have lived. Brown-Sequard, Bielschowsky, R a m h y Cajal, Tello, Marinesco, and Ranson, to name only a few, each contributed to some extent to an understanding of the basic problems involved. An extensive literature in the latter half of the nineteenth century developed around this fashionable research problem. Papers written during this period usually contained some of the most dramatic claims of success or the most dogmatic denials and negations. Many of these works, however, must be viewed critically because of the inadequacy of the histological procedures used. One of the earliest and more frequently quoted articles is the one of Dentan (1873). He performed lumbar cord severances in young dogs, but of four operated animals, two died after 2 days, one died after 3 days, and still another died after 7 days. Despite such poor postoperative success, Dentan reported complete active motility in the seven-day animal. Histologically he observed “normal” nerve fibers between the two stumps in the anterior cord, while in the region of the posterior cord, he described a connective tissue and glial scar containing no nerve fibers. His histological description coupled with his reported functional restoration by the seventh day after transection casts doubt on the operative procedure and the completeness of bansection. During the last quarter of the nineteenth century many German, Italian, and French investigators studied the results of spinal cord lesions in mammals. The works of only a few will be reviewed. Eichhorst and Naunyn (1874) crushed the spinal cord of rabbits and dogs. The gap was ‘infiltrated with neuroglial tissue after three to five weeks.” They found nerve fibers in the scar tissue but denied spinal cord regeneration believing that the fibers originated from the

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spinal ganglia. It has since been shown that the method of intradural spinal crushing is not satisfactory to insure a complete cord transection; additionally, it results in excessive traumatic degeneration. Schiefferdecker (1876) reported no signs of regeneration in the cord of spinal dogs up to 300 days following transection. He explained early functional restoration such as that described by Brown-Secjuard in the pigeon on the basis of spinal reflexes below the transection level. While Schiefferdecker examined traumatic and secondary degeneration most profoundly, the regenerative events were treated quite superficially. Kahler (1884) also refuted regeneration in the spinal cord of the dog and reasoned that the central nervous system contained no cells of Schwann. Taking a different view a decade later, Stroebe (1894) described fine nerve fibers within the scar tissue between the stumps of the transected spinal cord in rabbits. A few fibers appeared to bifurcate and cross the scar tissue to gain access to the opposite dorsal columns. Using the anilin blue staining method, Stroebe thought that transected spinal cord fibers commenced to regenerate. However, a true restitution of spinal cord tissue does not occur (“es aber zu einer eigentlichen Regeneration von Riickenmarksgewebe nicht kommt.”). Stroebe’s studies received important confirmation 12 years later by one of the most outstanding neurological scientists of the day, Max Bielschowsky. Working in Berlin under the direction of Oskar I’ogt, Bielschowsky (190s) described newly grown fine nerve fibers in the peripheral areas of brain tumors. He noted that these fibers had terminal boutons and other forms of terminal arborizations and, therefore, concluded that true regeneration of central fibers had occurred. In 1909 Bielschowsky extended his brain tumor studies to include similar observations on tumors of the spinal cord which apparently also attracted regenerated intraspinal fibers. At about this same time, Fickler (1901, 1905), Nageotte (1906), Marinesco and Minea (1906a,b) and the Spanish school headed by Cajal established once and for all the concept that regeneration to some degree was capable of occurring following lesions in the mammalian central nervous system. Rambn y Cajal (1906a, b ) transected the spinal cord of young cats and dogs and using his finer histological methods reported that large numbers of the severed intraspinal fibers sprouted new proc-

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esses with cones of growth similar to those observed in his studies with peripheral nerves. After 4 or 5 weeks, however, the numbers of regenerated fibers diminished, and he concluded that the processes of regeneration were followed by atrophy and absorption. In other experiments, he also described regeneration of fibers in the white matter of the cerebral cortex in newborn dogs. The work on degeneration and regeneration by Cajal and his associates in Madrid culminated in the publication of a most extensive and scholarly experimental treatise, which in many respects still stands as Cajal’s greatest work ( R a m h y Cajal, 1928). The experiments of Stroebe, Fickler and Cajal and the astute observations of Bielschowsky, thus, were especially responsible for the thesis that the regenerative efforts in the central nervous system of adult mammals resulted in abortive growth. In their opinions central nerve fibers commenced to regenerate, but for some reason, the newly formed sprouts would not continue across the transection site and make functional connections in the opposite stump.

B. THEORESOFFEREDTO EXPLAIN LIMITED REGENERATION IN THE CENTRAL NERVOUS SYSTEM One can ask: If regeneration of central nerve fibers commences, what factors are responsible for aborting the growth? In the first place, it must be established that replacement of lost neurons in the adult mammal does not occur to any significance as a result of a differentiation of new cells from existing, less differentiated elements. Nor does any neuronal division occur to any significant degree. This latter phenomenon is so rare that if it is ever considered to be encountered, investigators feel obliged immediately to report the findings (Altman, 1962). In mammals, it is generally stated that mitotic division of neurons ceases either during prenatal development or shortly after birth. Addison (1911) reported that mitosis did not occur in the rat cerebellum after the twenty-first postnatal day, and using tritiated thymidine, Sidman and Miale (1959) and Miale and Sidman (1961) found thymidine incorporation only through the tenth postnatal day in mouse cerebellum. Essentially the same facts have been reported for the spinal cord and for other areas in the central nervous system of mammals (Buchholtz, 1890; Sclavunos, 1899; Allen, 1912). It becomes evident that without the availability of new neurons, provided by differentiation and mitosis

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in lower forms, the central nervous system of adult mammals must depend on the ability of the severed fibers to sprout and to develop new processes, In other words, regeneration in the mammalian central nervous system means the regrowth of severed neuron processes rather than a restoration or restitution of new neuron cell bodies. This lack of cells, potentially capable of differentiation into new neurons, certainly places the mammalian brain and spinal cord to a distinct disadvantage with respect to functional repair in comparison to lower forms. Other investigators have compared central nervous system regeneration events with phenomena that occur in the peripheral nervous system, and have reasoned that in the brain and the spinal cord there are no Schwann cells and, hence, little or no regeneration. Yet the exact metabolic role of the Schwann cell in the peripheral nerve function is poorly understood and little evidence exists to support the idea that peripheral nerve fibers fail to regenerate in the absence of the Schwann cells. It must be pointed out that the neurilemma tubes and Schwann elements in the peripheral nervous system do afford the regrowing fibers with a means of parallel alignment and physical guidance according to the theories of Weiss ( 1934, 1936). In addition, regenerating peripheral nerve fibers guided through neurilemma tubes regenerate about 3 times faster than those fibers oriented away from these distal elements (S. C. Williams, 1930). Nevertheless, peripheral nerve fibers oriented away from neurilemma tubes still regenerate. The outgrowth of embryonic, maturing, and adult central neurons in tissue culture and the reports of so many investigators on limited regeneration of central fibers in vivo strongly indicate that the Schwann cell is not indispensable for some fiber growth to occur. Others have hypothesized that functional regeneration within the central nervous system does not occur because of damage inflicted on the neural tissue through disturbances in the vascular pattern following injury. Adequate vascularity is unquestionably an important feature in regeneration of tissue anywhere in the body. Hunter and Royle (1924) discussed this question following the production of lesions in the central nervous system of adult animals, pointing out that vascular disturbances following spinal cord transection may bring about an “isolation dystrophy” leading to chromatolysis in the ischemic zone. Hooker and Nicholas (1930) also

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felt that vascular disturbances potentially determined the postoperative results in their animals. They ascribed to this factor the primary reason for their failure to observe forceful regeneration of spinal cord fibers in rat fetuses. There is no question but that neural tissue is delicately sensitive to interference with its nutrition and homeostatic conditions, and it is common knowledge that neurons cease to function normally within a few seconds following alterations in circulation. An interesting thought in the light of present day electron microscopy was the hypothesis commented on by Clark (1943) that the density and texture of the tissues of the central nervous system are unsuitable for the growth of nerve fibers. It has been shown that brain tissue is much more compact than was once considered. Membrane physiologists postulated the existence of extracellular tissue spaces comparable to that seen in most other organs. Such spaces, however, do not exist (Schultz et al., 1957; Maynard et al., 1957) and the neuroglia with their cytoplasmic processes fill all of the interstices of the neuropil. The intracellular aqueous cytoplasm of certain neuroglia may act as the extraneuronal fluid of the brain. Perhaps the delicate tip of a regenerating fiber cannot travel far before it meets a glial membrane that offers resistance to further advance. This resistance may even become exaggerated if reactive neuroglia begin to divide to form glial scars following the production of lesions in the CNS. Might not abortive growth be primarily the result of the physical resistance of membranes rather than an incapability of the CNS fiber to grow? C. MORERECENTSTUDIESON CENTRALNERVOUS SYSTEM REGENERATION IN MAMMALS During the last three or four decades, a number of investigators, employing methods different from those of Cajal and his contemporaries, have broadened our views of the regenerative capacities in the CNS. Most of these experiments have been carried out in rodents and carnivores. The studies of Gerard and Koppanyi (1926), in which the spinal cord of rat fetuses in utero, neonatal rats, and young adult rats was transected, indicated the possibility of functional return, although histological proof of spinal cord regeneration was lacking. Hooker and Nicholas (1927, 1930) and NichoIas and Hooker (1928) using an electrocautery as well as a scapel to tran-

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sect the spinal cords of rat fetuses concluded that there was no evidence of functional regeneration even though “some fibers may begin to regenerate only to atrophy before becoming functional.” Shortly thereafter, Gerard and Grinker (1931) conceded that there was little crucial evidence of spinal cord regeneration in young rats and attributed the return of function in the hind limbs to the initiation of spinal reflexes below the level of transection. A further contribution by Gerard, nine years later (Sugar and Gerard, 1940), stimulated interest in the subject once again. These investigators demonstrated successful regeneration of intrinsic nerve fibers in transected spinal cords of rats, most of which had been operated on at an early age. They observed the return of spontaneous hind limb movements and then succeeded in obtaining contractions of the leg muscles upon electrical stimulation of the cerebral peduncles, Correlated with the physiological studies were histological preparations which demonstrated regenerated nerve fibers through the area of transection. Much of the functional return was noted during the second postoperative month. Bundles of regrown fibers bridged the lesion between spinal cord tracts of both cut stumps. In some animals, these authors placed muscle and nerve implants in the gaps at the site of transection, and they reported the greatest success when the implants were oriented in a longitudinal direction. Although some have questioned Sugar and Gerard’s positive observations of regeneration in the rat spinal cord (Bernard and Carpenter, 1950; Feigen et al., 1951) much confirmation for this earlier work exists in the thousands of animals studied by L. W. Freeman and his group over the past fifteen years (Freeman et al., 1949; Freeman, 1949, 1952a, b, 1954, 1955). In young rats, kittens and puppies it was concluded that functional regeneration could occur and that some of these processes could be influenced by meticulous postoperative care, drug administration ( Gokay and Freeman, 1952; Stokes and Freeman, 1951) or ionizing radiations (Turbes ef al., 1960). At about the same time that Freeman and his collaborators were studying regenerative features in the rat spinal cord, Windle and Chambers ( 1950a, b, 1951 ) described regeneration of nerve fibers in the transected spinal cord of adult cats and dogs which had been used in experiments designed to determine the central site of action of bacterial pyrogenic induced fever. The spinal cords of 4 animals

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living 20 to 59 days after the operation revealed new growth of intraspinal neurons into and across the cut. Confirmation and neurophysiological extension of these earlier studies by Windle and his collaborators soon followed (Clemente et al., 1951a; Windle et al., 1952b, 1953; Clemente and Windle, 1954; Scott and Clemente, 1951, 1952,1955). These were summarized in a monograph edited by Dr. Windle in 1955. It was shown that regenerated intraspinal fibers in adult cats were capable of anatomical regeneration, of traversing the site of transection, and of conducting impulses into the opposite stump for distances of up to 30 mm. Ascending tracts have also been shown to react similarly (Liu and Scott, 1958). In these studies significance was attached to the fact that pyrogen-treated animals revealed scars at the transection site which were more vascular and of a looser connective tissue matrix than in untreated animals. It was reasoned that these conditions presented to the regenerating fibers an environment more favorable for growth. In no animal, however, could it be established that transynaptic regeneration had resulted in useful functional regeneration. An interesting observation in these studies was the fact that pyrogen-treated animals showed a variable but consistent diminution in glial scarring at the lesion site which appeared to be beneficial to the growth potential of intraspinal nerve fibers. Recent advances in neurosurgical procedures have also been reported to be of benefit to nerve regeneration, both peripherally and in the spinal cord. Campbell and Bassett with their associates have described the use of a porous membrane-like filter sheet called Millipore, already well known to tissue culturists, to assist in the alignment of severed stumps of peripheral nerves (Campbell et al., 1956; Campbell and Bassett, 1957; Noback et al., 1958; Campbell et al., 1961). The linear arrangement of regenerated peripheral fibers and their success in the bridging of interstump gaps led to the use of these techniques in transected spinal cord studies in adult cats (Campbell et al., 1957a, b; Bassett et d.,1959). Encasing the spinal stumps with Millipore tended to orient regenerating spinal fibers longitudinally, and pial and glial cells migrated cephalad and caudad along the inner surface of the Millipore, thus, eliminating a dense scar between the cord stumps. Electrical stimulation of spinal cord fibers in these animals was capable of evoking conducted potentials through and beyond the site of transection (Thulin,

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1960a, b). In many respects the results reported by Thulin were similar to those of Scott and Clemente (1955), but once again the return of function below the lesion was never established. Experiments by Lance (1954) did not reveal even an attempt at regeneration of pyramidal fibers severed at the medullary level, despite the almost universal finding of at least abortive regeneration of central neurons by others. Lance noted no differences in scars of pyrogen-treated animals and controls with the exception of the 32day animals of his series. He suggested either a variance in regenerative capacity of different central neurons of a deterioriation of the pyrogenic substances which had been used in his experiments. Arteta (1956), using Pyrogens 5 and 3895 (Merck) demonstrated modifications in central scar formation with these substances. The cicatrix in the spinal cord lesions of his pyrogen-treated cats was made up of a matrix rich in reticular tissue and macrophages and “better vascularized than the controls.” The scars in the spinal cords of his treated animals resembled those of pyrogen-treated animals in the series of Windle and Chambers (195Oa) and Clemente and Windle ( 1954). Arteta { 1956) felt that regeneration of central nerve fibers was impossible because of the lack of an adequately arranged guiding system and not because central nerves were incapable of growth. The lack of neurilemma and of an “adequately arranged guiding system” in the spinal cord and brain certainly does not help a central regeneration process, but it cannot explain why neuroma formation is not observed in a severed spinal cord. It has been the author’s experience in the past that intrinsic spinal fibers do regenerate (Clemente and Windle, 1954), but even in the most successful animals the rate of regeneration was much slower than in peripheral nerve. Scott and Clemente (1955) were able to record impulses along regenerated spinal fibers only as far as 3 cm below a site of transection, even though animals were treated and allowed to live for as long as 17 months following complete spinal transection. Perhaps no sites existed on the postsynaptic neuron membrane at the time of need by the presynaptic regenerating fibers. A new means of producing controlled lesions in the central nervous system was recently described by Malis et al. (1957). By the use of monoenergetic, heavy, ionizing particles, such as those

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emitted from the 60-inch cyclotrons at Brookhaven and Berkeley, laminar lesions of the cerebral cortex were produced. Sharply defined narrow cortical zones could be completely destroyed with a minimum of injury above and below the lesions (Malis et al., 1960). Several weeks following radiation injury, abundant sprouting from adjacent cortical fibers was observed (Rose et al., 1960). Prolific regrowth of severed apical dendrites and other cortical fibers often created an “artificial zonal lamina” when higher radiation doses caused necrosis to occur in the more superficial cortical layers. These authors present the alternative hypothesis that their results may represent an expression of a normal continuous growth of all neurofibrils that may be characteristic of these cells rather than a mere regenerative response to injury. D. PERIPHERAL NERVEIMPLANTATION STUDIESAND CORTICAL GRAFTS

Cajal interpreted that success in peripheral nerve regeneration resulted from the existence of some neurohumor, probably emanating from the cells of Schwann, which positively influenced the outgrowing fibers in a neurotropic sense. Although he did not express that abortive regeneration in the central nervous system was due to a lack of Schwann cells, he suggested to Tello that grafts of peripheral nerves placed in the brain may attract regenerating central fibers. Tello (1911a, b) predegenerated pieces of sciatic nerve, implanted them into the cerebrum, and noted extensive growth of “central” fibers into the graft after 2 weeks. These signs of regeneration later vanished, however, as the implant was resorbed. Ortin and Arcuate (1913) and Cattaneo ( 1923) investigated the regenerative possibilities of optic nerve fibers under conditions somewhat similar to Tello. Clark ( 1942) used homographs and inserted predegenerated peripheral nerve stumps into the brains of adult rabbits, but concluded that the fine fibers which were observed reinnervating the grafts came from meningeal nerve branches or perivascular nerves. He was unable to convince himself that intrinsic central nervous system fibers had grown into the graft. In other animals (1943) he severed peripheral nerves and placed the central end (regenerating stump) into the brain. Although in many animals the regenerating peripheral nerve fibers remained within the confines of the implant,

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he did observe growth of peripheral fibers into brain tissue when an inflammatory process had occurred around the implant. Also. peripheral fibers grew well when the stumps were inserted into the ventricular system. Success in this experiment has been achieved, however, when the temporal or mandibular branch of the facial nerve was inserted into the brain (Clemente et al., 1951b; Windle ct nl., 1952a; Clemente, 1952, 1955, 1958). By using drugs which decreased the amount of scarring around the implanted stumps, regenerated peripheral nerve fibers were observed to blend with central fibers and cells. It was felt that these experiments confirmed the hypothesis that the central nervous system is capable of maintaining regenerating fibers within its substance. There were indications that primitive mesodermal elements and reticular cells were somewhat beneficial to regenerating nerve fibers, whereas, neuroglial proliferation presented impenetrable scars to regrowing fibers. This view is also shared by Noback et al. (1958, 1959, 1962). Turbes and Freeman (1958) reported on peripheral nerve/spinal cord anastomoses made 1-2 months following complete spinal cord transection in adult dogs. Intercostal nerve trunks were dissected rostra1 to the lesion site and then severed. The proximal nerve stump was then implanted into the spinal cord caudal to the transection site. They claimed that 2 weeks after insertion of the nerve trunk. most of the animals attempted to stand and walk. In 5 dogs this progress was reversed by subsequent surgical sectioning of the inserted nerve. These same authors claim to have evidence of the reestablishment of functional transynaptic connections with motor neurons in the ventral cord (Turbes and Freeman, 1961; Jacoby et al., 1960). The fate of implanted nonneural grafts and of the implantation of spinal ganglia into the central nervous system has recently been reviewed by Glees (1955). It had been noted by Erikson and Glees ( 1953) that grafts of skin implanted into the cerebral cortex of rabbits at times contained regenerated cortical nerve fibers that had grown into the graft from the surrounding brain tissue. On the other hand, muscle grafts appeared to degenerate after a while and become replaced by connective tissue. If muscle grafts survive, however, large numbers of intracortical nerve fibers are observed to penetrate the scar surrounding the graft, perhaps in an effort to reinnervate it (Nathaniel and Clemente, 1959). These findings should

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be viewed in the light of the recent experiments of Rose et al. (1960) in which a prolific growth of cortical fibers was described. E. THE BIOCHEMICAL SEARCH

That biochemical substances exist which are capable of inducing or attracting the growth of nerve fibers has been a question that has interested experimental neurologists since the days of Cajal and Forssman. It was felt by these investigators that degenerating nerve tissues possessed these qualities, although the experiments of Weiss and his collaborators (especially those of Weiss and Taylor, 1944) cast serious doubt on the neurotropic nature of degenerating nerve. Weiss’ evidence, however, should not be interpreted as meaning that other biological systems may not possess nerve growth promoting humors. As a matter of fact, recent evidence points to just the reverse. It was found that when certain mouse sarcomas were transplanted to the body wall (Bueker, 1948) or into the allantoic membrane of the 3-4-day chick embryo (Levi-Montalcini, 1952; LeviMontalcini and Hamburger, 1953) the sensory and sympathetic systems of the host were radically affected. These appeared to be stimulated into excessive growth both with respect to increases in cell numbers and increases in neuronal size. Large numbers of nerve fibers were induced to grow, far in excess of those seen in control animals. These authors then reported that the sarcoma growth-substance was diffusable and effective in vitro. Cultures of chick spinal and sympathetic ganglia, when placed close to sarcoma explants, showed excessive numbers of nerve fibers radiating in all directions from the nerve cell culture, whereas in controls only normal growth was observed (Levi-Montalcini et al., 1954). It has since been found that even more potent nerve growth promoting properties exist in the poisonous venom of the moccasin snake, Agkistrodun piscivms ( Cohen and Levi-Montalcini, 1956; Levi-Montalcini and Cohen, 1956) and in the salivary glands of the mouse and rat (LeviMontalcini, 1958; Cohen, 1958). The growth substance is a protein and has been purified, and it appears to act directly on the nerve cell. These authors have determined that the presence of glucose or mannose is required for continued nerve growth and of the necessity of at least one amino acid, phenylalanine. Scott (1963) has indicated

some success in the use of this substance in the transected spinal cord of kittens, Exactly how the growth factor stimulates the nerve fiber, however, is not known as yet. The fascinating observations of Levi-Montalcini and her c01laborators on tumor and salivary gland extracts is a progressive step which might be considered to have started from the observation of Bielschowsky (1906) over 50 years ago. He observed from neuropathological material that nerve fibers grew into the edges of brain and spinal cord tumors. Duncan and Bellegie (1948) made similar observations after they had transplanted pieces of rat sarcoma into the pia-glial membrane and at the severed end of the spinal cord in rats. Inferred from studies on collateral peripheral nerve regeneration, nerve fibers appear to have a dynamic association with their peripheral end organs such that the fibers may compensatorily respond into new growth, following destruction of neighboring axons (Edds, 1953). The evidence indicates that the sprouting is due to the action of a humoral agent released by adjacent degenerating nerve fibers or from the cells of Schwann. Thus, reinnervation of partially denervated muscle and restoration of function by collateral regeneration in autonomic ganglia have been described (Murray and Thompson, 1957a, b ) . Anatomical and physiological evidence has also been put forward that collateral sprouting occurs in the spinal cord below the level of a partial section, presumably as a response to the injury (Liu and Chambers, 1958; McCouch et d., 1958; Teasdale et d.,1958). Attempts to modify this experiment by severing the dorsal roots in kittens and studying changes in the monosynaptic reflex in the operated segments after the animals had become adult, however, did not reveal evidence of functional colIateraI sprouts (Eccles d d.,1962). This latter evidence does not mean, however, that collateral sprouting had not occurred earlier. Yet another agent has been considered effective in stimulating nerve regeneration. A factor isolated from the white matter of the brain by von Muralt and his associates (Koechlin and von Muralt, 1945, 1947; Jent, 1945; Jent d al., 1945; Koechlin, 1955) was shown capable of increasing the rate of regeneration of corneal nerve fibers. Konig (1953) and Martini and Pattay ( 1954) felt that malononitrile and succinonitrile were also able to increase the speed of peripheral nerve regrowth and they explained it on the basis that malononitrile

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tends to increase the rate of neuronal nucleic acid production (Hydhn and Hartelius, 1948). Thus, the search continues for substances which are capable of stimulating nerve growth or of inhibiting scar formation. An understanding, however, of neuronal metabolism at the cellular level is basic to the problem. Windle (1962) points out that the answer may lie in the comparative anatomy and neurochemistry of lower forms where central regeneration comes about as an innate matter of fact with none of us realizing the biochemical stimulants involved in neural differentiation. REFERENCES Addison, W. (1911). 1. Comp. Neurol. 21, 459. Allen, E. (1912). 3. Comp. Neurol. 22, 547. Altman, J. (1962). Science 135,1127. Andersson, E., and Ask, 0. (1933). Acta Ophthalmol. 11,411. Arteta, J. L. (1956). 3. Comp. Neurol. llos, 171. Ask, F. ( 1926). Acta Ophthulmol.3, 12. Ask, F., and Andersson, E. (1927). Acta Ophthulmol.4, 97. Attardi, D. G., and Speny, R. W. (1963). Exptl. Neurol. 7, 46. Baffoni, G. M. (1952). Rend. accad. nuzl. Lincei sci. j%. e mat. e nut. 8, 189. Barfurth, D. ( 1888). Anat. Anz. 3,403. Barfurth, D. (1891). Arch. mikroskop. Anat. u. Entwicklungmech. 37, 406. Barnard, J. W., and Carpenter, W. (1950). 3. Neurophysiol. 13,223. Bassett, C. A. L., Campbell, J. B., and Husby, J. (1959). Exptl. Neurol. 4, 386. Bell, E. T. (1906). Arch. mikroskop. Anut. u. Entwicklungsmech.68, 279. Bell, E. T. (1907). Arch. Entwicklungsmech.Organ. 23,457. Bielschowsky, M. (1906). J. Psychol. u. Neurol. 7, 101. Bielschowsky, M. (1909). 1. Psychol. u. Neurol. 14, 131. Blatt, N. (1924). Arch. Ophthulmol.Graefe’s 114, 47. Born, G. (1896). Arch. Entwicklungsmech.Organ. 4,349. Born, G. (1897). Arch. Entwicklungsmech.Organ. 4 517. Braus, H. (1906). Morphol. Jahrb.35, 509. Brown-Sequard, C. E. (1849). Compt. rend. SOC. biol. 1, 17. Brown-Sequard, C. E. (1850). Compt. rend. SOC. biol. 2,3. Brown-Sequard, C. E. ( 1851 ). Compt. rend. SOC. biol. $77. Brown-Sequard, C. E. (1892). Arch. physiol. norm. pathol. Ser. V , 4, 410. Buchholz, Dr. (1890). Neurol. Zentr. 9, 140. Bueker, E. D. (1943). 3. Erptl. Zool.93,99. Bueker, E. D. (1944). Science 100, 169. Bueker, E. D. (1945). 1. Comp. Neurol. 82,335. Bueker, E. D. (1948). Anat. Record 102,369. Burr, H. S. (1916). 3. Comp. Neurol. 26,203. Campbell, J. B., and Bassett, C. A. L. (1957). Surg. Forum 7,570.

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NEUROBIOLOGY OF PHENCYCLIDINE (SERNYL). A DRUG WITH A N UNUSUAL SPECTRUM OF PHARMACOLOGICAL ACTIVITY' By Edward

F . Domino

Department of Pharmacology. University of Michigan. Ann Arbor. Michigan

I . Introduction . . . . . . . . . . . . . . I1. Chemical Structure of Phencyclidine and a Related Derivative 111. Neuro-PsychopharmacologicalActions . . . . . . . A Gross Behavior of Man . . . . . . . . . . B. Gross Behavior of Animals . . . . . . . . . C . Conditioned Reflex Behavior in Animals . . . . . D . Sensory and Motor Reflexes . . . . . . . . . E . Alterations of the Electroencephalogram . . . . . F . Alterations of Evoked Potentials . . . . . . . IV . Cardiovascular and Respiratory Actions . . . . . . A . Effects in Man and Various Animals . . . . . . B. Effects on Dog Blood Pressure and Peripheral Resistance . V. Metabolic Actions . . . . . . . . . . . . A . Effects on Body Temperature . . . . . . . . B. Effects on Oxygen Consumption in Vivo . . . . . C. Effects on Oxygen Consumption in Vitro . . . . . D. Metabolic Fate . . . . . . . . . . . . VI . Discussion . . . . . . . . . . . . . . VII . Summary . . . . . . . . . . . . . . . References . . . . . . . . . . . . . .

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I . introduction

Frequently the development of a new drug provides the pharmacologist and clinician with a number of surprises. The compound 'Supported in part by grant MY-2653-C4 from the United States Public Health Service.

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phencyclidine (Sernyl) is no exception. The drug shows mixed stimulant and depressant properties in animals (Chen et al., 1959). Differences in its actions in various species are marked. As one ascends the phylogenetic scale it seems that the agent is effective in smaller dosage. Although phencyclidine and its analog, cyclohexamine, have desirable properties as anesthetic medications in man (Greifenstein et al., 1958; Lear et al., 1959), the high incidence of stormy behavioral side-effects upon recovery from anesthesia reduces their potential use in anesthesiology. Phencyclidine is known to be a remarkable psychotomimetic in man when given in small doses intravenously (Luby et al., 1959). As an experimental model for drug-induced psychosis, phencyclidine may have certain advantages over LSD-25 and mescaline. As described by Luby et al. (1959) and Rosenbaum et al. (1959), the intravenous administration of phencyclidine mimics more of the primary psychopathology of the schizophrenias as compared to LSD-25 and mescaline which tend to mimic more of the secondary symptoms. It is the purpose of this report to review some of the available literature describing the unusual actions of phencyclidine and to provide additional data from the author’s laboratory much of which has not been published previously. Although ideally it would be invaluable to be able to compare systematically the effects of phencyclidine, LSD-25, and mescaline on selected variables which are thought to be related to their psychotomimetic actions, this is not yet possibIe. The emphasis in this report, therefore, will be on the pharmacology of phencyclidine. When analogous data on LSD25 or mescaline is available in the literature or otherwise, it will be referred to in order to provide a tentative basis of comparison. It is hoped that such a presentation, even though it suffers from a lack of integration at times, will serve as a stimulus to further research with this perplexing drug. II. Chemical Structure of Phencyclidine and a Related Derivative

Phencyclidine is 1-( l-phencyclohexyl ) piperidine hydrochloride. It is also known as Sernyl or CI-395. It is a white, stable solid with a melting point of 234236°C. It is readily soluble in water. The structural formulas of phencyclidine and a related derivative, cyclohexamine (CI-4001, are shown in structures ( I ) and (11).

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Ill. Neuro-PsychophurmacologicalActions

A. GROSSBEHAVIOR OF MAN

The effects of phencyclidine, like any other drug, are dose dependent. The agent has been given orally, intramuscularly, and intravenously to both normal human volunteers and a large variety of patients. Phencyclidine as well as its congeners including cyclohexamine and several more active compounds have been undergoing clinical trials for several years. Already well over a thousand or more humans have received phencyclidine without developing any permanent ill effects. The drug so far appears to be relatively safe. An extensive clinical literature exists on the therapeutic uses of these compounds involving: ( b )pre- and postoperative analgesia; ( b ) control of intractable pain; ( c ) surgical anesthesia; and ( d ) the treatment of mental disorders. Much of the psychiatric literature deals with the use of phencyclidine as an experimental tool in producing a model psychosis. Inasmuch as the latter area is of particular interest to the writer, that literature will be described in detail. During the early human trials with phencyclidine note-worthy psychiatric side-effects were observed. Most human volunteers who took phencyclidine orally in doses up to 7.5 mg per day described the effect as drunkenness. Some reported blurring of vision, visual hallucinations, and delusions. With larger doses, a loss of balance and mental confusion were observed. A hangover effect was also described by some. Generally, oral doses above 10 mg per day caused an impairment of mental function. Schizophrenics seemed to

be quite sensitive and showed a profound disorganization. This impression of increased sensitivity to phencyclidine appears quite the reverse of the general impression that schizophrenics are more resistant than normals to LSD-2-51 Luby et al. (1959) described the effects of phencyclidine given in subanesthetic doses (0.1 mg/kg) as an intravenous infusion in 5% glucose over a 12-minute period to 9 normal subjects and 9 psychiatric patients. Neurologically all subjects showed rotatory nystagmus, ataxia, and altered gait. Diminution of pain, touch, and position sense were uniformly observed, as was a mild diminution in auditory and visual acuity. An impressive alteration in body image, feelings of estrangement or isolation, negativism, hostility, apathy, drowsiness, inebriation, hypnogenic state, and repetitive motor behavior were observed. The drug uniformly intensified the primary symptoms of a small number of schizophrenics. Meyer et al. (1959) emphasized that the disturbance in sensory input produced by phencyclidine resembled sensory deprivation. Luby gt al. (1959) also hypothesized that the impairment of sensory input accounted €or the drug-induced psychiatric state. Rosenbaum et al. (1959) compared the effects of phencyclidine (0.1 mg/kg, i.v.), LSD-25 (1 pg/kg, orally) and amobarbital and amphetamine (500 and 15 mg, i.v. ) on reaction times, rotary pursuit, and weight discrimination in schizophrenic and nonschizophrenic subjects. Reaction times were slowed in the untreated schizophrenics and phencyclidine drugged subjects in the nonshock state. The nonshock reaction times, in contrast, after LSD-25 and amobarbital, were significantly faster. With shock motivation, the reaction times of both the schizophrenics and the phencyclidine-treated subjects were appreciably shortened. In contrast LSD-25 caused no significant change from the predrug level in either the shock or nonshock reaction time. Amobarbital produced a significant slowing of the reaction time under both the shock and nonshock conditions. The disturbances in the nonshock reaction time did not, however, approach the schizophrenic level. On the rotary pursuit test, the phencyclidine-treated subjects showed a significant drop in performance which closely approximated the rather flat motor curve of the schizophrenics. Amobarbital produced no significant change in motor function, while LSD-25 improved it. Similarly, on weight discrimination, the phencyclidine-treated group resembled the poor performance of the

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schizophrenics, in contrast to LSD-25 and amobarbital. These authors concluded that phencyclidine was the only drug which altered performance in the same direction as schizophrenia, They suggested that the primary deficits of schizophrenics (attention and motor function) and subjects treated with phencyclidine resulted from disturbances in proprioceptive feedback. Gershon and Olariu ( 1960) compared the effects of phencyclidine (0.25 mg/kg), LSD25 (5 ,ug/kg), mescaline ( 5 pg/kg) , and Ditran (0.2 mg/kg) given orally to various psychiatric patients. These investigators did not observe many of the psychological effects of phencyclidine reported by Luby et al. (1959) who gave the drug intravenously. Gershon and Olariu also were less impressed with the psychological effects of LSD-25 and mescaline in these patients. In striking contrast Ditran produced marked psychiatric symptoms in both schizophrenic and nonschizophrenic subjects. Interestingly, these authors reported that sodium succinate (12 gm, i.v.) appeared to antagonize the stuporous response to phencyclidine without affecting the nystagmus. Levy et aE. (1960) compared the effects of phencyclidine and cyclohexamine in doses of 0.05-0.2 mg/kg intravenously, intramuscularly, and orally in various types of psychiatric patients. Intravenous injections produced the most striking effects within a few minutes, while intramuscular injections were less marked, Oral administration appeared the least effective. This observation probably accounts for the less impressive effects of orally administered phencyclidine described by Gershon and Olariu ( 1960). Phencyclidine produced more psychophysiological disturbance than cyclohexamine. In 4 schizophrenics Levy et al. (1960) showed that phencyclidine produced an increase in schizophrenic symptoms including a disturbance of body image, depersonalization, and exaggeration of thought blockade. In contrast, cyclohexamine appeared to alleviate some of these symptoms. Chlorpromazine (50 mg, i.m.) seemed to antagonize the psychotomimetic effects of phencyclidine. The observations of Lear et al. (1959) suggest that cyclohexamine also produces psychic disturbances qualitatively similar to phencyclidine. In a study of its use as an intravenous anesthetic, postanesthetic emergence delirium was commonly observed. Johnstone et al. (1959) also studied the effects of phencyclidine as an intravenous anesthetic. The usefulness of the drug was limited be-

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cause of the acute toxic psychosis during recovery. These investigators noted that the psychotic reaction was encountered most frequently in young or middle-aged males. The most violent forms followed intravenous doses of 20 mg total of phencyclidine (approximately 0.25 mg/kg) , The milder degrees of agitation produced by phencyclidine resembled the effects of ethyl alcohol. Davies and Beech (1960) also determined the effects of 0.0750.1 mg/kg phencyclidine given intravenously to 12 normal volunteers. Many of the psychological features described by Luby et uZ. (1959) were observed. These included changes in body image, estrangement, disorganization of thought, negativism and hostility, drowsiness and apathy, hypnogenic state, and a feeling of inebriation. Vomiting was occasionally noted. Various psychological tests were also used to determine more objectively the effects of phencyclidine. In a task designed to measure size judgements, there was a tendency with or without phencyclidine to overestimate size. After the drug, three out of four subjects showed marked tendencies to increase the area covered in handwriting. Tapping speed was significantly reduced. The duration of after-effect on the Archimedes SpiraI was also reduced by phencyclidine as has been reported for barbiturates by others, The drug lowered the rate of light flicker to produce the percept of fusion in the same direction as produced by barbiturates. Normal subjects tended to overestimate time intervals, but phencyclidine-treated subjects tended to underestimate time intervals. The drug also tended to impair learning and recall of paired words, as well as the ability to define proverbs. Davies and Beech (1960) felt that although phencyclidine did have effects on certain tests similar to the barbiturates, it also produced alterations which were similar to the schizophrenic process. They did not feel, however, that the drug could be called a schizophrenomimetic agent. Ban et al. { 1961) are of a similar view. These investigators compared the effects of 0.01-0.1 mg/kg of phencyclidine given intravenously and various other drugs on 55 patients in a mental hospital. Both objective psychological and subjective data were obtained. These investigators felt that phencyclidine did produce specific symptoms of schizophrenia, while LSD-25 and mescaline produced the expected drug- or personality-specific symptoms. Therefore, they are in essential agreement with Luby et nl. (1959) that phencycli-

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dine intensifies the primary symptoms of schizophrenia. The drug was also shown to prolong simple reaction time. Other psychological testing procedures were not altered significantly. In 3 alcohol-dependent patients, a decreased reaction time occurred with increased tapping speed. In these patients the critical flicker-fusion frequency and after-image disappearance level were diminished in agreement with the other studies quoted above. Bakker and Amini (1961) studied the effects of 5-12 mg of phencyclidine given intravenously to 7 normal subjects. Both subjective and objective tests were used for determining drug effect. In general, phencyclidine slowed or depressed performance of various tasks. Concentration was severely disturbed, as was learning and retention of new information, The authors concluded that phencyclidine interferes with those functions which integrate interoceptive and exteroceptive input in which goal-directed action becomes possible. Bakker and Amini (1961) felt that the compound produced an organic picture of psychosis in which only some of the symptoms were similar to spontaneously occurring psychoses. An extremely interesting interaction between phencyclidine and sensory deprivation has been described by Cohen et at. (1960). The combination produced a considerable decrease in psychotomimetic effects. The subjects tended to be calm, felt more in control of themselves and experienced a state of “utter nothingness” or “emptiness.” Cohen et at. (1962) extended their previous findings on the comparative psychotomimetic effects of phencyclidine, LSD-25, and amobarbital (see Rosenbaum et al., 1959) by studying these agents on symbolic and sequential thinking. Only the phencyclidine-treated subjects had inferior scores which approximated those of schizophrenics. These investigators felt their data was consistent with the hypothesis that phencyclidine produces a thinking disorder similar to chronic schizophrenia. Very recently Helrich and Atwood (1962) have obtained some evidence that haloperidol sharply reduces the agitation and disorientation of postoperative patients given phencyclidine. Chen and Ensor (1962) using rats have been able to show an antagonistic or additive effect of phencyclidine and haloperidol on motor activity and extensor seizures, depending upon the dose of haloperidol employed.

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B. GROSSBEHAVIOR OF ANIMALS Chen et al. (1959) were the first to study the effects of phencyclidine in a wide variety of animals. Signs of gross CNS stimulation and depression were noted which varied considerably depending upon the species and dosage. Excitant hyperactivity predominates in mice and rats while sedation or a calming effect predominates in pigeons, guinea pigs, hamsters, rabbits, cats, dogs, and monkeys. Larger doses of phencyclidine may produce a cataleptoid response or general anesthesia, while still higher doses produce seizures in pigeons, guinea pigs, dogs, and monkeys. Convulsions are not evident in fish, frogs, hamsters, rabbits, and only occasionally were seen in cats. Chen and Weston (1960) described in detail the effects of phencyclidine in doses of 0.3-15 mg/kg, given intramuscularly to Macaca mulatta. These investigators compared phencyclidine with a wide variety of other centrally acting drugs, including reserpine, chlorpromazine, bulbocapnine, LSD-25, bufotenine, meprobamate, and phenobarbital. Phencyclidine in doses of less than 1.0 mg/kg produced mild sedation or tameness. Semicoma and stupor occurred at doses of 2.5 mg/kg. The level of surgical anesthesia was evident at 5 mg/kg, and convulsions at 15 mg/kg. These investigators concluded that phencyclidine has a spectrum of activity different from all of the other compounds tested. Although phencyclidine produces a cataleptic state which is practically indistinguishable from bulbocapnine, somewhat larger doses are anesthetic in contrast to bulbocapnine. Chen and Weston (1960) were impressed with the apparent depression of sensory response of these animals in contrast to the activity of the motor system, which gave a cataleptic response and evidence of normal skeletal muscle tone. Subsequently a number of investigators have determined the gross behavioral effects of phencyclidine in various animals, The drug has been used very successfully to tranquilize baboons and monkeys for purposes of handling. C. CONDITIONED REFLEXBEHAVIOR IN A N I ~ I A L S

1. Reuiew of the Litmature Adey and Dunlop (1960) found that both phencyclidine and cyclohexamine in doses of 1 3 mg/kg given intraperitoneally sup-

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pressed or seriously interfered with a learned approach in a T-maze for 8-24 hours in cats with chronically implanted electrodes in the brain. During the predrug controls the hippocampus showed a characteristic 6-cps discharge during the approach performance. Such rhythmic discharge was depressed or abolished by both agents. This was accompanied by a failure of goal-directed behavior. With recovery from the drug, the rhythmic discharge reappeared concurrently with normal approach behavior.

2. Rat Conditioned Avoidance Our own studies using phencyclidine in rats trained for a conditioned avoidance response show it disrupts learned behavior although in a nonspecific manner. Twenty-four male albino rats of 200-400 gm were trained to avoid an electric shock to the grid floor of a box by jumping to a pole suspended from its ceiling, similar to the apparatus described by Cook and Weidley (1957). The animals were trained to a criterion of 95-100% successful avoidance responses. The animals, in groups of six, were given doses of 1,2,4, and 8 mg/kg of phencyclidine subcutaneously. The rats were observed before and 1hour after drug administration. Increasing doses of phencyclidine progressively blocked the conditioned avoidance response. An almost straight-line relationship exists between dose and per cent avoidance blockade. The effects of phencyclidine also were determined on escape response to the applied electroshock in those animals that did not show an avoidance response. The drug caused an almost comparable degree of blockade of the escape response. It would thus appear that the depressant effects of phencyclidine on avoidance and escape behavior are similar. Following phencyclidine administration the rats showed considerable gross motor agitation for periods as long as 2 hours. When placed in the avoidance situation, the animals appeared disorganized and blind. They would jump haphazardly and miss the pole. After application of the electroshock to the grid floor, the animals would squeal excessively and behave in an agitated and disorganized manner. It appeared that phencyclidine had no appreciable analgesic effects in the doses used. Inasmuch as chlorpromazine blocks the depressant effects of LSD-25 on the same conditioned avoidance procedure in rats (Cook and Weidley, 1957) and because Levy et al. (1960) felt that chlor-

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promazine alleviated some of the effects of phencyclidine in man, it was thought pertinent to study the effects of chlorpromazine on phencyclidine-depressed avoidance behavior. Since 4 mg/kg of phencyclidine is about the 75Hevel blocking dose for avoidance, it seemed reasonable to combine this agent with chlorpromazine in a close of 2 mg/kg. The latter dose of chlorpromazine has some minimal depressant effects on conditioned avoidance behavior. Similarly, a minimally effective dose of LSD-25 was used. Groups of 12 rats at one week intervals were administered subcutaneously equal volumes of: saline plus saline; chlorpromazine plus saline; phencyclidine plus saline; LSD-25 plus saline; phencyclidine plus chlorpromazine; and phencyclidine plus LSD-25. All animds were studied before and 1 hour after the administration of the various drug combinations. A summary of the results obtained is presented in Fig. 1. Before the administration of the two doses of saline, the 100 u)

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FIG. 1. Effects of chlorpromazine, phencyclidine, and LSD-25 on conditioned avoidance behavior in the albino rat. Chlorpromazine (Chlor.) was given in a dose of 2 mg/kg, phencyclidine (Sernyl) in a dose of 4 mg/kg,

LSD-25 in a dose of 0.4 mg/kg. All drugs were given subcutaneously. The bar height represents the mean of 12 animals and the short vertical line the standard error for each.

animals had 100%avoidance behavior (as illustrated by the diagonally shaded bars). After the administration of saline, avoidance

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behavior was depressed very slightly (stippled bars). Similarly, after the administration of 2 mg/kg of chlorpromazine and saline, minimal depression of avoidance behavior was observed. After the administration of 4 mg/kg of phencyclidine plus saline, however, there was a marked depression in avoidance behavior. LSD-25 in a dose of 0.4mg/kg plus saline had a minimal depressant effect. The combination of phencyclidine plus chlorpromazine, or phencyclidine plus LSD-25 caused the greatest depression of avoidance behavior. Both combinations showed additive or synergistic effects. D. SENSORY AND MOTORREFLEXES 1. Review of the Literature Meyer et al. (1959) described the neurological effects of intravenous phencyclidine in various surgical and neurological patients. Doses of 7.5 mg produced a significant sensory loss. Pin-prick was no longer painful. It appeared that the response to all forms of sensory stimulation was slowed or decreased. After a dose of 8 mg one patient was unable to read time; he could recognize common objects, but slowly. Doses of 9.5 mg of phencyclidine commonly produced vertical nystagmus and bilateral ptosis. With doses of 10.5 mg common objects could not be identified by vision or touch, although motor movement was present. In larger amounts the patient became comatose with nystagmus, both horizontal and vertical, and depression of corneal, pupillary light reflexes, and response to any form of stimulation. The tendon reflexes were enhanced but the plantar reflexes blocked. Meyer et al. (1959) were so impressed with the sensory deficits produced by phencyclidine that they suggested that it induces sensory deprivation. Davies and Beech (1960) observed that 0.075-0.1 mg/kg of phencyclidine produced definite neurological changes in each of 12 normal volunteers. The subjects showed a diminution of pain, touch, and proprioception. All showed nystagmus and ataxia, and one became diplopic. Ban et al. (1961) also noted that phencyclidine in doses of .01-0.1 mg/kg intravenously caused varying degrees of loss of sensory discrimination as well as paraesthesia. The deep tendon reflexes including the biceps, triceps, patellar, and Achilles tendon reflexes were uniformly increased by the drug but not in proportion to dose. Variable degrees of nystagmus occurred in 10%of the patients. All patients,

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however, complained of the symptoms vertigo, unsteady “slapping” gait, and swaying; nausea was observed in 90% of the cases. Morgenstern et al. (1962) determined the effects of 7.5 mg of phencyclidine given orally to 18 healthy volunteers. They measured sensory thresholds with 7 tests based on: perimetry, audiometry, visual acuity, taste thresholds, touch thresholds, two-point discrimination, and position sense. Over the first 2 hours of testing, the drug-treated group showed generally an increase in all sensory thresholds, while control subjects showed no change or a slight reduction in sensory threshold. Furthermore, although all sensory modalities were altered, two-point discrimination and esthesiometry were most affected, while perimetry, audiometry, position sense, taste, and visual acuity were all less impaired, equally. Of special theoretical importance, the deficits in sensory modalities were clear before the psychological effects of phencyclidine. Morgenstern et al. (1962) suggest that the psychological symptoms produced by the drug may be the result of a partial sensory deprivation. Chen et al. (1959) have described the extensive variability in the neurological manifestation of phencyclidine in various animal species. The depressant effects as observed in cats and monkeys were clearly associated with a decrease in the animals’ responses to noxious stimuli. The corneal, pupillary, and patellar reflexes were unimpaired. 2. Effects of Phencyclidine on the Pntetlar and Linguomundibulur Reflexes Our own experience with the effects of phencyclidine on the patellar and linguomandibular reflexes, and the modification of the patellar reflex by electrical stimuIation of the bulbar facilitatory and inhibitory areas in n-chloralose anesthetized cats is in agreement with the published literature. The effects of phencyclidine werc determined as per cent alteration of the basal patellar and linguomandibular reflex amplitude. The drug was administered intravenously on an accumulative dose schedule every 5 minutes to 6 cats and the data pooled. The dose schedule was 0.25, 0.5, 1.0, 2.0, 4.0, and 6.0 mg/kg. Doses of 0.25-6.0mg/kg progressively reduce the linguomandibular reflex from normal to approximately 3% of the control. With doses of 0.25 and 0.5 mg/kg of phencyclidine the amplitude of the patellar reflex increased slightly. Doses of 1.0-6.0

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mg/kg progressively decreased the patellar reflex to approximately 44% of the control. Increasing doses of phencyclidine progressively depressed bulbar inhibition of the patellar reflex. In contrast, bulbar facilitation of the patellar reflex was much less affected. When expressed as the per cent of facilitatory increment of the patellar reflex, bulbar facilitation was not reduced and at the larger doses was enhanced. Bulbar inhibition of the patellar reflex was consistently depressed with increasing doses of phencyclidine. 3. Effects of Phencyclidine on the Effects of Stimulation of the Motm Cortex of Monkeys with Chronic Electrodes

The effects of phencyclidine on electrical stimulation of the motor cortex of monkeys was determined in several animals. The end-point for stimulation of the motor cortex was taken to be a flexion, usually of the right leg, followed by minimal clonic activity. In doses of 0.5 mg/kg given intravenously, phencyclidine clearly elevated the threshold for evoked motor activity. Similarly the duration of after-discharge was reduced. In some animals, catatonic-like postures were obtained after phencyclidine following minimal electrical stimulation of the motor cortex. E. ALTERATIONS OF

THE

ELECTROENCEPHALOGRAM

1. Review of the LiterduTe Greifenstein et al. (1958) made serial EEG recordings on 6 surgical patients given an intravenous infusion of phencyclidine. Definite slowing, most pronounced in 0 activity, was observed in all cases. A decrease of EEG fast wave activity occurred after 2-3 mg of phencyclidine. After 7-10 mg definite, diffuse EEG slowing was noted. The slowing predominated in the occipital, temporal, and parietal regions. These EEG effects were clearly different from those produced by sleep or barbiturates. Similar findings were reported by Meyer et d. (1959). Rodin et al. (1959) described further the EEG effects of low doses of phencyclidine infusion in normal and psychiatric patients. Three different dose levels from 0.03-0.2 mg/kg were used. Phencyclidine in the larger doses produced profound EEG slowing. A stepwise reduction of CY frequency, and induced 0 and 8 wave activity was noted depending upon the dose. Rodin et al. emphasized that in the doses used phencyclidine differed con-

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siderably from other anesthetic or psychotomimetic drugs. Furthermore, the psychotomimetic effects of phencyclidine were observed frequently in the absence of definitive EEG changes. When EEG changes were observed, the psychic manifestations nearly always preceded them. Chen et al. (1959) described the effects of phencyclidine on the EEG of the “acute high-spinal” cat. Doses of 0.1-0.25 mg/kg given intravenously ( a “calming” dose) produced little change in the low voltage, fast frequency EEG. Doses of 0.5-1.0 mg/kg which produced catalepsy in intact animals caused high voltage spikes which appeared intermittently in the occipital, temporal, and frontal cortical areas, in that order. Anesthetic doses (3-5 mg/kg) produced high amplitude EEG activity in all cortical areas. From the published records, it can be noted that after doses of 10 mg/kg suppression-burst activity was very marked in the bipolar parietal, temporal, and occipital areas, but less evident in the frontal area. As described earlier Adey and Dunlop (1960) showed that, in trained cats with chronically implanted electrodes, phencyclidine and cy clohexamine in doses of 1-3 mg/kg intraperitoneally abolished the 6 cps hippocampal rhythm characteristic of approach behavior in a T-maze. Furthermore, marked spike-like activity was noted in the hippocampus and especially the pyriform cortex. Spikes in the amygdala were infrequent, but the 40 cps bursts characteristic of the alert animal were depressed. In acute post-ether gallamine-immobilized animals, phencyclidine-induced hippocampal spiking was rarely seen, in contrast to the animal with chronically implanted electrodes. Slow frequency stimulation (3-5 cps) of the nucleus ventralis anterior evoked rhythmic trains of hippocampal slow waves as well as recruiting responses in the suprasylvian cortex. After 3 mg/kg of cyclohexamine given intraperitoneally, the hippocampal response was almost abolished while the cortical response remained unaltered. Slow frequency stimulation (1 cps) of the rostra1 midbrain reticular formation procluced small amplitude responses in both the suprasylvian gyrus and hippocampus. Cyclohexamine in doses of 3 mg/kg intraperitoneally produced a marked alteration of these responses. High-frequency bursts which appeared in the cortex were phase locked with the first few stimuli, while the small evoked hippocampal response was depressed after the first few stimuli. With reticular stimulation rates of 5 cps, cyclohexamine depressed the

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hippocampal evoked responses. Following rapid rates of reticular stimulation ( 3040 cps ) cyclohexamine-induced cortical seizure spikes were converted to low-voltage fast-frequency activity, but similar stimulation of nucleus ventralis anterior markedly augmented the regularity of the seizure discharge. Himwich ct al. (1958) have reported that in rabbits phencyclidine produced high-amplitude spike-like waves, appearing first in the cerebral cortex and, with larger doses, involving the rhinencephalic structures. They concluded the drug must act primarily on the cerebral cortex. Van Meter et al. (1960) attempted to provide further support for such a notion. Doses of 1.0-2.0 mg/kg intravenously of phencyclidine produced a slight but definite increase in the number of spikes and slow waves recorded from both the isolated cerebral hemisphere and isolated motor cortex. In doses of 0.5 mg/kg intravenously, phencyclidine also increased the electrical threshold for recruiting responses in the rabbit motor cortex. 2. Studies on the Dog and Mankey In our own laboratory we have determined the EEG effects of phencyclidine in various chronic animal preparations. A total of 5 male beagle-like mongrels were prepared with chronically implanted electrodes in various structures of the brain. Bipolar stainless-steel wire electrodes were used. The techniques used to implant the electrodes were similar to those described by Domino and Ueki ( 1959). Four Macaca mulatta and two M . cynomo2ogu.s monkeys of both sexes were prepared also with chronically implanted bipolar electrodes in various areas of the brain. The techniques used were similar to those in the dog with minor variations as described by Domino and Ueki (1960). When the animals completely recovered from surgery, they were placed in a relatively quiet, closed compartment with a one-way window €or visual observations and EEG recordings. The dogs were restrained in an appropriate stockadetype frame. The monkeys were restrained in a “Walter Reed” type plastic chair. After a variable period of time the animals frequently would drowse in the restraining apparatus. A Model I11 Grass electroencephalograph was used for electrical recordings. Whenever possible the brain sites were confirmed histologically by the Hess iron-deposition technique to obtain the Prussian blue and/or green color at the electrode tips. Nerve cells were counterstained with

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thionine (see Domino, 1955,for details). Photic driving responses were elicited with high-intensity flashes from a General Radio stroboscope driven by a Grass stimulator at a frequency of 10 cps for a period of 5 seconds. Light flashes were presented randomly to the restrained, unanesthetized monkeys at intervals of 3-5 minutes. For control purposes, some animals were run before and after various psychotomimetic agents with and without the installation of several drops of 1% atropine sulfate into both conjunctival sacs. a. Effects of Phencyclidine on the EEG of Unanesthetized Dogs with Chronic Electrodes. The EEG effects of phencyclidine were determined in 5 dogs. With doses of 0.1-0.25 mg/kg given intravenously, the neocortical structures showed minimal to no EEG changes. With doses of 0.5 mg/kg of phencyclidine, the neacortical EEC varied considerably, depending upon the individual animal and the time after drug administration. In some animals, minimal fast-frequency, moderately high amplitude EEG changes were observed, In other animals theta- to alpha-like activity or generalized &waves were present particularly in the association areas. A most unusual alternating pattern of low-voltage fast-frequency activity lasting for a few seconds followed by a similar but shorter period of &wave activity was also observed. For doses of 1.0 mg/kg of phencyclidine, the EEG generally showed high-voltage &wave activity. Following doses of 1.0 mg/kg to 9.0 mg/kg, grand ma1 seizures were obtained in several of the dogs, both in the EEG as well as behaviorally. Relatively small doses of phencyclidine produced depression of the normal electrical bursts, synchronous with respiration in the olfactory bulb and medial amygdala, at a time when the neocortical EEG showed only a slight increase in low-voltage fast-frequency waves. The reduction of the electrical bursts in rhinencephalic structures was not accompanied by marked respiratory depression. An example of these EEG effects is illustrated in Fig. 2. In panel A is shown the control EEG activity of a normal dog standing comfortably in the restraining apparatus. The usual low voltage, fast frequency EEG pattern of an awake animal was observed in the neocortical leads. Electrical bursts synchronous with inspiration were present in the olfactory bulb. Within 1minute after 0.5 mg/kg of phencyclidine given intravenously, the dog appeared oblivious

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of his surroundings. Although respiration was not particularly depressed, the electrical bursts were completely absent in the olfactory bulb (see Fig. 2B). The neocortical structures showed a slight increase in the amplitude of the low-voltage fast-frequency waves. Within 5 minutes after the administration of phencyclidine, complex changes were observed in the electrical activity of the neocortex. Particularly in the frontal and parietal association areas periods of very fast frequency activity (30-60 cps) alternated with 8-waves. This pattern is shown in Fig. 2C, in the EEG leads from area 7 and 8. Some theta- to alpha-like activity is also present. At this time, the bursts in the olfactory bulb were completely suppressed and replaced by delta- to theta-like activity. Twenty minutes after the administration of phencyclidine some fast waves were observed in the olfactory bulb, however, typical respiratory bursts were still absent. In the visual area, a peculiar fast-frequency lowvoltage burst was followed by &waves and alpha-like activity. This alternated in cycles. Within an hour the animal showed some recovery, both behaviorally as well as in the EEG. As illustrated in Fig. 2E, the neocortical activity at this time was quite similar to control. Some low voltage, fast frequency bursts were evident in the olfactory bulb. Two hours after the administration of phencyclidine the dog had recovered almost completely, both behaviorally as well as in its EEG (compare Fig, 2F to 2A). h. Effects of Phencyclidine on the EEG of Monkeys with Chronic Electrodes. A total of 6 monkeys were each given phencyclidine at approximately weekly intervals in doses of 0.1-0.5 mg/kg intravenously. In doses of 0.1425 mg/kg, phencyclidine produced mild gross behavioral effects and minimal EEG changes to none in neocortical structures. Grossly the animals exhibited a mild ataxia and drunken state. Occasionally, there was a slight increase in the amplitude of the low-voltage fast-frequency activity. Sometimes this pattern was modulated by %wave activity of moderate amplitude. In doses of 0.5-1.0mg/kg, phencyclidine produced clear-cut gross behavioral changes and marked EEG effects. These resembled in part the findings in dogs. Marked individual variations were noted. A t these larger dose levels, some animals showed predominantly 6wave activity. The alternating low-voltage fast-frequency and 8-

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FIG.2. EEG effects of phencyclidine in the dog. A. control EEG of an awake dog. B. 1 minute after 0.5 mg/kg of phencyclidine given intravenously. C . 5 minutes later. D. 20 minutes later. E. 1 hour later. F. 2 hours later. Atl: cortical archa I). A l : cortical area 1. A4: cortical area 4. A N : cortical area su)

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ixir 19. A17: cortical area 17. OB1 and OB2: anterior and posterior olfactory bulb leads. RESP: respiration. Inspiration is downward. All EEG recordings were bipolar. Voltage calibration: 100 microvolts 321

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wave burst pattern previously observed in the dog was also noted in the monkey. An example of this activity is illustrated in Fig. 3. In 3A is shown the normal low-voltage fast-frequency EEG pattern of an awake monkey sitting comfortably in a restraining chair. In the eighth channel, clear-cut respiratory bursts synchronous with inspiration are observed. This respiratory burst pattern is observed most clearly in the alert animal and decreases considerably during sleep (Domino and Ueki, 1960). Within 5-10 minutes after the intravenous administration of 0.5 mg/kg of phencyclidine, alternating low-voltage fast waves and 6 activity was observed in most of the neocortical leads. The amygdala lead at this time showed a marked diminution of the typical respiratory bursts observed in the control period. The monkey appeared quite catatonic with its eyes open and blinking occasionally. The corneal and patellar reflexes were easily elicited, The animal, however, gave no particular response to ordinarily painful pinches. Within approximately 1 hour after the administration of phencyclidine, the monkey showed definite signs of recovery. Its EEG at this time showed a predominantly 8 pattern in the more frontal areas of the brain, while the other neocortical leads were returning toward normal. Occasional bursts of theta-like activity were observed. Respiratory bursts again reappeared in the amygdala-pyriform cortex lead. In approximately 2-3 hours, the animal appeared recovered both behaviorally as well as in its EEG (see Fig. 3C). With doses of less than 1 mg/kg, at no time was convulsive activity observed in the monkey behaviorally or in the EEG. This contrasts with our findings in the dog. c. Effects of Phencyclidine 012 the Dog Isoluted Cortex. The effects of phencyclidine in doses of 0.1-0.5 mg/kg given intravenously were determined on the threshold and duration of cortical after-discharge elicited in the isolated suprasylvian gyrus. After recovery from ether anesthesia the animals were immobilized with decamethonium and maintained on artificial respiration. Although considerable variability was observed in the threshold and duration of cortical after-discharge, the effects of phencyclidine in doses of 0.5 mg/kg given intravenously were obvious. These consisted of an those of Fig, 2. A6: cortical area 6. A.H.: anterior hypothalamus. P.H.: posterior hypothalamus. RET: mesencephalic reticular formation. AMG: medial amygdala to pyriform cortex.

FIG. 4. Effects of phencyclidine on EEG photic driving in the monkey. A. control record of an awake monkey. B. photic driving response after the instillation of 2 drops of 1%atropine sulfate into each conjunctival sac. C. 10 minutes after the administration of 0.5 mg/kg of phencyclidine given intravenously. D. approx. 25 minutes later. E. 1 hour later. F. 2 hours later.

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Symbols and voltage calibration are similar to previous figures. Post, Hypo.: posterior hypothalamus. Light flashes were recorded from a photocell placed behind the animal. 325

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increase in the tlireshold and a decrease in the duration of evoked afterdischarge. Phencyclidine also altered the spontaneous electrical activity in the isolated cortex. It has been reported to produce higher voltage, slow waves in the rabbit isolated motor cortex as well (\'an Meter et al., 1960).

F. ALTER~TIOSSOF EVOKEDPOTENTIALS In our laboratoly we have been especially concerned with neuropharmacological evidence that phencyclidine impairs the responses of the brain to various sensory inputs. As described above, even low doses of phencyclidine markedly depress the olfactory bulb and amygclala responses to the normal stimulus of aidow in the nostrils that accompanies respiration. It was of interest to determine if other sensory modalities were also affected. Therefore, EEG photic driving responses were elicited in the monkey and the effect of phencyclidine on these potentials was compared with those produced by LSD-25 and mescaline. 1. Effects of Phencyclidine 071 EEG Phutic Driving in the Monkey Phencyclidine caused a reduction in EEG photic driving in doses of 0.5 mg/kg given intravenously. Normally, flashes of high intensity light at 3 frequency of 10 cps for 5 seconds produced definite c.1-oked responses in the visual areas. Considerable animal variation i t a s observed in the degree of photic driving. Usually the best responsc~swere obtained in the classical visual system including the lateral geniculate and cortical area 17. Photic driving responses were also recorded in areas 18 and 19 and in the reticular formation of the brainstem. Occasionally the animals closed their eyelids during the high intensity fight flashes. Even with the eyes closed, photic driving responses were observed. In order to prevent excessive variability in photic driving responses associated with marked movements of the head away from the light source, control experiments were performed in which the head was partially fixed in place. Controls involving the instillation of 1%atropine sulfate into the conjunctival sac of both eyes were used. Dilatation of both pupils by the local administration of atropine into the conjunctival sac had either no effect or slightly increased the amplitude of photic driving, as illustrated in Fig. 4A and B. Within 10 minutes after the administration of 0.5 mg/kg phencvclidine given intravenously, the animal

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appeared stuporous and catatonic, At this time, the EEG showed an increase in the low-voltage fast-frequency activity especially in area 8. Photic driving responses were considerably reduced (see Fig. 4C). Within 20 minutes after phencyclidine administration some channels of the EEG showed considearble %wave activity and others low-voltage fast-frequency waves. In this animal, photic driving responses now were almost completely obliterated (see Fig. 4D). In about 1 hour, photic driving responses were again observed although they were considerably altered from those obtained in the control period. In some animals there was a tendency for a simplification of the photic driving response. During recovery from phencyclidine, the responses frequently appeared enhanced. Fairly complete recovery usually occurred within 2-3 hours after phencyclidine (compare Fig. 4A, B, and F ) . Somewhat similar findings were observed in a total of 6 monkeys, 4 Macaca mulatta and 2 M . cynumologus. In doses of 0.1 mg/kg of phencyclidine sometimes an increase in photic driving was observed. This was especially evident if the animals relaxed. In doses of 0.5 mg/kg of phencyclidine given intravenously, however, the responses were depressed. 2. Efects

of LSD-25 cm Photic Driving Respmes in the Monkey

The effects of LSD-25 in doses of 1-25 pg/kg given intravenously were determined in monkeys with chronically implanted electrodes in various cortical and subcortical areas. Photic driving responses were elicited in the same manner as described previously for phencyclidine. Light flashes of 10 cps for 5 seconds were presented every 3-5 minutes. Photic driving responses were most clearly observed in the visual areas, particularly area 17. In some animals, doses of LSD-25 as low as 2 g / k g affected photic driving responses. The most consistent effects in all animals were observed in doses of 10 fig/kg or more. Figure 5A shows a control electroencephalogram of the photic responses recorded in area 17 and the reticular formation following high intensity light flashes. Marked variability was observed in the extent of the photic responses depending upon whether the animal had its eyes open, closed, etc., as described previously. Following the administration of 10 pg/kg of LSD-25 intravenously, considerable flattening was observed in the lowvoltage fast-frequency EEG. This occurred rather quickly within 2-5 minutes after the intravenous administration of LSD-25. Usually

3 FIG.5. Effects of LSD-25 on EEG photic driving in the monkey. A. control EEG tracing. B. 5 minutes after 10 Pg/kg of LSD-25 given intravenously. C. 30 minutes later. D. 1 hour later. Symbols and voltage calibration are similar to those of the previous figures. MR: mesencephalic reticular formation. PRT:

SEC. I

pretectum. CC: corpus callosum. A7: cortical area. 7. S: septum. P: photic stimulus recorded from a photocell placed behind the animal. 329

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after 10 minutes, the photic driving responses were characteristically reduced in amplitude and the potentials altered with multiple waves following the individual light flashes. Photic driving responses appeared to be reduced in the mesencephalic reticular leads slightly before those in the visual area (see Fig. 5B). However, within 10 minutes and lasting for approximately 1-2 hours or more, the photic driving responses were considerably altered in all areas recorded as is illustrated in Fig. 5C. Partial recovery usually occurred within 1-2 hours, However, multiple frequency responses were noted for periods as long as 12 hours. Weekly administration of LSD-25 produced some tolerance to these effects on photic driving. Control procedures including dilatation of the pupils with local instillation of 1% atropine sulfate solution, the intravenous administration of 100 pg/kg of brom-LSD, or 0.5-1 mg/kg of d-amphetamine did not reproduce the LSD-25 induced alterations in photic driving. 3 . E8ect.s of Mescaline on Photic Driving Responses in the Monkey

The effects of mescaline on photic driving were not remarkable compared to those produced by LSD-25 or phencyclidine. In doses of 10 mg/kg given intravenously, mescaline produced minimal depression of the amplitude of the photic driving response. In some animals the principal action of mescaline was to induce a series of more complicated waves following each period of photic stimulation. The animal frequently blinked during this period. Within 2 hours, these mescaline-induced alterations returned toward normal. However, complete recovery did not occur for several more hours. Throughout this period the spontaneous EEG patterns in neocortical and subcortical structures remained essentially the same. 4. Eflects of Pheimjctidine on Single Evoked Responses in the Cat ViSwLl cortex

Electrical activity evoked in the visual cortex to single light flashes in both eyes was recorded in 6 cats paralyzed with decamethonium. Considerably variability was observed in the amplitude of the primary evoked response. The evoked response was markedly modulated by spontaneous EEG activity. Following the administration of phencyclidine in doses of 0.5 mg/kg given intravenously, both the initial positive and negative portions of the primary response were slightly reduced. In general, the amplitude of the initial negative response was reduced more than the initial positive re-

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sponse. Increasing doses of phencyclidine up to 5 mg/kg diminished the evoked potential but never obliterated it. In fact, animals that were completely anesthetized with 30 mg/kg of pentobarbital showed evoked responses that were depressed in amplitude. Administration of phencyclidine to such preparations had no further depressant effect on the amplitudes of the primary response. Under general anesthesia with pentobarbital, very reproducible evoked responses were obtained but these were resistant to depression by phencyclidine.

5. EtTects of Phencyclidine on Sciatic Nerve Responses Eooked in the Rabbit Sensory C d e x Single shock stimulation of the sciatic nerve evoked a large primary response in the sensory cortex of rabbits anesthetized with 35 mg/kg of pentobarbital given intraperitoneally. This dose of pentobarbital was sufficient to cause light general anesthesia. During the recording procedure local application of 2% xylocaine to the skin edges was administered as necessary. With doses of 0.1 ing/kg of phencyclidine given intravenously, the initial positive and negative portions of the primary response were not appreciably affected. Similar observations were made in a series of 6 control rabbits given an equal volume of 0.9%sodium chloride. In doses of 0.5 mg/kg, however, phencyclidine clearly caused a depression of the negative portion of the primary response. In many animals, the initial positive response was also slightly reduced. When increasing doses of phencyclidine were administered, the negative portion of the primary response decreased. Even with very large doses of phencyclidine up to 5.0 mg/kg, the initial positive portion of the primary response was never completely obliterated. In doses of 10 pg/kg given intravenously, LSD-25 had no appreciable effect on the initial positive response. A slight decrease in the height of the initial negative response was observed, usually within a half an hour after its administration. Within one hour after the administration of LSD25 the evoked responses approached control amplitudes. 6. Cmparatiue Efects of Phencyclidine on Sciatic and P h i c Eooked Reqonses in the Rabbit Cerebral Cmex

In view of the differential effect of phencyclidine in depressing touch and two-point discrimination more than visual acuity in man (see Morgenstern d al., 196Z), it was important to determine if a

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similar effect could be obtained in an animal. Therefore, lightly pentobarbital-anesthetized rabbits, similar to those described above, were prepared for recording simultaneously the sciatic nerve evoked response in the somatic sensory cortex, and the photically evoked response in the visual cortex. Two AEL stimulators were so arranged that the stimulator for the sciatic nerve also controlled the discharge of a second stimulator used to elicit the light flash. The latter’s stimulus pulse was delayed 50 mseconds for discharge of the AEL photic unit. The delay between the two sensory inputs was introduced to reduce stimulus interaction within the central nervous system, and to allow unobstructive visualization of the evoked responses on a cathode ray oscilloscope. With each animal, a series of control evoked responses were recorded from the contralateral cortex before and after the intravenous administration of 0.5 mg/kg phencyclidine. Both stimuli were at an above threshold but sub-

CONTROL

AFTER 0.5 MWKG SERNYL

RECOVERY

SENSORY VISUAL

SENSORY VISUAL

I00 U V l O l V 25 MILLISEGIDW

FIG. 6. Comparative effects of phencyclidine on the evoked sciatic nerve and photic responses in the rabbit cerebral cortex. Two sets of evoked responses in each primary sensory area are illustrated for each treatment. Phencyclidine (Semyl) was given intravenously in a dose of 0.5 mg/kg and the potentials recorded 5 minutes later. Recovery occurred in 1-1s hours. A positive potential is downward. Calibrations are as indicated. Light flash was delayed 50 msec.

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maximal level such that the variability of the evoked response was minimal. Figure 6 shows the results obtained in one typical animal. Two sets of responses for each treatment are shown to illustrate some of the variability of the evoked responses. A typical positivenegative primary complex may be observed for both sensory inputs. After 0.5 mg/kg phencyclidine, the sciatic nerve primary complex was clearly more reduced than the visually evoked response. In a series of 7 such animals the positive primary complex was reduced but not statistically significantly for both the sciatic nerve as well as the visual evoked response. In contrast, the negative portion of the sciatic nerve primary response was significantly depressed more than that of the visual negative primary response. In two other series of 6 or 7 animals in which the responses were evoked separately, phencyclidine in a dose of 0.5 mg i.v. significantly depressed both the positive and negative portions of the sciatic nerve primary complex. It did not, however, signscantly depress the visual positive primary response. The visual negative primary response was significantly depressed but not as much as the sciatic negative primary response. IV. Cardiovascular and Respiratory Actions

A. EFFECTS IN MAN AND VARIOUS ANIMALS

Greifenstein et al. (1958) have described the cardiovascular and respiratory effects of phencyclidine in man. The drug consistently increased both the systolic and diastolic blood pressure. Doses of 0.06 mg/kg of phencyclidine intravenously produced an increase of approximately 8 mm Hg in systolic and diastolic blood pressure. Doses of 0.25 mg/kg produced a 26-mm increase in the systolic and a 19-mm rise in diastolic blood pressure. The pulse rate increased slightly but not as consistently. No arrhythmias were noted. A slight increase in mean blood pressure has also been observed by Chen et al. (1959) following the intravenous administration of low doses of phencyclidine to pentobarbital-anesthetized dogs. Ganglionic ( hexamethonium ) and adrenergic blocking ( l-naphthylmethylethylp-bromethylmethylamine HBr; SY-28 ) agents were ineffective in antagonizing the phencyclidine pressor response. Large doses of phencyclidine produced a slight fall in blood pressure, bradycardia, and occasionally arrhythmias. The drug depressed the isolated rab-

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bit heart. Phencyclidine in doses of 1-4 mg/kg intravenously to pentobarbital-anesthetized dogs did not suppress the pressor effects of epinephrine and dimethylphenylpiperazine ( DMPP ) , nor the depressor effects of acetylcholine and histamine. I n fact, the pressor effects of epinephrine and DMPP were potentiated (Chen et aZ., 1959). Greifenstein et al. (1958) showed that phencyclidine in doses of O.W.25 mg/kg given intravenously to patients usually increased the minute and tidal volume as well as the respiratory rate. In animals receiving a “cataleptic” to an “anesthetic” dose of phencyclidine no striking alteration of respiration was noted (Chen et aZ., 1959). Very large doses produced respiratory depression.

B. EFFECTSox Doc, BLOODPRESSUREAND PERIPHERALRESISTANCE 1. Fheiacyclidiiae Modification of the B b d Pressure Response to Varlous Amines in Anesthetized Dogs

The effects of phencyclidine were determined in our laboratory on the blood pressure response of epinephrine, norepinephrine, and serotonin in 14 dogs anesthetized with pentobarbital sodium. Epinephrine ( I g / k g ) , norepinephrine ( 1 g / k g ) , and serotonin (15 !tg/kg) were given intravenously in a random fashion at approximately 5 minute intervals. Usually after two or more replicates of Pach agent, when fairly reproducible vasopressor responses were obtained, phencyclidine was administered intravenously in a dose of 0.5 mg/kg. After the administration of phencyclidine a transient pressor response was observed which lasted approximately 10-15 minutes. Following administration of the drug it was noted that the pressor response to epinephrine, norepinephrine and serotonin were enhanced for as long as one hour even though the baseline blood pressure returned to normal. The mean blood pressure increase rt: S.E. to 1 &kg of epinephrine before phencyclidine was 30.4 k 3.1 mm Hg. After phencyclidine administration the mean blood pressure increase was 43.4 k 4.0 mm Hg. Similarly, the mean pressor response to norepinephrine administration before phencyclidine was 40.4 +- 4.4 and after 53.4t 5.0 mm Hg. Before phencyclidine administration, the mean pressor response to serotonin was 31.3 5.1 and after 60.7 t 11.0 mm Hg. Although the data

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indicate that after phencyclidine was given the pressor responses to epinephrine, norepinephrine and serotonin were increased, considerable individual variation was seen. A majority of the animals showed at least 10-mm increments in the pressor responses to epinephrine, norepinephrine, and serotonin. There were, however, notable individual differences. Some animals showed marked enhancement of their pressor responses to epinephrine and norepinephrine with minimal enhancement of serotonin. Other animals showed precisely the opposite---serotonin pressor responses were markedly enhanced with minimal enhancement of epinephrine and norepinephrine. In other animals, all three agents clearly showed enhanced effects, Because of these marked individual variations it was thought pertinent to subject the data to an analysis of variance by the method of unweighted means. This analysis showed significant interaction, and significant differences between dogs prior to and after the administration of phencyclidine. A harmonic mean of the number of observations per cell was used which gave F ratios as follows: The pre-phencyclidine analysis showed that the F ratio for the source of variation between dogs was 16.3, between drugs 2.74, and for interaction was 7.34. Thus, before the administration of phencyclidine there were significant individual variations between dogs in their responses to epinephrine, norepinephrine, and serotonin. In 6 of the 14 dogs studied, the phencyclidine was replaced by an equal volume of O.% saline. A simple paired comparison Student’s t test indicated that after phencyclidine administration the pressor responses to epinephrine, norepinephrine, and serotonin were significantly enhanced (P < 0.01). After the administration of saline, however, the pressor responses to these three agents were not significantly enhanced. In 6 animals the administration of 1.0 mg/kg of chlorpromazine consistently reduced the enhanced pressor response to epinephrine and serotonin. In general, the pressor responses to norepinephrine were also reduced but not as consistently. The above effects are clearly illustrated in the blood pressure tracings in Fig. 7 taken from one experiment. After 0.5 mg/kg of phencyclidine the pressor effects of epinephrine, norepinephrine, and serotonin were enhanced. After 1 mg/kg of chlorpromazine, the mean arterial blood pressure was reduced slightly. Epinephrine

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“reversal” was evident. The serotonin pressor response was markedly reduced and converted to primarily a depressor response. The norepinephrine pressor response was still clearly present. In three animals, 20 g / k g LSD-25 intravenously reduced the enhanced responses to serotonin which occurred following phencyclidine pretreatment. The effects of phencyclidine on small doses of serotonin ( 2 1 0 pg/kg) which caused primarily depressor responses was not analyzed statistically. It appeared, however, that phencyclidine did not enhance the depressor response of serotonin as it did the pressor response obtained with larger (15-30 pg/kg) doses of serotonin.

2. Effects of Phemjclidine on the Peripheral Resistance of the Dog H i d Limb A series of 5 pentobarbital-anesthetized dogs were prepared for perfusion of their hind limbs via the descending aorta. The peripheral resistance was measured before and after the intra-arterial administration of 0.5 and 1.0 mg total of phencyclidine. The drug produced a diphasic change in peripheral resistance which consisted of a very transient dilator followed by a marked vasoconstrictor response. Doses of 0.5 mg of phencyclidine caused a mean dilator response t S.E. of 2.5 mm 42.5, and a mean constrictor response t S.E.of 21.5 15.6 m m Hg. Doses of 1.0 mg of phencyclidine caused a mean dilator response -i- S.E. of 14.4 zk 4.6 and a mean constrictor response 3- S.E. of 30.6 k 2.4 mm Hg. The vasoconstrictor response persisted after the administration of the ganglionic blocking drug trimethadinium, It can be concluded that phencyclidine produces a direct peripheral vasoconstriction in the perfused dog hind-limb preparation. FIG. 7. Effects of phencyclidine on the pressor responses to epinephrine, norepinephrine, and serotonin in the pentobarbital anesthetized dog. EPI: I-epinephrine. NOREPI: 1-norepinephrine. 5-HT: serotonin. CPZ: chlorpromazine. All were given intravenously. Top row: Control situation. Middle row: After 0.5 mg/kg Sernyl. Bottom row: After 1 mg/kg chlorpromazine. Although not illustrated, phencyclidine (Semyl) caused a transient pressor response which lasted approximately 10 minutes. Responses to epinephrine, norepinephrine, and serotonin were obtained 1545 minutes after phencyclidine administration. Subsequently chlorpromazine was administered as a possible antagonist. Time base markings represent 1 minute intervals.

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V. Metabolic Actions

A. EFFECTS OK BODYTEMPEFUTURE

LSD-25 is known to cause a profound elevation in the rectal temperature of rabbits (see Cerletti, 1956). It was of interest to determine the effects of phencyclidine on body temperature alone and in combination with LSD-25 in the rabbit. Four treatment groups of albino rabbits were utilized. Twelve different rabbits were used per group in order to obtain data which could be analyzed statistically. The first group received simultaneously intravenous injections of 10 g / k g of LSD-25 and 1.0 mg/kg of phencyclidine. The second group received 10 pg/kg of LSD-25 and a volume of saline equal to the phencyclidine injection. The third group received 1.0 mg/kg of phencyclidine and a volume of saline equivalent to the LSD-25 injection. The fourth group received two injections of saline in volumes equivalent to the LSD-25 and phencyclidine injections. The mean rectal temperature of the rabbits subjected to these various treatments is illustrated in Fig. 8. It can

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FIG. 8. Effects of LSD-25, phencyclidine and saline in various combinations on the rectal temperature of albino rabbits. Short vertical lines represent f the standard error. LSD-25 was given in a dose of 10 pg/kg and phencyclidine in a dose of 1.0 mg/kg, intravenously. Sodium chloride (0.9%in water) was given in volumes equal to the drugs administered.

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be observed that in general the control rectal temperature prior to any of the drug injections was approximately 398°C. Although there were marked individual variations with regard to the control rectal temperature, the mean temperature of groups of 12 rabbits was remarkably constant. Following the administration of 10 g / k g of LSD-25 and 0.9% sodium chloride, the mean rectal temperature markedly increased within one half hour. A peak temperature rise to 41.2 rrt 0.17"C (that is, 1.4"C) was observed. This elevated temperature gradually decreased with time. Four and a half hours after the injection of LSD-25, the temperature was still slightly elevated. Simultaneous injection of phencyclidine plus LSD-25 caused an elevation in temperature but this was not nearly as marked as in the case of LSD-25 plus saline. Saline administration alone resulted in a gradual reduction in rectal temperature although the fall was not statistically significant. Similarly, phencyclidine and saline caused a slightly greater fall in rectal temperature, although the data was not significantly different from saline alone. It was concluded that phencyclidine partially antagonized the temperature elevation due to LSD-25 in albino rabbits. It is of interest that this applies only to small doses of LSD-25. Chen has shown that phencyclidine does not significantly lower the elevated body temperature, if 100 pg/kg of LSD-25 was administered to the rabbits.

+

B. EFFECTSON OXYGEN CONSUMPTION in Vivo The oxygen consumption of groups of 8 albino rats was determined for periods of approximately one half hour before and after the administration subcutaneously of 4 mg/kg, phencyclidine, and 0.4 mg/kg LSD-25. Oxygen consumption was measured from 15-60 minutes after drug administration. A desiccator jar surrounded by water to maintain a relatively constant environmental temperature was used. Total oxygen consumption was expressed in milliliters per kilogram per minute at standard conditions. The summarized data is presented in Fig. 9. Oxygen consumption generally fell with time. This was probably due to initial excitation due to handling the animals. In the control period prior to drug administration the average oxygen consumption for a group of 8 rats was 21.95 ml/kg/minute. After the subcutaneous administration of 0.9%sodium chloride the mean oxygen consumption decreased to

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T

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5

z

I0

ON

WINE

SERNYL

LSD-25

SER+NYL LSD-25

CHLOR.

SERPYL

CH$R.

CHLOR

SERNYL

FIG.9. Effects of various drugs OIL total oxygen consumption. Oxygen consumption is expressed at standard conditions. The height of the bar represents the mean and the small vertical line -t S.E. Diagonally shaded bar is the controi and the stippled bar, after treatment. All drugs were given subcutaneously and tested 15-60 minutes later. (See text for details.)

26.2 ml/kg/minute. Phencyclidine consistently and significantly elevated the oxygen consumption in rats (see Fig. 9). On the other hand, LSD-25 caused a significant fall in oxygen consumption similar to that occurring following the administration of saline. The simultaneous administration of 4 mg/kg of phencyclidine and 0.4 mg/kg of LSD-25 prevented any significant change in the oxygen consumption from control levels. Chlorpromazine in a dose of 2 mg/kg subcutaneously lowered the oxygen consumption when given alone. When it was given together with phencyclidine (Sernyl Chlor.) or one half hour before phencyclidine (Chlor. Sernyl), it did not prevent the increase in total oxygen consumption (see Fig. 9). This Iatter observation is especially interesting because chlorpromazine depresses the marked motor agitation induced by phencyclidine in the rat. It therefore appears unlikely that the increase in oxygen consumption is due solely to the motor agitation induced by phencyclidine. Furthermore, we have been able to show that phencyclidine increases slightly the oxygen consumption of guinea pigs, a species in which it does not produce motor agitation.

+

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C . EFFECTSON OXYGEN CONSUMPTION in Vitro M phencyclidine increased Lees (1961) demonstrated that the oxygen consumption of both rat liver homogenates and mitochondria approximately 20%.In the same concentrations the drug decreased the P/O ratios for succinate. Phencyclidine also increased mitochondria1 swelling. A more detailed report of this investigation recently became available ( Lees, 1962). Phencyclidine increased the oxygen consumption of rat liver homogenates with a variety of other substrates beside succinate. These include a-ketoglutarate, P-hydroxybutyrate, and citrate. The oxidation of malate was not significantly affected unless large concentrations of phencyclidine were used, in which case the oxidation of malate was inhibited. Phencyclidine was an effective uncoupler of oxidative phosphorylation in concentrations of 1 to 5 x M . In vivo pretreatment of the animals with 10 mg/kg of phencyclidine given intraperitoneally produced inconclusive changes in the in vitro oxidation of succinate by liver homogenates. Lees also studied the in uitm effects of some analogs of phencyclidine. Cyclohexamine and N-ethyl-l-cyclohexylcyclohexylamine had a stimulatory effect but were less potent than phencyclidine. Lees noted that these analogs of phencyclidine are thought to have less psychotomimetic effects in man.

D. METABOLICFATE The relatively short duration of action of phencyclidine in various species suggests that it is rapidly metabolized. Ober et al. (1963) have studied the metabolic fate of phencyclidine with C14

uniformly distributed in the phenyl ring, as well as phencyclidine with tritium in the 3 and 4 positions of the piperidine ring. The monkey rapidly and extensively metabolizes phencyclidine to various hydroxylated derivatives which are excreted in the urine as conjugates. Different species produce the same urinary metabolites but in different amounts. Man, as well as most other species, excrete mainly the mono-4-hydroxy piperidine derivative while the cat (phencyclidine is unusually long acting in the cat) excretes the drug primarily unchanged. Based on data in animals, the relatively weak pharmacologic activity of the metabolites suggests that they do not contribute significantly to the pharmacologic activity of phencyclidine.

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VI. Discussion

There is little doubt that phencyclidine possesses a unique pharmacological spectrum of activity. The compound is of special interest to the neurobiologist. Low doses of phencyclidine clearly increase the thresholds for various sensory inputs in man and animals. The drug does not appear to block sensory input at a peripheral level. For example, muscle spindle afferent discharge is probably intact for the deep tendon reflexes are not depressed unless massive doses are used. Phencyclidine clearly does alter the response of the central nervous system to various sensory inputs in both man and animals. These effects occur in subanesthetic as well as in anesthetic doses and appear to involve all senses. In animals such as the cat, dog, and monkey, phencyclidine depresses the electrical bursts synchronous with respiration that are present in the olfactory bulb and amygdala. The depression of the electrical burst activity does not appear to be due to respiratory depression. Electrophysiological evidence also suggests that visual input as recorded from the primary area of the cerebral cortex is also altered by phencyclidne. For example, phencyclidine depresses the photic driving response in monkeys. The depression is manifested as decrease in the voltage of the photic driving response with a lack of synchronous one-to-one following. These effects persist for approximately 1 hour after pliencylidine administration, although in some animals the photic driving responses did not return to normal for as long as 2 hours. The effects of phencyclidine on EEG photic driving appear to be quite different from those due to LSD-25 and mescaline. LSD-25 produces a reduction in the amplitude of the photic driving response and an increase in the number of secondary and tertiary waves following the initial photic stimulus. Thus, LSD-25 tends to complicate the photic driving pattern and at the same time reduces its amplitude. The reduction in amplitude of the photic driving response is compatible with the findings of Evarts (1957) of a blocking effect of LSD-25 at the lateral geniculate. The doses of LSD-25 used in the experiments of Evarts, however, were much larger than those used in the monkey photic driving experiments, The effects of LSD-25 on photic driving could also be due to an action at cortical levels are described by Purpura, Marsazzi, and Hart, etc. (see Rinkel and Denber, 1958). The effects of mescal-

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ine on photic driving were not as dramatic as that of LSD-25 and phencyclidine. On the basis of acute experiments in cats in which evoked responses in the visual cortex were obtained to single flashes of light, it is obvious that phencyclidine does not completely block visual input. The experiments in the cat were difficult to quantitate because of marked spontaneous EEG modulation of the evoked response. Nevertheless, the effect of phencyclidine was to reduce the negative and positive components of the primary response. At no time did phencyclidine completely block the initial positive response. In fact, in barbiturate-anesthetized cats the effects of phencyclidine on the primary response were negligible in spite of the fact that massive doses were administered. It is obvious that the sweeping generalization that phencyclidine is a specific sensory blocker is completely inaccurate. Low threshold stimulation of the sciatic nerve probably elicits the sensation of touch and proprioception. It is, therefore, of interest that phencyclidine reduced the sciatic nerve evoked response in rabbits more than the visual evoked response, The negative portion of the primary complex appeared to be reduced more than the positive, although both may be involved. The observation that the visual evoked response in the rabbit appears to be more resistant to phencyclidine than the sciatic evoked response tends to parallel the findings of Morgenstern et aE. (1!362) using man who showed that touch and two-point discrimination are most affected by phencyclidine. The rabbit data are also compatible with the findings of Luby et al. (1959) for man that proprioception is also depressed. It would appear that to some extent all sensations are affected by phencyclidine. This is in marked contrast to the other psychotomimetic drugs. A crucial question is at what level does the depression of sensory input occur? Polysynaptic reflexes ( linguomandibular and plantar) are more depressed by phencyclidine than monosynaptic reflexes (patellar). Presumably, therefore, some spinal cord and brainstem interneurons are depressed by this drug. Some selectivity of depressant effects is evident in that bulbar inhibitory pathways appear more depressed than bulbar facilitatory ones in contrast to drugs like chlorpromazine ( Hudson and Domino, 1!36l).Ascending brainstem reticular pathways may also be affected. There is evidence that

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phencyclidine may depress thalamic as well as cortical neurons. The actions of phencyclidine on the hippocampus are complex. It produces depression as well as electrical signs of stimulation. In fact the EEC alterations produced by phencyclidine at various levels of the central nervous system vary considerably. The EEG effects of phencyclidine depend upon the dose administered and the species employed. The dog may show minimal neocortical EEG changes to none in doses comparable to those producing psychotomimetic phenomena in man. With large doses of phencyclidine that produce marked behavioral depression of the animal one can observe 6 - to &wave activity in the neocortical EEG. Prior to the development of slow waves a moderate-voltage fastfrequency EEG can be observed frequently in some leads, particularly in the more frontal and parietal areas. The EEG effects of phencyclidine in the monkey are equally bizarre depending upon the close administered. It is to be emphasized that depression of photic driving responses in the monkeys were usually obtained with doses that exceed those which cause psychotomimetic effects in man. After small doses of either phencyclidine or LSD-25, it frequently appeared that as the animal relaxed, photic driving responses were even improved. It has been found ( Rodin and Luby, personal communication, 1960) that phencyclidine has no consistent effects on photic driving in man. Castaut et aE. (1953) have reported a shift of photic driving responses into nonvisual areas in a number of human subjects taking LSD-25. The effect with LSD-25 in monkeys was not similar in the majority of animals. Only an occasional animal after LSD-25 showed a shift of photic driving responses to nonvisual areas that previously did not show these responses. Chen et al. (1959) reported that phencyclidine caused a marked increase in locomotor activity in the rat. In our studies on conditioned avoidance and escape responses in rats trained to jump to a pole a similar increase in locomotor activity was observed. The rats exhibited a state of drunken excitement. With large doses of phencyclidine this state clearly interfered with both avoidance and escape behavior. Following the onset of the conditioned stimulus ( a buzzer) the animals would jump wildly trying to reach the pole. Similarly when an electric shock was applied to the grid floor, the animals exhibited a comparable pattern of behavior. It was obvious

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that phencyclidine produced considerable confusion although it is unclear whether this was solely due to alteration of visual cues. The experiments of Adey and Dunlop (1960) would suggest that phencyclidine disrupts learned behavior in the cat in part by an action on the hippocampus. In any event, chlorpromazine as well as LSD25 in doses, which in themselves had minimal effects on conditioned avoidance behavior, markedly potentiated phencyclidine-induced suppression of learned behavior. After the administration of chlorpromazine the phencyclidine injected rats seemed less agitated. In spite of a reduction of locomotor activity their conditioned behavior was much worse. The effects of phencyclidine on the mean arterial blood pressure of anesthetized dogs was comparable to that observed in man, that is, the drug caused a slight increase in mean arterial blood pressure. Although the increase in mean arterial blood pressure persisted for approximately 10 minutes after phencyclidine was administered, the pressor responses to epinephrine, norepinephrine, and serotonin were considerably enhanced for as long as 1 hour. Considerable individual animal variation was observed. After phencyclidine some dogs appeared to be primarily catecholamine reactors, showing negligible potentiation of the serotonin pressor response. Other animals appeared to be primarily serotonin reactors, showing negligible enhancement of the catecholamine pressor responses. Still other animals following phencyclidine showed an enhanced pressor response to all of the amines. In pooling all of the data these individual animal variations were lost. It appears that phencyclidine in most animals can enhance the pressor responses to all three agents. These results were analyzed statistically and were found to be highly significant. Chlorpromazine reduced the enhanced epinephrine response as well as the enhanced serotonin pressor response. The responses to norepinephrine frequently were not reduced. On the other hand LSD-25 reduced the phencyclidine-enhanced serotonin response. The significance of the peripheral cardiovascular effects of phencyclidine on catecholamines and serotonin is difficult to evaluate. Probably the peripheral pressor response to phencyclidine administration that is observed in man as well as animals can be explained in part on the basis of potentiation of the effects of these amines. It is of interest that phencyclidine antagonized the hyper-

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thermic action of LSD-25 in rabbits, although this may be a nonspecific antagonism unrelated to a serotonin mechanism. To date, there is no published information to suggest that phencyclidine affects the actions of the various amines on the central nervous system. Such experiments would be interesting in view of the relationship of other psychotomimetic and psychototherapeutic drugs to the central actions of catecholamines and/or serotonin. The rnctabolic effects of phencyclidine may be the result of uncoupling of oxidation from phosphorylation, similar to dinitrophenol, or result from a potentiation of the metabolic actions of catecholamines. In any event, it \!.odd appear that a great deal more work is necessary, particularly using a wide variety of animal species hefore much can be said which is not pure speculation. VII. Summary

The pharmacological actions of phencyclidine appear to be complex. The effects depend upon the species and dose. Evidence is available that in subanesthetic doses the drug alters the reactivity of the central nervous system to various sensory inputs. It would appear the drug acts at spinal cord, brainstem, diencephalic, and cerebral cortical levels. Schizophrenic subjects appear to be quite sensitive to phencyclidine and become much worse, in contrast to their behavior after LSD-25 and mescaline. There is little question that phencyclidine possesses a pharmacological spectrum of activity uniquely different from other psychotomimetic or anesthetic drugs. AC~SVOWLEUCMEXTS

The author would like to thank Drs. G . Chen, R. Fleniing, and D. McCarthy of the Department of Pharmacology, Parke Davis and Company for generous supplies of phencyclidine, and Dr. C . Stevens for making available the summary files and reprints of the Department of Clinical Investigation, Parke Davis and Company Research Laboratories, Ann Arbor, ?\f ichigan. REFERENCES Adey, W. R., and Dunlop, C. W. (1960). J. Pharmacol. Exptl. Therqi. 130, 418. Bakker, C. B., and Amini, F. B. (1961 1. Comp. Psychint. 2, 269. Ban, T. A., Lohrenz, J. J., and Lehmonn, H. E. (1961). Can. Psychiat. Awoc. J . 6, 150. Cerletti, A. (1956). I n “Neuropharmacolog” ( H. A. Abramson, e d . ) , Trans. of 2nd Conf., pp. 9-84.Josiah hlacy, Jr. Foundation, New York.

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Chen, G., and Ensor, C. (1962). Pharmacologist 4,155. Chen, G. M., and Weston, J. K. (1960). Anesthesia and Analgesia 39, 132. Chen, G., Ensor, C., Russell, D., and Bohner, B. (1959). J. Phurmacol. Erptl. Therap. 127, 241. Cohen, B. D., Luby, E. D., Rosenbaum, G., and Gottlieb, J. S. (1960). Cump. Psychiat. 1, 345. Cohen, B. D., Rosenbaum, G., Luby, E. D., and Gottlieb, J. S. (1962). A.M.A. Arch. Gen. Psychiat. 6,395. Cook, L., and Weidley, E. (1957). Ann. N . Y. Acad. Sci. 66, 740. Davies, B. M., and Beech, H. R. (1960). J. Mental Sci. 106, 912. Domino, E. F. (1955).J. Pharmacol. Exptl. Therap. 115,449. Domino, E. F., and Ueki, S . (1959). J. Pharmucol. Exptl. Therap. 127, 288. Domino, E. F., and Ueki, S . (1960). Electroencephalog. and Clin. Neurophysiol. 12, 635. Evarts, E. V. (1957). Ann. N . Y. Acad. Sci. 66,479. Castaut, H., Ferrer, S., Castelis, C., Lesevre, N., and Lushnat, K. (1953). Confinia Neurol. 13, 102. Cershon, S., and Olariu, J. (1960). J . Neuropsychiat. 2,283. Creifenstein, F. E., DeVault, M., Yoshitake, J., and Gajewski, J. E. (1958). Anesthesia and Analgesia 37, 283. Helrich, M., and Atwood, J. M. (1962). Phurmcologist 4,155. Himwich, H. E., Van Meter, W. G., and Owens, H. F. (1958). In “Neuropsychopharmacology” ( P. B. Bradley, ed. ) Elsevier, Amsterdam. Hudson, R. D., and Domino, E. F. (1961). Federation Proc. 20, 307. Johnstone, M., Evans, V., and Baigel, S. (1959). Brit. J. Anaesthesia 31, 433. Lear, E., Suntay, R., Pallin, I. M., and Chiron, A. E. (1959). Anesthesiology 20, 330. Lees, H. ( 1961 ). Federation Proc. 20, 306. Lees, H. (1962). Biochm. P h a m o l . 11, 1115. Levy, L., Cameron, D. E., and Aitken, R. C. B. (1960). Am. J. Psychiat. 116, 843. Luby, E. D., Cohen, B. D., Rosenbaum, G., Gottlieb, J. S., and Kelley, R. (1959). A.M.A. Arch. Neurol. Psychiat. 81, 363. Meyer, J. S., Greifenstein, F., and DeVault, M. (1959). J. Nervous Mental Diseuse 129,54. Morgenstern, F. S . , Beech, H. R., and Davies, B. M. (1962). Psychopharmcologia 3, 193. Ober, R. E., Gwynn, G. W., Chang, T., McCarthy, D. A., and Glazko, A. J. (1963). Federation Proc. 22, 539. Rinkel, M., and Denber, H. C. B., eds. (1958). “Chemical Concepts of Psychosis,” 485 pp. McDowell, Oblensky, New York. Rodin, E. A., Luby, E. D., and Meyer, J. S. (1959). Electroencephalog. and CEin. Neurophysiol. 11, 796. Rosenbaum, G., Cohen, B. D., Luby, E. D., Gottlieb, J. S., and Yelen, D. (1959). A.M.A. Arch. Gen. Psychiat. 1, 651. Van Meter, W. G., Owens, H. F., and Himwich, H. E. (1960). J. Neuropyschiat. 1, 129.

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Ynle University School of Medicine. N e w Haven. Connecticut

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I . Introduction I1. Conditioned Reflexes. Instrumental Responses. and Free Behavior I11. Study of Free Behavior . . . . . . . . . . . . A . Methodology for Recording. Analysis and Quantification of Behavior . . . . . . . . . . . . . . . B. Methodology for Remote-Control Stimulation of the Brain (Electrical and Chemical) . . . . . . . . . . C. Definition and Classification of Behavior . . . . . . IV. Study of Evoked Behavior . . . . . . . . . . . A . Brain Stimulation Triggers Physiological Mechanisms . . B. Interaction between Spontaneous and Evoked Activity . . C . Flexibility of Evoked Behavior . . . . . . . . . D. Fatigability . . . . . . . . . . . . . . E . Lasting Effects . . . . . . . . . . . . . F. Reliability . . . . . . . . . . . . . . G. Variability . . . . . . . . . . . . . . V. Types of Behavior Evoked in Free Situations . . . . . . A. Motor Responses . . . . . . . . . . . . . B. Sequential Behavior . . . . . . . . . . . . C. Offensive-Defensive Behavior . . . . . . . . . . . . . . . . . . . . D . Behavioral Inhibition VI . Fragmental Organization of Behavior . . . . . . . . A . Experimental Data and Theory . . . . . . . . . B. Postulates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vII . Summary References . . . . . . . . . . . . . . .

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349 351 353

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356

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362

363 374 374 376 378 379 380 382 383 385 386 394 395 413 430 430 434 436 438

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I Introduction

Among the methods used to investigate the neurophysiological basis of behavior. perhaps the most direct and dramatic is electrical stimulation of the brain . Results are obtained immediately. are repeatable. and evoked and spontaneous responses often flow in 349

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JOSk M . R. DELCADO

close correlation with each other and with mutual dependence. In the last decade, methods for implantation of electrodes in the brain have multiplied and improved, and already a remarkable body of knowledge has accumulated on the effects of cerebral stimulation on autonomic reactions, conditioning, instrumental responses, selfstimulation, electrical activity and other important aspects (Brazier, 1960, 1961; Jasper et al., 1958; Jasper and Smirnov, 1960; Roe and Simpson, 1958; Sheer, l961a; Simon et uZ., 1961; Wolstenholme and OConnor, 1958). We know that animals may seek or avoid excitation of specific cerebral regions, that direct stimulation of the brain may induce conditioning, may determine learning, and may evoke a wide variety of behavioral effects, such as tameness, ferocity, vocalization, sexual activity, stereotyped walking, changes in social hierarchy, and modification of food intake. Therapeutic implantation of electrodes in humans has made it possible to demonstrate that increases in friendliness, changes in output of words, inhibition of thoughts, hallucinations, fear, dcju vu, memories, hostility, changes in sexual orientation and other effects may be evoked by direct stimulation of the brain (Higgins et aZ., 1956; Ramey and ODoherty, 1960; see also Section \’I of Sheer, 1961a) In spite of this impressive list of evoked phenomena, direct stimulation of the brain must be considered a crude method for the exploration of cerebral functions, and our understanding of the results is rather limited (Teuber, 1961). Reliability of evoked effects has been controversial (Penfield and Welch, 1949; Sherrington, 1947). Recently it was claimed that electrical stimulation produces little that resembles the normal (Cobb, 1961). Most studies have been descriptive without providing quantification of observed evoked effects, and, as stated by Carpenter (1960), “Clearly there is a need to progress from qualitative descriptions . . . to the formation of quantitative expressions which accurately represent behavior (and) social interactions. . , .” Long-term investigations are rare, and fatigability has been explored to only a limited extent. Little is known about interaction between spontaneous and evoked behavior, or about possible brief or permanent modifications of spontaneous behavior induced by brain stimulation. The influence of brain excitation on social behavior is an almost unexplored subject. While observation of total behavior has been the purpose of field studies, which milst be carried out under difficult and sometimes ~

FREE BMAVIOR AND BRAIN STIMULATION

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hazardous conditions, in the laboratory, where investigators may have animal colonies at their disposal day and night, they have chosen to observe only a few patterns of behavior for very short daily periods. In studies of rhesus groups in captivity (Brody and Rosvold, 1952; McDowell, 1953; Rosvold et al., 1954; Mirsky, 1955; Mirsky et al., 1957), observation periods were of only 10-60 minutes, and only one colony was studied for as much as 2 hours daily. The need for a thorough and objective collection of data on all forms of behavior has been mentioned by several authors (Kliiver, 1933; Scott, 1958; Yerkes and Yerkes, 1935). The one author who attempted to record spontaneous normal group behavior ( McDowell, 1953), commented that other investigators had selected particular monkey activities such as mating, and had neglected much of the “total behavior picture.” A series of recent methodological developments has made possible the continuous recording and the quantification of the total spontaneous behavior of animal colonies, providing a base line which may be compared with effects evoked by cerebral stimulation. In addition, the invention of transistors permits the construction of minute and practical instruments for the establishment of wireless two-way contact between animals and investigators, and allows stimulation of the brain in completely free animals. A review of methods, problems, results and possibilities is presented in this paper, which has the following specific aims: (1) to describe methodology for cinemanalysis, telerecording, and telestimulation in order to study free behavior during brain stimulation; (2) to demonstrate that spontaneous activities may be recorded, identified and quantified, allowing the systematic study of free and evoked behavior on both individual and social levels; ( 3 ) to discuss the types and significance of behavior evoked by brain stimulation in unrestrained subjects; and ( 4 ) to present a theory of “fragmental organization of behavior.” II. Conditioned Reflexes, Instrumental Responses, and Free Behavior

Two of the classical techniques for the analysis of cerebralbehavior relations are conditioned reflexes and instrumental responses. As is well known, in conditioning, a normal reaction (e.g., salivary secretion), which responds to a normal stimulus (e.g., the sight of food), may be experimentally elicited by a neutral stimulus

352

JOSE hf. H. DELGADO

(the sound of a bell), if previously bell and food have been repeatedly paired. In the instrumental response, the animal learns a task (e.g., pressing a bar) in order to avoid an unpleasant sensation (e.g., electric shock), or to get a reward (e.g., food). A sensory cue such as a buzzer usually precedes the punishment or reward, and is able to evoke the learned response by itself. Both of these methods yield results which are easy to quantify, and with many variants, they have been basic in our present knowledge of cerebral-behavioral relations. In recent years they have also been used in combination with electrical stimulation of the brain. However, in order to interpret and evaluate the results obtained with conditioning and with iiistrumental responses, the following considerations, which apply to one or both of these techniques, should be kept in mind: ( 1) A selection of experimental subjects is necessary, rejecting those who do not learn, or who have spontaneous behavior unsuitable for the test because they are noisy, destructive, distractible, or too lazy; ( 2 ) Learning of a specific type of response is required; ( 3 ) The test explores one single type of learned behavior; ( 4 ) Usually the performance of the test is a “yes” or “no” proposition and in only a few cases is there a multiple choice. (5) The tests require the performance of acts which, in general, are not found in spontaneous life, such as lifting cups or pressing bars; ( 6 ) The tests are carried on with animals under restraint, or confined in small cages or special chairs; ( 7 ) The tests occupy only a short amount of time from the total span of the animal’s life; and (8) Training and handling of the animals may modify their normal behavioral responses. Another methodological approach for the investigation of cerebral-behavioral relations is the study of the continuous flow of spontaneous activities and the analysis of modifications induced by brain stimulation. This type of study may be called “tusk-free,’’ because no specific performance is demanded of the animals, or “free beIzaeior,” because u7liat the animals choose to do is observed, rather than what they do when confronted with instn-mental t Lsts. I’s::allv the animals are free on a stage or inside a cage in the company of other animals. The term “free behavior” is probably the most descriptive, and will be used throughout this paper with the understanding that in some experiments, electrical stimulation of the brain may evoke a predictable response, leaving little freedom of

FREE BEHAVIOR AND BRAIN STIMULATION

353

choice to the animal. During spontaneous behavior, the animal is reacting to natural external or internal stimuli ( Krushinskii, 1962). Brain stimulation is an experimental variable which may produce an effect by itself (evoked reaction), or may modify the free responses to natural stimuli. Both evoked and free behavior interact with each other (see Section IV, B ) . In the experimentation within behavioral spontaneity lie the merits and handicaps of this type of study. Most of the forementioned limitations inherent in conditioning and instrumental response techniques do not apply to the study of free behavior. It should be emphasized that, since the subjects are not expected to do a special task, there are no entrance requirements, and none are rejected from the experiment. As indicated by Nissen ( 1951), instrumental responses explore “what animals can do”-a specific type of learning, of adaptive behavior, and frequently leave “only one definite way of reaction open to the animal,” ( Katz, 1937), while spontaneous behavior shows what animals normally do in a situation of complete freedom of conduct. Each technique gives different and complementary types of information. Laboratory life admittedly cannot be equated with life in the field, and the codnement of animals in cages may produce behavioral changes (Carpenter, 1942a; Clark and Birch, 1945; Grossack, 1953; Kinnaman, 1902; Scott, 1958; Yerkes and Yerkes, 1935; Zuckerman, 1932). However, the systematic study of brain stimulation would not be possible in the field. According to Lorenz ( 1950), it is easier to investigate behavior patterns in a cage than in the field because the absence of natural surroundings and releasing situations shphfy the study. Maslow (1936) thinks that in the laboratory situation, we have “not so much the introduction of new factors, as the exclusion of many variables and uncontrollable factors.” Ill. Study of Free Behavior

Having the animals under some restraint is advantageous for the study of electrical activity of the brain, autonomic reactions, instnimental responses, and other effects. Restraint, however, imposes obvious handicaps because it inhibits behavior and makes impossible the display of a good number of individual and social activities. Stimulation of the same point may give different results in the animal with or without restraint. For example, in one monkey seated

354

JOSk M. R. DJXGAW

on a chair, excitation of the nucleus medialis dorsalis of the thalamus evoked movement of the head, restlessness, and vocalization; the same stimulation in the animal when free in the colony evoked a reliable sequence with head movement, walking, jumping, hanging on the wall, and walking back to the starting point. Most of the information discussed in the present paper refers to free-moving animals. The different degrees of experimental freedom in neurophysiological studies of awake subjects may be classified as follows: Restrained animals 1. Body and limbs secured to a board. This method is useful mainly for recordings. 2. In a chair or harness. Dogs in Pavlovian experiments or monkeys in chairs (Lilly, 1958a; Mason, 1958). The animal has limited mobility and may feed himself (Fig. 5 ) . 3. On a platform or stage, prevented from escape by a belt or collar and a chain, through which electrical connections may he attached ( Bursten and Delgado, 1958). Unrestrained animals 1. On an enclosed stage, in a small cage or an aquarium. Leads connect the animal with instruments, allowing considerable freedom of movement ( Hess, 1932; Lilly, 1961;Olds, 1960). 2. Completely free-canying its own stimulator or transmitter. Mobility limited by the size of the living quarters. Social studies are possible (Delgado, 1959b, 1962) (Figs. 1, 2, and others). 3. Field studies. The subject is completely free in natural habitat, carrying a harness with instrumentation and contact is established by radio or by programming mechanism (Craighead and Craighead, 1963 ) . In addition to animal studies, information about cerebral stimulation has been obtained from human patients who have electrodes implanted in their brains for diagnostic or therapeutic purposes for days or months (Bickford et al., 1960; Delgado and Hamlin, 1958, 1WO; Heath, 1954; Sem Jacobsen, 1959; Sherwood, 1960; Spiegel and Wycis, 1963; also see Section VI of Sheer, 1961a). These studies have exceptional value because cerebral areas may be explored while the patient is comfortably sitting down in an armchair outside the operating room, and is reading, talking, or engaged in other spontaneous activities.

FFUE BEHAVIOR AND BRAIN SITMULATION

355

In general, animal experiments may be classified in two groups: 1. Direct study of the responses evoked by brain stimulation, which are causally related to the excited cerebral structures. In this case, it is assumed that similar responses observed during spontaneous activity are probably related to similar cerebral structures. This type of study has been very rewarding, and will be reviewed in section V. It should be mentioned, however, that in many investigations, the background of spontaneous activity is considered to be of only secondary importance, or even undesirable, and most of the attention is paid to the phenomena emerging from this background. Individual factors, which are obviously important, as recognized long ago by Pavlov (1957), are seldom taken into consideration. 2. The investigation is focussed precisely on spontaneous activities in order to analyze the cerebral structures which may play a role in each behavioral category. An established colony of animals provides a continuous source of natural stimuli and responses upon which the variables of brain stimulation are assessed, Evoked reactions may be investigated at both individual and social levels. Gestures, facial expressions, or sounds of the animals which may be difficult for the human observer to interpret, are often clarified by studying how other animals of the group react to these communications. In addition, a social situation is evidently necessary for the investigation of social effects. Anthropologists and zoologists have been interested in studying the behavior of groups. Few neurophysiologists, however, have ventured into this field, in spite of the fact that in animals, and certainly in man, some of the most interesting functions and dysfunctions of the brain are related to social activities. There are several reasons for the reserve of many investigators. Perhaps the main one is methodological, because it is difficult to stimulate the brain without restraining and disturbing the experimental subject. Recent developments of methods for biological telemetry and telestimulation, discussed in the following sections, may help to solve these technical handicaps. Other problems are the identification and classification of the great variety of manifestations of spontaneous behavior, the need for objective recording and quantification of behavioral categories and the possible existence of too many variables. These problems are discussed in the following sections.

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JOSk M. R . DELGADO

4. METHODOLOGY FOR RECORDING,ANALYSIS AND QUANTIFICATION OF BEHAVIOR Behavior is expressed through motor manifestations which are difficult to register. The great variability of possible motor patterns within each behavioral category defies the use of recording instruments, and requires a sophisticated analyzer. At present, there is no substitute for the process of human observation. Unfortunately this introduces a factor of distortion dependent on the personal qualities of the investigator, training, interest, fatigue, and other variables. The onIy choices are when and how the human observer is going to look at the animal, and the method he is going to use to register the interpretation of his visual perception. 1. Direct Observation

The usual procedure is to write a protocoI during the observation of the experiment. Efficiency may be increased by scoring the results on special sheets (Rosvold et al., 1954), or by dictating the observations on a tape. Valuable results have been obtained in this way, but these methods would not be suitable for the following types of experiments: ( a ) continuous observation for hours or days; ( b ) timing of brief delays in evoked responses, or the study of fast or complicated phenomena; ( c ) simultaneous analysis of many responses; ( d ) simultaneous studies of individual and social relations in animal colonies; ( e ) investigation of night behavior. In some of these cases, instrumental help may be necessary. To increase the number and aspects of units of behavior to be analyzed, an Aronson keyboard with a bank of microswitches has been used by several authors. As many as 40 items may be scored while continuously viewing the subject (Clark et at., 1954; SchneirIa and Rosenblatt, 1961).The switches may punch a paper tape for automatic analysis of results with an IBM 1620 computer (Tobach et al., 1962). With this instrument, it is also possible to obtain the simultaneous recording of data generated directly by the animal. and interpreted by the observer. This observer-to-computer system, abbreviated to A.T.S.L.,' is an important advance for the quantification of spontaneous behavior and has already proved its usefulness. 'Initials of the originators of the method: Aronson, Tobach, Schneirla, and Larson.

FREE BEHAVIOR AND BRAIN STIMULATION

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However, as it depends on direct observation of the animal, most of the limitations mentioned above still apply. The other alternative is not to study behavior in status nascendi, but to record it by means of cinematography.

2. Cinemamlysis Recordings of spontaneous or evoked behavior by means of moving pictures has been used incidentally by many authors and systematically by a few investigators (Bartorelli and Wyss, 1942; Bianchetti, 1942; Gesell, 1935; Halverson, 1931; Hess, 1932; Schatzmann, 1943). The forerunner of photographic study of behavior was Muybridge, excerpts from whose monumental 11 volumes recording different activities of animals and men have recently been republished ( 1955a, b ) . Depending on the type of experiment, slow motion (64-128 pictures per second, or even faster), normal motion (16-24 pictures per second), or time-lapse photography ( 1 picture every few seconds) have been used. Cinematography of behavior has several advantages over direct observation: ( a ) Records are permanent and objective; ( b ) As the movie camera does not fatigue, recordings may be prolonged for hours, or for days with time-lapse photography; ( c ) Night behavior may be recorded in the absence of visible light with infrared film; ( d ) The experimental time may be compressed or expanded by changing the recording rate and the speed of film projection; ( e ) The same experiment may be repeatedly analyzed by the same or by different observers; ( f ) Simultaneous study of the behavior of several animals, their social relations, and their temporal and spatial factors is possible; ( g ) If new facts are discovered in the course of the investigation, it is feasible to re-examine previous experiments. Cinematography shares some problems with direct observation: ( I ) Identification of behavioral categories requires the personal interpretation of the observer, introducing a possible factor of error; ( 2 ) There is no agreement on the classification of behavioral categories, and different authors use different words and concepts. ( 3 ) Some patterns, such as grooming or mounting, are easy to identify, but the border line between others, such as playing and fighting, or sitting and “balling,” are difficult to define. This technique has some handicaps as compared with direct observation: ( I ) The pictures on the film do not reveal the animals as clearly as when seen di-

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JOSA M. R. DELGADO

rectly; ( 2 ) When time-lapse photography is used, there is a loss of information depending on the rate at which the pictures are taken. Meaningful records of spontaneous daily behavior must be taken for many days or weeks, and thus require the use of time-lapse photography, Perhaps some investigators have refrained from using time-lapse techniques for reasons of economy. The set-up is costly, but the observers’ time is even more expensive, and, as the film may be viewed at different speeds, the unnecessary “prolonged watching during non-active periods‘‘ (Allee, 1938) can be passed over at great speed. Our initial studies were done with a colony of 8 cats ( Alonso de Florida and Delgado, 1958), and since then, more extensive investigations have been carried on in monkey colonies, each studied for several years ( Delgado, 1962b, 1963a). The colony was permanently housed in a special air-conditioned sound-proof room. The colony cage, shown in Fig. 4, measures 7 x 3 x 3 feet. It was illuminated by neon tubes (1.6 units of light measured with a photometer), with a constant 12 hour cycle of “day and night.” The recording equipment consisted of a 16-mm solenoid-activated Bell-and-Howell motion picture camera, using black-and-white Tri-X film, which takes a clear photograph with moderate illumination. Night recording was done with high-speed Kodak infrared film, using photofloods with infrared filter. For a detailed study of night patterns, colored photographs were obtained every 15 minutes with an electronic flash. The analysis of 16,000-20,000 pictures taken daily was facilitated by automation. With the aid of a time-and-motion study projector, the films were projected automatically in bIocks of 10 frames onto a viewing screen. Behavior of each animal was identified by symbols (Table I ) typed with an electric IBM output typewriter connected to a bank of electric counters, which totals the entries for each behavioral unit every 1000 frames ( 3 3 minutes). The protocols for each film (Table 11) consist of typewritten COTumns of symbols recorded during each ten-frame period (20 seconds of behavior), subtotaled every 10oO frames, and totaled for the whole film for each behavioral category and for each animal (Tables I and 111). The time involved in the analysis of each film depended on the purpose of the experiment. For example, if only “mounting” was being considered, the full day of recording for 6 animals was analyzed in less than 1 hour. Investigation of all categories for all

TABLE I BEHAVIOR OF MONKEYNO. 4 ~~

Behavio+

-

STATIC a ) lndivfdual

Mean I

53 I2 7790

0-30 3040-14408

0-255

997

234-2340

5634

2260-8330

2976

16216830

2086

560-5300

180

50-450

3ok

57-589

1539

815-2315

2941

5-38 1000-5780

2313

368-3490

1845

688-2630

0 19

R RUNNINCAWAY

0-210

38

15

Y PLAYING

Range

0-0 6-32

v Avomm

0.8

0-3

Q THREATENING

0.5

0-3

c CRwcHm

0

0-0

X A'ITACKING

2.2

04

x A'ITACKED

2 PREsBNrING M MouNIlNG

I: MOUMgD

-

V

2-70

6.2

2-14

-1800 aecorda

0.1

0-1

(30 min.)

0.1

0-1

a Balling: sleeping alone, Nestling: social sleep of two or more monkeys together. Grooming: digital or oral examination of fur or skin. Presenting: hindquarters raised toward another monkey, tail raised. The other categories are self-explanatory. Cinemanalysis of 10 control days during 4 months of study. Figures represent seconds with the exceptions of categories U,R,V, X, x, Z, M, and K which represent number of occurrences. 359

360

JOSk M. R. DELCADO

~

StJ

so

No.

SO.

?TO.

1

2

3

4

5

1Iorike.i. -____I_.

-

1'1vie 3 IOPSI

I: S

s S S

:3:li

S 05 05 05 05

05 05 4 :OO

No. 6

DPJ DJ HWV5 M6JS JP SJV3 OG 08 06 06 06 06 N6 S6

P P

P

8

Y

S

JFVV6,4 JF JP

S€U

SK2 JS

SFU F

8

S

F F

G1 G1 G1 G1 GI G1 G3 G3 G3 S N2,3,5,6 N2,3,5,6

Y F F F

F

R

?a

05 05 05

I3

36

5.7

13

?r'2,3,5,6 S2,3,5,6

N2,3,5,6 K2,3,5,6

I3 13

I3

JS P

F F F I" S 8

S R E

S

S

E I'J

R3 K2 S S G2 G2 G2 G2 G2 G2 N2 N2 N2 N2 N2,3,5,6 N2,3,5,6

Rehnviord categories rworded during 10 photographs (20 see). Each column represents n mvnkey of the colon)-. Examples arc given of 3 different periods of 2 minutm each. Xumbers represent monkeys pirticipting in social behavior; letters represmt mtegories as explained in Table I. For example: 0 5 of fist cdumn = hI0nke.y No. 1 groomed by Monkey No. 5. DPJ of second column = Monkey No. 2 drinking, picking, and moving hody.

the animals with detailed analysis of temporal, spatial, anatomical, and social factors of a single day took about 1 month. More information and results are explained in Section 111, C and a film explaining methodology and results is available ( Delgado, 196Oa). 3. Telemetry

The expressive or behavioral aspect of emotion has been studied mainIy by measuring autonomic changes such as skin resistance, heart rate, respiration, skin temperature, etc (Dunbar, 1946; Lindsley, 1951) . Attempts at remote recording of neural activity were made several years ago (Breakell et al., 1949; Prast and Blinn, 1949), and new methods have recently been published (Baust et

FREE BEHAVIOR AND BRAIN STIMULATION

361

al., 1961; Filimonov, 1960; Fischler et aZ., 1960, 1961; Morrell, 1959; Shipton, 1960; Sperry et al., 1961). One of the side-products of human interest in the conquest of outer space has been great advances in telemetering techniques, and, as shown at the recent symposium (Slater, 1963), it is possible today to track the ftight of birds (Griffin, 1963; Singer, 1963), to locate grizzly bears (Craighead and Craighead, 1963), to detect the proximity between a baby monkey and its mother (Jensen, 1963), and to record a variety of autonomic responses such as respiration, electrical activity of the heart, and movement of the intestines (Eliassen, 1963; England and Pasamanick, 1961; Fuller, 1950; Geddes, 1963; Holter, 1961; Lord et al., 1962; Mackay, 1961; Smith and Baldwin, 1961; Zworykin, 1957). Interesting results, including clinical data, have already been obtained, but biological telemetry is still in the developmental stage. We may expect that in the immediate future a widespread utilization of constantly improving methods will increase our present meager knowledge of autonomic functions during spontaneous free behavior. The main index of dynamic behavior is the general motor activity of the organism, which has been recorded by several authors using photocells, changes in capacitance induced by animal proximity, triggering of microswitches, and variants of these methods (Cole and Glees, 1957; Davis, 1957; Dews, 1953). These techniques, however, could not detect the individual activity of several animals forming part of a colony, and to solve this problem the author has developed one instrument in collaboration with Dr. J. A. J, Stolwijk ( Delgado, 1963b) consisting of a frequency modulated transmitter, which has a drop of mercury as an inertia sensory device. The transmitter is activated only when the animal moves, and in this way the life of the battery is about 6 months. The instrument is carried on a belt by the monkeys. Each animal has a different frequency which is picked up by a separate FM receiver. The multiplex output of the tuner is connected with a special amplifier to activate electric counters and event recorders. Mobility is analyzed in this way at both individual and social levels. Simultaneous telerecording of movements and time-lapse photography permits the correlation of behavioral categories with animal mobility. In this way, we have established the basis for auto-

362

JOSk M. R. DELGADO

matic recording and quantification of individual and social periods of rest and activity, which has been useful for behavioral and psychopharmacological investigations ( Delgado, 1963a). B. METHODOLOGY FOR REMOTE-CONTROL STIMULATION OF THE BRAIN( E L E ~ C AAND L CHEMICAL) Connecting leads between the brain and the stimulator are used without much difficulty in behavioral studies carried out on lower animals, such as the rat (Olds, 1958), and the cat (Hess, 1932). Monkeys are more skillful and destructive, and stimulation of their brains requires that connecting leads be heavily protected, or what is better, that wireless methods be used (Fig. 4). Remote-control techniques for cerebral stimulation have been described by several authors (Chaffee and Light, 1934-1935; Clark and Ward, 1937; Delgado, 1959b; Fender, 1937; Gengerelli, 1961; Gengerelli and Kallejian, 1950; Greer and Riggle, 1957; Harris, 1946-1947; Loucks, 1934; Mauro et al., 1950, and Verzeano and French, 1953). These techniques, which we have reviewed elsewhere (Delgado, 1959b, 1961) have the following handicaps: in general, only one point of the brain may be excited; direct monitoring of stimulation parameters is not possible; the electrical activity of the brain cannot be recorded; and the intensity of the stimulation depends on the signal received, which may vary with changes in the orientation of the receiving antenna. In our opinion, it is preferable to implant an array of intracerebral electrodes having external sockets, according to some of the available techniques (Hess, 1932; Sheatz, 1961; see other bibliography in Delgado, 1961) . In this way many cerebral points are available and electrical recordings and monitoring of stimulation are possible with animals under some restraint. Later any selected contact of the external socket can be connected by means of subcutaneous leads (Delgado, 1963b) with the stimulator, which is carried by the animal around its neck, or on a harness on its back. We have described two types of remote-control stimulator. One ( Delgado, 1959b) has a programming mechanism constructed in a modified wrist watch, which activates a miniature two-transistor multivibrator, as shown in several figures of this paper (Figs. 1, 2, and others). The physical characteristics of the stimulation are exponentially descending pulses: 20-100 cps; 0.2.1 msec of pulse duration; 0-22 volts. In general, cathodal monopolar stimulations are used, but bipolar may also be employed. Programming and in-

FREE BEHAVIOR AND BRAIN STIMULATION

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tensity of the stimulations are preset before each experiment. The usual schedule is to stimulate for 5 seconds once every minute for 1 hour, starting and ending at a predetermined time. The other instrument activates a miniature multivibrator by means of a superheterodyne receiver which has 4 stages of amplification with 2 pv sensitivity, and which measures 1x 2 x 3 inches and weights 3.75 ounces (Delgado, 1963b).The receiver operates in the 27-Mc band, and is crystal tuned to avoid drift and to facilitate the use of several channels for the same or for different animals. As the only purpose of the receiver is to close a circuit to activate the stimulator, the intensity of excitation is independent of the intensity of the received signal, and reliability of performance is in this way insured. One advantage is that the instrument is constructed of commercially available units, which are inexpensive and reliable. For chemical injections the same receiver activates a “chemitrode pump,” which has two compartments of about 2-ml capacity, separated from each other by an elastic membrane. One side contains the substance to be injected into the brain, and the other contains a solution of hydrazine and two platinum electrodes. Gas, produced by passing a current through the hydrazine compartment, is the driving force which pushes the elastic membrane, injecting the drug into the brain at a rate adjustable between 0.1 ml per minute to 0.2 ml per day or less. The intracerebral injections are made through “chemitrodes” permanently implanted in the skull. The chemitrode is an assembly of two very fine tubings and two electrical contacts, which permits the injection and collection of liquids to and from the brain, and also permits electrical stimulation or recording of the same area (Delgado et al., 1962; Hoebel and Teitelbaum, 1962). Other simpler techniques for chemical stimulation of the brain by remote control have been described by Delgado (1955b), by Olds and Olds (1958), and by Cordeau (1962). Methods for intracerebral injections of chemicals in restrained animals have been developed by several authors (Feldberg and Sherwood, 1953; Gaddum, 1961; Haley and McCormick, 1957; Kogan, 1956, MacLean, 1957). C. DEFINITION AND CLASSIFICATION OF BEHAVIOR The study of free behavior is possible because of the recurrence of typical patterns of responses to naturally occurring stimuli, which can be observed, recorded identified, and isolated in time and space.

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JOSk M. R. DELGADO

In general, responses are highly complex, involving muscles and glands in a determined sequence, and are accompanied by biochemical, histological, physiological, psychological and, in the case of groups, sociological phenomena. The ideal of simultaneous studies of all levels of reaction is unattainable, and the investigator must concentrate on only a few of many aspects. Behavior in general may be identified by postural and motor patterns, and by autonomic reactions. Behavioral categories may be defined objectively by anatomical descriptions of postures and movements, without prejudging their functional meaning, The gain in objectivity, however, does not justify the necessary length of the description, which, in some cases, could add to the confusion. For example, grooming in the monkey may be done with the hand or with the mouth, licking, biting, picking or pawing different parts of the body. It is more practical, therefore, to utilize the usual behavioral terminology providing that we are aware of its limitations. In the study of behavior, the following aspects may be considered: 1. Behnuimal Cuteguries Continuously occurring behavior must be broken down by the investigator in order to classify and to quantify its manifestations. Each behavioral unit should describe the observed reality in some clear and simple way which could be used by many investigators. Recognition of these units presupposes an interpretation by the observer, which is the greatest weakness in behavioral studies because of the introduction of a factor of personal distortion. Possible variability of interpretation may be determined experimentally by comparing the results of different investigators who analyze the same data. For this purpose, cinematographic recording of behavior offers great advantages. A survey of the literature reveals a great variety in the criteria of different authors for classification of behavior. Discussing the axis of behavioral comparisons, Nissen ( 1958) grouped the categories into three classes: ( a ) ficnctionul or finalistic, which indicates biological utility of behavior (food getting); ( b ) descriptive, which emphasizes the mechanisms by which a purpose is achieved (locomotion); and ( c ) exphnutory, which may be philogenetic, physiological, or may try to establish general laws. Behavioral rating scales and lists of definitions of behavioral

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categories based on observations and on experimental studies have been published by several authors. Carpenter ( 1934, 1940, 1942), in his pioneer series of field studies, described the main patterns of individual and social behavior of several species of monkeys, giving basic data on sexual behavior, dominance, grooming, vocalization, play, eating, and so forth, but he made no attempt to classify systematically the daily activity. Behavioral patterns observed during free life of monkeys in their natural habitat have been carefully described by Imanishi (1960). In these studies, which have been prolonged up to 7 years with one group, the animals are attracted to a feeding area where they can be studied in detail. Special mention should be made of the study of a rhesus colony on Monkey Hill (Chance, 1956), and of a recent paper by Hinde and Rowel1 ( 1962), which includes many excellent descriptions and photographs of different types of behavior. Kaufman and Rosenblum (personal communication, 1962), who are studying large colonies of bonnet and pigtail macaques, have devised a list of 54 behavioral categories which is both precise and comprehensive, and should yield important results when coordinated with computer analysis techniques. In most laboratories, research has been oriented towards analysis of specsc activities, such as dominance, sex, or grooming, and few attempts have been made to classify and quantify the daily activities of animal life. Dominance has been studied by Maslow and Flanzbaum ( 1936), who observed pairs of monkeys for periods of 20 minutes, during which the animals were scored in 17 different, carefully defined categories, such as feeding, genital inspection, presentation, dorsiventral mounting, ventroventral copulatory behavior, attempts to mount, and so forth. Warden and Galt (1943) used a scale of 4 stages of dominance, namely, zero, slight, strong, and complete. Infant monkey behavior and mother-infant relations with surrogate mothers has been extensively investigated by Harlow (1960). In the study of the iduence of amygdalectomy on social behavior of the rhesus monkey, Rosvold et al. (1954) evaluated dominance and aggression according to a series of acts, principally related to food-getting. Mirsky (1955) analyzed the influence of sexual hormones on social behavior of the rhesus monkey during peanut tests. The observed categories included dominant acts, peanut-getting acts, and subordinate acts. Hebb (1949) studied the

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behavior of the chimpanzee towards a familiar caretaker, a disguised “timid man, a disguised “angry” man, and to different objects. In his detailed study of companion preference and dominance in chimpanzees, Nowlis ( 1941) classified behavior in 17 different categories which form 3 groups: social behavior, contact or noncontact social behavior, and aggressive or nonaggressive behavior. The broadest classification has been made by Scott (1958), which could be applied to different species of animals, and which included the following categories: (1) ingestive, (2) shelter-seeking, ( 3 ) agonistic, ( 4 ) sexual, (5) care-giving, ( 6 ) care-soliciting, ( 7 ) eliminative, (8) allelomimetic, and ( 9 ) investigative behavior. In an attempt to combine the behavioral categories of Scott and the other systems described by Bales (1951) for small human groups, Thompson (1958) divided responses into two main categories: “positive and negative, depending on whether they make for more or less group unity.” “Allelomimetic,” “epimeletic,” (care giving ) “et-epimeletic,” (care soliciting) and “sexual” behavior of Scott’s classification are considered positive factors because they tend to increase the unity of the group, while fighting and investigation or exploration tend to increase the dispersion of the group, and would be negative factors. One of the few studies to quantify spontaneous behavior has been done by Norton and de Beer (1956) in the cat. Four categories were considered: sociability, contentment, excitement, and hostility, and each one was rated according to a group of acts. Sociability, for example, included “jumps up, mewing, tail up, comes forward and alert.” The multiplicity of classifications reflects the different interests of the investigators, the difficulties of the subject, and also the need of a general theoretical basis (Rashevsky, 1951; Rodriguez Delgado and Delgado, 1962; Russell et d., 1954). After several years of observation and analysis of Macacus rheas colonies we developed the classification of behavioral categories shown in Table I. Terminology and description followed as closely as possible the existing literature ( Zuckerman, 1932; Carpenter, 1942a, b; Hines, 1942; Lmanishi, 1960; Hinde and Rowell, 1962). We chose descriptive rather than psychological terms in order to establish a distinction between the observed reality and its interpretation. Mounting, for example, is considered only as a typical pattern, easy to describe, to recognize, and to quantify. Later, each mounting may be considered

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in its particular context to determine whether it occurred as an expression of sexual appetite, playful behavior, dominance, or something else. In our classification, behavior is divided as follows: ( a ) Static m postural units which are defined and identihd by anatomical relations of the different parts of the animal’s body in relation to itself, i.e., “balling” during sleep. This type of category can be recognized by one single photograph because it does not involve movement. ( b ) Dynamic units can be identified and defined by a temporospatial sequence of changing anatomical relations, i.e., the animal climbing the wall of its cage. Recording of these units requires a series of photographs with time intervals related to the duration and the speed of the spatial change. Both static and dynamic units are considered at ( a ) individual and ( b ) social levels and each group is divided into specific categories, as shown in Table I. Definitions and discussion of our classification and its application to other studies may be found elsewhere (Delgado, 1962,1963a).

2. Units of Measurement After behavioral units are identified, the time and the place of their occurrence may be measured. This process is based on logical and mathematical techniques and requires the establishment and the definition of the corresponding units of measurement. Mathematical expression of behavior offers the possibility of statistical analysis and the advantage of using precise figures which may be compared in different experimental conditions or in different animals, as shown in Tables I and 111. At the same time, it creates the risk of distortion, because heterogeneous qualities of behavior may be included in the same figures. Mathematical expression also may give a false sensation of accuracy to a process of measurement which depends on the previous identification of categories by the observer. Evaluation of the data and the establishment of correct criteria for behavioral homogeniety are, therefore, of paramount importance. a. Temporal Units. Time is one of the basic and irreversible coordinates of behavior. Both static and dynamic categories develop through time. Temporal analysis permits the knowledge of the duration of each category, its rate of repetition, its distribution through the experimental period, and how each subject distributes its avail-

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able time among different patterns of behavior. Some investigators have used a metronome or a stop-watch to investigate the duration and sequence of behavioral categories. Results, however, are more accurate with the use of cinematography. In this case, the movie camera is a mechanical observer, which makes a permanent record of the behavior (one photograph) during each fixed period of time (photographic interval). Different intervals of observation permit the study of different aspects of behavior. M7ith a slow-motion camera, Hess (1957) has been able to measure the latency of motor response to cortical stimulation, which is about one-fortieth of a second, but this requires the taking of 128 frames per second, and 100 feet of film are used every half minute. For long-term studies of daily behavior, time-lapse photography may be used. The proper photographic interval must be determined for each type of experiment. \\'hen the time scale is compressed by taking one picture every few seconds, the resting periods of the 'animals may be analyzed at great speed, but data on short-lasting activities may be lost. Experimentally, we have seen that the amount of information recorded on film taken at 24 frames per second is superior to that gained by direct observation, mainly because the film may be slowed down, and also may be reviewed several times. In our studies of monkey colonies, after comparing results obtained from movies at 24 frames per second with the information obtained from timelapse photography taken e v e n 1, 2, 3, and 5 seconds, a two-second interval between frames was chosen as the best compromise. At this speed, five frames are equal to 10 seconds of time, and more than 8 hours can be covered with the regular four-hundred-foot roll of film. During the night the monkeys' activity diminishes, and infrared pictures taken at the rate of one ever!. 10 seconds proved to be suitable. b. Spatial Units. Behavioral responses develop in the three-dimensional space that forms the sphere of action of each animal. Spatial units can be easily identified by using a three-dimensional grill plotting each space by means of letters and numbers, as in playing chess. Studying the correlation between behavior and space, we can consider: Location of each subject during the performance of each behavioral unit, for example, an animal sleeping in the right rear corner of the cage. Disphceinerzt through space, for example, a monkey walking from Point A to Point B. Combining temporal

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and spatial units, we may determine the rate of change, and the duration of the periods. For example, a monkey may walk between Points A and B at a speed of 4 meters/second, or an animal may hang from the left side wall for 2 minutes, In addition, territoriality and spatial relations among the members of a colony may be determined. For example, the boss monkey usually occupies more than 50%of the cage area, with the other 5 members of the colony crowding into the rest. Space has great significance and importance in the performance of behavior, and some behavioral categories are recorded with an enormous preference for certain locations in the cage. For example, all the monkeys sleep 80% of the time in the right and left rear corners of the cage. 3. Evaluation of Data

The analysis of each behavioral category requires the evaluation of its anatomical, temporal, and spatial characteristics, and a correlation of the collected data. The criterion for sorting homogeneous information depends on the purpose of the research. Cats and monkeys are homogeneous considered as mammals, but heterogeneous as “primates.” Balling and nestling in monkeys are homogeneous as sleep patterns, but are heterogeneous as social categories. The details of data analysis also depend on the type of research. In some cases, we analyzed the time and the sequence of grooming for each part of the body, but in most cases, all kinds of grooming were grouped together as one unit. One of the advantages of the permanent photographic recording of behavior is that it is possible to select new data from previous experiments-something which cannot be done with the usual observation techniques. In general, the following aspects were analyzed in our experiments: (1) Diflment patterns included in each behavioral unit, for example, drinking may be accomplished by taking water with the hand, with the tongue, by lapping or by sucking. Different strategies for the “making” (beginning) and “breaking (ending) of each category were studied. ( 2 ) Duration of each period. Some categories, e.g., attacking, lasted only a few seconds, while others, e.g., nestling, lasted without interruption for many minutes. The mean durations and the ranges for lying, balling, and nestling in 6 different monkeys are shown in Table 111. ( 3 ) Number of periods. Each category appeared a variable number of times each day. For example, Monkey

TABLE I11 DAILYBEHAVIOR OF A COLONY OF 6 MONKYYY .

hlo11key: -

KO.

-

1,yirig Number Duration Total timi. --

0 3

1.3

0-2 73 10-100

0-4 34 10-130 44 0-190

22

Total time -

-

__.

Uuratiori

-____-

Nestling Number lhratiori

Total timcz

No. 4

- - - I - - I - - - _ - _ _ _

(1-100

Balliiig Number

No. 3

No. 2

I

7 7 1-18 84

2 i 0-7 20 10-70

53 0410

__

-

10-140

-

220 0-800 _ - _ -

xcr

(;

4 i 0 20 ii1 10-480 1 F, I 0-480

--__-I_____________

0 6 0-2 110 10-240 cti 0-240 -

1.5 0-4 35 10-180 252 0-140

- _

so.5

4.5 1-8 30 10-140 134 30-340

~---__.I-__

10 6-16 542 60-3180 5422 4200-9020

17 8-32 285 10-24 12 4850 2184-7600

0.7 0-5 120 10-2 1 0 360 0-1550 __

20-30 12 0-30 -

8 5-21 485 10-2120 3880 1560-7100

1.8 0-5 25 10-50 45 0-80

0.4 0-2 30

-

. _

20 4 4 i

258 10-2412 5634 2260-8330

- -

_-__ 18 7-Si 282 10-2412 4638 2770-6490

7 7 2-27 56 10-430 428 60 1210

__

--

18 11-27 300 10-2220 4988 2310-7480

a Data from 10 control days during 4 months of study for the categories of lying, balling, and nestling. Number of occurrences, duration of each occurrence, and total time arc shown for each category. The first figure represents the mean, the other two figures separated by a dash represent the range.

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no. 8 of our colony had, each day, 2-8 long periods of balling (10-26 minutes each), and up to 20 shorter periods of balling (less than 2 minutes each). (See also Table 111). ( 4 ) Total daily time. Multiplying the duration of each period by the total number of periods, the total time for each behavioral category was obtained. This data showed how a monkey spent his day, as well as the relationship between all categories (Table I ) . (5) Distribution through time. Some categories, such as walking, appeared rather uniformly distributed throughout the day, while others, such as nestling, were concentrated at midmorning and midafternoon. (6) Distribution in space. There is great asymmetry in the spatial distribution of determined types of behavior. For example, Monkey no. 5 sat in the right rear center space more often than in any other area of the cage. ( 7 ) Social spread. Grooming, nestling, and eating were generally observed simultaneously in several members of the colony, while yawning or hanging usually appeared in only one single animal. The performance of some behavioral categories by some animals seems to induce others to do the same. (8) Behavioral correlations. Some categories seem to have a special relationship, often preceding or following each other: for example, grooming, nestling, and mounting. Some relations, such as in grooming, were reciprocal, with alternation of the active and passive roles. (9) Social contact. Likes and dislikes were shown by the amount of contact or avoidance in social Categories. In one colony of 6 monkeys, combinations of some pairs were never observed (Monkey nos. 2 and 7, 4 and 6, and 4 and 7 ) . Other combinations of pairs were recorded in less than 10%of the time, and 4 other pairs (Monkey nos. 7 and 8,4and $ 2 and 6, and 2 and 4 ) predominated to a great degree. Monkey no. 8 nestled with Monkey no. 7, but not with any other member of the colony. The cluster of monkey nos. 2, 4, 5, 6, and 7 was the most usual combination, being observed 86%of the time. ( 1 0 ) Night behauisr. Most of the night the colony slept, forming one or two clusters. Sleep was interrupted by several periods of general movement, including walking, picking, climbing and hanging. Fighting, threatening, presenting, and mounting were never observed (Table IV) . All these aspects of the categories determined the behavioral profile of each monkey and had a determined range of variability. Results obtained beyond this range during the introduction of ex-

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JOSk X I . R. DELGADO

TABLE IV 6

S I G H T fiEHAVlOR I N COLONY OF

Time

Nestling, 1 group --

.

>lONKEYS'

Nestling, 2 groups General movement

~~_.______

7:30 A.hI. 9:00 A.X.

88 10

_.

8

TI __

-.

._

25)

-

1.5 2

104 -.

1 :00 & l . l I .

__ __ -.

5 . .

119

.-. .

28

-_ 5:00 A.31.

2

-

-.

3 : o o A.M.

-

_.

-.

11 :00 P.M.

- ~.

.-

-

9 ._.

._

1

~ ~ . .

-

.. .

2.5

15 -

.-.

75 -

-

If6

-.

595

95

4 -

i:00 B.XI.

TOTAL

30

Sight behavior expressed in minutes. During the night the monkeys emtiraw each other, forming one or two clusters broken up by short periods of 0

activity. Fighting, threatening, presenting, and mounting were never observed.

perimental variables such as brain stimulation or the administration of drugs could be considered significant.

4. Range of Variabilitg While short samples do yield spec& results in some special types of studies, as when a male is presented with food or with a new female, these single-purpose experiments should be differentiated from studies of an established colony of animals in which short daily samples of, for example, 10-20 minutes would reveal random behavior of no predictable type. Naturally, the longer the sample, the more the rhythm of group life is revealed, and approximate periods of play, feeding and resting become clear. The ideal of a continuous day and night recording of behavior is feasible

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with the use of time-lapse photography. In our studies of monkey colonies, eight hours of daily recording proved to be practical. Table I shows the mean and the range for 10 control days during periods of 4 months for all behavioral categories of one typical monkey. Table I11 shows the mean and the range taken during the same control days for 6 different monkeys, expressing the number of occurrences, the duration of each occurrence, and the total time of 3 different behavioral categories. These tables are presented to exemplify the range of variability in each individual monkey and also in different monkeys. Studies of other behavioral categories may be found elsewhere ( Delgado, 1962). Every day all monkeys groom, walk around the cage, sleep, pick, and sit. Every day, balling time was low in some animals (monkey no. 4 ) , and high in other cases (monkey no. 8). Hanging time differed greatly from monkey no. 6 to monkey no. 1. Monkeys nos. 1, 2, 3, 4, and 7 showed a lying time below 4 minutes daily. Monkey nos. 3 and 4 spent a small amount of time balling. A small increase in the lying or balling time in these animals was, therefore, significant. Monkey no. 8 showed a different distribution of behavior from the other animals because its lying and balling time were high, its nestling time was low, and its active grooming was below the average of the rest of the colony. The behavioral category of yawning, which was easy to quantify, was present in the males, and nearly absent in the females, It was completely inhibited by reserpine, chlorpomazine and pentobarbital, and slightly increased by iproniazid. Social relations showed clear preferences. Monkey no. 1, for example, often had grooming relations with monkey no. 2 and no. 3; less frequently with monkey no. 4 and no. 6, and never with monkey no. 5. Mounting also showed individual characteristics and preferences. The day a new female (no. 7 ) was introduced into the colony, the number of mountings by monkey no. 3 increased from 4.5 to 124. Removal of Monkey no. 3 (the boss) from this colony increased the mountings of monkey no. 4 (the other male) from 1 per day up to 33. The predictable occurrence of behavioral categories within a determined range has also been demonstrated by Norton and de Beer (1956) in the cat. A stable quantitative base of social interaction was established, and was used to assay the effects of several pharmacological agents. Reliability of social behavior in the cat

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was also established in the studies of Alonso de Florida and Delgado (1958), in which amygdala stimulation increased playful and contactual activities with significance calculated by ranking procedures to P < 0.05. It is beyond the scope of this review to analyze in detail our experimental data (Delgado, 1962, 1963a; Rodriguez Delgado and Delgado, 1962), but we would like to demonstrate by presenting part of this material, that it is possible to classify spontaneous behavior, to quantify its occurrence, and to analyze each behavioral category, determining precisely the following characteristics: number of periods, duration, total daily time, distribution in time and in space, social spread, behavioral correlation, and the social relations with individual likes and dislikes. In summary: Spontaneous behavior may be objectively recorded, analyzed in detail, and quantified in seconds, in fulfillment of hopes expressed some time ago by Skinner ( 1938). IV. Study of Evoked Behavior

In free behavior studies, the variable of brain stimulation is introduced during the continuous flowing of spontaneous activities. The observed effects, therefore, contain factors of both spontaneous and evoked reactions. Before reviewing the specific types of evoked behavior, it is advantageous to discuss the functional significance of brain stimulations. How phq siological-or antiphysiological-are they? How reliable? How adaptable? How fatigable? Can they interact with free behavior? A.

BRAIN STIMULATION

TRIGGERS PHYSIOI.OCICAL MECHANISMS

Several authors doubt that electrical stimulation of the brain should be considered physiological. Anderson et al. (1958), thinks that it is difficult to compare normal behavior with evoked effects, mentioning the complications of operative trauma, experimental conditions, and the nonspecificity of electrical stimulation. Hess (1957) has pointed out that “. . . electrical stimulus, unlike physiological excitation, unselectively affects all elements of a similar threshold that fie within the radius of action of the electrodes.” Cobb ( 1961) considers the greatest oversimplification the belief “among those not educated in physiology, that the electrical stimulation of a nerve or brain center, closely resembles normal neuronal

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stimulation. Electrical stimulation, however, produces little that resembles the normal.” Terzuolo and Adey (1960) state that in the majority of cases, cortical stimulation “has failed to elicit anything but fragments of skilled movements.” It is reasonable to question the normality of a brain implanted with electrodes, and also to doubt that electrical discharges of perhaps several volts applied to one point can be compared with the finesse and complexity of a large number of convergent and divergent connections with multisynaptic circuits, relays, and delays, working at the millivolt level. Most investigators would agree that many responses in animals and also in humans are simple contractions of a small group of muscles-certainly very far from the harmonious coordination of voluntary movements. In these cases, we must accept the fact that brain stimulation has not produced physiological effects, but only fragments of them (see Section VI). There is, however, a great variety of evoked responses in animals ( such as circling movements, yawning, sequential activity, aggressive behavior, sleep) and in humans (such as increase in verbalization) which are very similar to spontaneous manifestations (see Section V ) . To interpret the mechanism of action of electrical stimulation, we must consider that present procedures are still very crude, and therefore could not be expected to obtain results even remotely comparable to those of spontaneous activity unless physiological mechanisms were triggered by the stimulation and were the real cause of the evoked effect. A simple motor act, like the flexion of a limb, requires the sending of motor impulses to many muscles, the processing of propioceptive information from many regions, the adjustment of servomechanisms and the activation of many other phenomena. If a stimulation can activate a group of neurons, produce salivary secretion, or start muscular contractions, we cannot hold the applied electricity responsible for the complicated sequence of electrical, chemical, thermal, and mechanical changes involved in these reactions. All these mechanisms are pre-established in the organism, and the applied electrical excitation is only the depolarizing trigger initiating a process which, once started, is independent of the trigger. Evoked behavior is like a chain reaction in which the final result depends more on the structure of the components than on the trigger. Electrical stimulation cannot “create” behavior but can only activate pre-existing patterns.

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JOSk M. R. DELGADO

In sun~nmy,the author considers the electrical stimulation only as a trigger of physiological mechanisms which may be more or less complex, depending on the stimulated area of the brain. If this is true, the evoked behavior must have the qualities of the spontaneous reactions, and both evoked and spontaneous acts may be able to interact with each other. B. I N T E R ~ C T BETWEEX I O ~ SPOSTAXEOUS AXD EJ~OKED ACTIVITY The position of the head and the posture of the body are continuously regulated by proprioceptive and vestibular stimuli, and in any movement, voluntary or induced, this system of stabilization is activated, and is in part responsible for the performance of motor behavior, As described by Hess (1957), excitation of the subthalamus in the cat evoked rotation of the head in the frontal plane. If the frequency of the stimulating pulses is slow ( S cps), and the intensity is small, it is possible to see corrective movements between the pulses, striving to bring the head back to its normal position. \Vith stronger stimuli, there are no corrective movements, and the body follows the rotation of the head. In this case, when the animal rotates until it is on its back, there is one quick jerk, and the animal springs to its feet. The reason is that, “when the animal is on its back, the artificial and natural vestibular and proprioceptive stimuli no longer compete and summate to produce a similarly directed movement around the longitudinal axis” ( Hess, 1957). During conditioning esperiments, cats often try to suppress motor movements evoked by cerebral stimulation, and are “actually capable in many cases of suppressing it” (Grastyh et al., 1956). Also in the cat, an algebraic summation between voluntary and evoked movements has been described ( Delgado, 195%). In several experiments, electrical stimulation of the sulcus presilvius evoked lifting of the contralateral foreleg. \Vhen pieces of fish were offered from a proper angle, a Finiilar movement was made by the animal in the attempt to take the food with its paw. Then the fish was offered to the cat simultaneously with stimulation of its brain, resulting in an overshooting, with greater amplitude of movement of the foreleg than that observed in either case alone, and with the animal being unable to reach the food until a series of trial-and-error corrective movements were made. In other experiments, the same area was stimulated while the cat was airborne in a voluntary jump from the observation

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stage down to the floor. The result depended on the intensity of the stimulation. At threshold levels, or slightly above, the evoked motor effect was completely inhibited by the voluntary jump, and the animal landed with perfect coordination of movement. When intensities of 2-3 times that of threshold were employed, the coordination of the voluntary jump was disrupted. The forelimb was improperly used and the animal fell on the floor. Similar phenomena have been observed in monkeys stimulated by radio control in the colony. Near-threshold stimulation of the centrum medianum which produced ipsilateral turning of the head in the horizontal plane, showed effects which were increased or decreased if stimultaneously a noise was made at either side of the animal. The response was completely inhibited if the animal was threatened by the observer or by another monkey. In other experiments, also on monkeys, chewing was evoked by stimulation of the rostral amygdala (100 cps, 1 msec pulse duration, 0.5 mA). A similar pattern of chewing was observed while the animal was eating a piece of apple. If the amygdala was stimulated at this moment, the speed of chewing (number of movements per unit of time) was increased 2040%. If food was offered during electrically evoked chewing, the animal was able to pause in order to open its mouth and bite the food, which was then chewed with a behavorial pattern indistinguishable from normal. Another instance of coordination between spontaneous and evoked behavior occurred when the inferior part of the sulcus presylvius was stimulated in the cat. Licking was consistently evoked and the animal looked for some application of the evoked effect, licking the floor, our hands, a piece of food, or its own fur (Delgado, 1952b). The act of licking was reliable: its pattern, its purpose, and the postural adaptation of the animal varied according to the experimental surroundings and the will of the animal. Another example is shown in Fig. 1. In some cases brain stimulation did not evoke a determined effect, but only modified the reaction of the organism to normallyoccurring stimuli, producing a change in behavioral tuning (see Section V ) . If hunger was increased, the pattern of eating was not modified; only the amount of food consumed was augmented. If rage was evoked, the pattern and direction of the attacks depended on the location and reactions of other animals. As mentioned by von

378

JOSk 35. H. DELGADO

Holst and von Saint Paul (1962) in their studies on roosters, evoked aggressiveness was manifested by attacking other birds, while if the animal was alone, the only observable effect was motor restlessness. This unspecific reaction was also observed when other areas of the brain, associated with hunger, thirst, or courship, were stimulated in environments which did not permit expression of the complete sequence of behavior. In srcmmury: Voluntary and evoked movements may interact with each other, having algebraic summation. Complete mutual inhibition is possible, depending on the relative strength of the corresponding stimuli. In some cases the only effect of the cerebral stimulation is to modify the response of the organism to normally owurring stimuli.

C. FLEXIBILITY OF E\-OKED BEHAWOH One of the characteristics ot spontaneous activity is the ability to adapt its performance to unforeseen circumstances in order to reach a goal. This requires a processing of sensory information, feedback mechanisms, and also a capacity for instantaneous readjustment of the central commanding organization ( Paillard, 1960; Ruch, 1951). Many of the simple motor responses evoked by brain stimulation (see Section 1 7 , A ) are stereotyped and are little affected by changes in the experimental situation. In contrast, many other eboked responses show considerable flexibility of performance. The inhibition of evoked effects during a threatening situation, is an example of adaptation, but more direct evidence can be presented. As shown in the cat (Delgado, 1952b), stimulation of the middle part of the left sulcus presilvius produced a turning of the head to the right, following in general a horizontal plane. Placing obstacles, such as a hand or a book, in the path of the evoked movement, induced the animal to modify its performance by raising its head in order to avoid the obstacle, and then continuing the movement of head turning to the right. Flexibility and adaptation were also clearly demonstrated when aggressiveness was increased in cats and in monkeys by cerebral stimulation (Figs. 2 and 4). The offensive pattern depended on the relation between the stimulated animal and the rest of the members of the colony. In general, the aggressiveness was directed against a particular animal, and the motor pattern was continuously being adapted to the unpredictable jumps and

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changes in the position of the animal pursued. It was evident that in this case the cerebral stimulation did not evoke a predetermined motor performance, but a motivation to attack, which was served by the motor skill already existing in the animal. In monkeys alone or under restraint, central gray and thalamic stimulation were manifested mainly as restlessness. I n summary: Some evoked simple motor responses are stereotyped, while other types of evoked behavioral reactions show flexibility and adaption to different experimental situations.

D. FATIGABILITY Different postures of the body may be maintained for long periods of time, and voluntary motor activity may be continued for minutes or hours without fatigue, as shown by the flight of birds, and by the succession of behavioral activities of daily life. On the other hand, it is well known that electrical stimulation of area 4 evokes a motor response that fatigues in a few seconds, and no further response can be elicited until the cortex regains its initial excitability after a recovery period. Brain fatigability has been little studied, in spite of its great interest, and we do not know why the motor cortex ceases to respond. The quick fatigability of area 4 is an experimental factor which could be interpreted as an indication that its electrical stimulation cannot be compared with its normal physiological activation. Another interpretation is that the motor cortex is perhaps not responsible for the onset and organization of voluntary movement, being only one of severaI substations of the efferent motor command. In this case, it would be natural that electrical stimulation of the motor cortex activates only a fragment of the motor response, lacking important supporting integration for the persistance of the motion. More experimentation is needed to clarify these possibilities. That the whole brain shows a fatigability similar to that of the motor cortex is a widely accepted belief probably based on undue generalization of classical studies on the motor cortex. Recent experimentation, however, modifies this concept. As we have shown in the monkey (Delgado, 1959a), cerebral structures can be classified into three groups: ( a ) Quick fatigue. The evoked effect disappears in a few seconds, and about one minute’s rest is necessary in order for the area to recover its initial excitability. It is well known

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that this is the case in areas 4 and 6, and also in some points in the amygdala and in pathways from area 6. ( b ) Slow fatigue. The evoked effect is maintained for minutes, and slowly fades away in less than 1 hour. For example, continuous stimulation of the right putamen evoked contralateral turning of the head and body with some flexion of the contralateral arm, which did not fatigue in 30 minutes. Increased aggressiveness, induced by stimulation of thalamus, hypothalamus, and other structures showed no signs of fatigue after 20 minutes. ( c ) No fatigue. The evoked effect is prolonged as long as the stimulation is applied. This has been demonstrated by stimulation of the hypothalamus, producing ipsilateral contraction of the pupil for as long as 72 hours without interruption. In other experiments on monkeys. electrical stimulation of the nucleus reticulark for 5 seconds once every minute for 1 hour daily for 15 days, with a total of about 900 stimulations, produced contralateral tiirnings of the head, dilatation of both pupils, mastication and lowtoned vocalization, repeated consistently throughout the whole period of stimulation. Lack of fatigability has also been clearly demonstrated in experiments on self-stimulation. As shown by Olds (1958), rats may excite their brains (self-stimulation1) for hours or for days, becoming physically exhausted before the hypothalamus sliows signs of fatigue. In summary: Electrical stimulation of the motor cortex produces a rather quick fatigability contrasting with the greater endurance of the spontaneous motor functions. The concept of quick fatigability of this area must not be generalized, because other areas of the brain, such as the putamen, show slow fatigability (about 30 minutes) and still other areas, such as the hypothalamus and thalamus, show no fatigue at all after long-term stimulations.

E. L..isnsc EFFECTS Postural tonus, size of the pupils and respiratory movements are maintained within physiological oscillations for the life of the individual, and it is conceivable that some evoked effects could also be maintained for days or months by means of direct stimulation of the brain. In the majority of cases, the duration of the evoked effect de-

' The rat has intracerebral electrodes connected by long wires to a stimulator, and learns to press repeatedly a pedal which triggers the electrical excitation of its own brain.

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pends on the duration of the cerebral stimulation, with a relationship between local fatigability and endurance of the evoked effect. Poststimulation effects and rebound have been described in many experiments, but their duration is usually of only a few seconds. The literature about lasting effects is meager (see review of Hughes, 1958), but the following information is available: 1. One single ten-second stimulation of the basolateral amygdala inhibited alimentary conditioned reactions, and the animal walked away from the food tray, refusing to eat even when food was put into its mouth. The effect lasted for minutes or hours, and one animal refused to eat for 3 days. General behavior did not seem to be affected, and the animals played, rested, purred, and licked as usual ( Fonberg and Delgado, 1961) , 2. Intermittent radio stimulation of the cingulate gyrus in the cat for 5 seconds every 15 seconds for 24 hours produced almost continuous circling, with frequent vocalization and attempts to escape from the cage, which lasted for the 24 hours of the experiment, without, however, modifying food intake. This effect contrasted with the peaceful behavior of the animal during the 10 previous control days ( Fonberg and Delgado, 1961) . 3. In a colony of 8 cats with behavior recorded by time-lapse photography, experiments were done on animals under some restraint outside of the colony, stimulating the amygdala for one-half second every 5 seconds for 1 hour daily, for 1-15 days. The animals were returned to the colony immediately after the stimulation. On stimulation days, cinemanalysis revealed an increase in playful behavior, which was statistically significant. This effect was reversible, and could be duplicated in the same animal. The pattern of play was not modified, only the amount of time spent in this activity ( Alonso de Florida and Delgado, 1958). It should be emphasized that, in these experiments, the behavior of the animal was not recorded during the one-hour stimulation period, but only after its return to the colony. The increase in playful behavior, therefore, was not the direct result of the cerebral stimulation, but was an effect which outlasted the stimulation period. In these experiments, contactual activity also seemed to be increased, while other behavioral categories, such as walking and aggressiveness, were not modified. Local electrical activity was also modified. In contrast, similar prolonged stimulation of the internal capsule, falx cerebri, and anterior hypothalamus, did not produce lasting behavioral modifications. In summy: In the majority of cases, duration of the evoked

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effect was related to the duration of the stimulation and to the fatigability of the brain. In some cases, stimulation for a few seconds evoked effects which lasted for hours or for days. Intermittent stimulation of the amygdala produced lasting, reversible and reproducible changes in the social behavior of the cat.

F. RELL~BILI~’I’ The belief that electrical stimulation of the brain produces unreliable results is based mainly on the work of Sherrington (1947), who described “instability of cortical points” in animals. This concept was supported by the studies of Penfield and Welch (1949) in humans, who claimed that excitation of one point of the motor cortex could evoke flexion or extension of a limb in an unpredictable way, depending on several factors difficult to control. The concept of cortical instability has been recognized in modern textbooks of physiology (Lovatt Evans, 1956). To understand the above-mentioned findings, we must realize that in “acute” experiments or in surgery, the cerebral cortex is exposed and there are many variables such as anesthesia, position, and pressure of the contacts, hydration of the cortex, impedance of the tissue, local circulation, and temperature, which are probably responsible for the iinpredictability of the results. The above-mentioned variables are better controled in animals n7ith permanentlv implanted electrodes. However, even in this case, according to Ruchwald and Ervin (1957), the most important factors to determine the evoked effect would be the stimulation parameters and the “instantaneous organization of the brain as determined by its metabolic state, its recent history and the background of sensory input.” Most investigators would agree that results of cerebral stimulation certainly may change if the parameters of stimulation are modified (see bibliographv in MihaiIoviir and Delgado, 19S6), or if the experimental conditions are altered. Some of the important factors are proprioceptive information (Gellhorn, 1953), and the state of the animals, whether asleep or awake, under restraint or free, alone or in a group, and so forth. However, within the relative uniformity of the laboratorv medium, the results of electrical stimulation of the brain showed ;emarkable stability ( Delgado, 1955a, 1959a). The same cerebral points, as, for example, in the rhinal fissure, were

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stimulated in completely free monkeys 60 times in 1 hour, and the experiment was repeated on different days. Time-lapse recordings of spontaneous and evoked behavior furnished particularly clear evidence of the stability of results (Delgado, 1963a). Reliability of the effects obtained by cerebral stimulation was described some time ago by Clark and Ward (1937), and is supported by a large number of publications on self-stimulation, in which rats and cats may excite their own brains several thousand times daily for months (Brady, 1958; Olds, 1960), and by experiments on cerebral stimulation in conjunction with learning, conditioning, and positive and negative reinforcement (Sheer, 1961a). In summary: Instead of the principle of cortical instability, the concept of stability of cerebral points should be accepted with the understanding that results will differ if experimental conditions change. G. VARIABILJTY

Electrical stimulation of the brain in unrestrained animals presupposes previous implantation of intracerebral electrodes with contacts located in predetermined areas of the brain. For reliability of results in different animals, it is necessary to have good stereotaxic maps and techniques (see references in Sheer, 1961). However, there are two important limitations which should be emphasized: anatomical variability, and the less known but equally important physiological variability. 1. Anatomical Variability Surgical application of stereotaxic techniques to humans required an accurate knowledge of spatial location and interrelation of craniocerebral structures. Bone landmarks were initially utilized, until their lack of consistency became apparent. Spiegel and Wycis (1952) described a range of 50 mm in the relation between the pineal body and the vertical interaural plane, and similar results were found by Amador et al. (1959) in the relation between the midcommissural point and the interaural plane. Talairach et al. (1950) suggested that the ventricular system could be used to locate the position of the deep nuclei, with the anterior and posterior commissures being the most precise reference points ( Talairach, 1954). Unfortunately a difference of a few millimeters still exists

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in the relation between commissures and other intracerebral structures ( Schaltenbrand and Bailey, 1959). Amador et al. (1959) suggested that the success of the maps of Clarke and Henderson (1911, 1914) and Ingram et al. (1932), “lie, at least in part, in the relatively slight variability of the skull in their experimental animals.” Later experimentation has shown that anatomical variability in animals is not so slight. In the series of 20 monkeys, on which the excellent and well-known stereotaxic map of Olszewski (1952) is based, there is a difference of 3.5mm in the location of the horizontal plane in different animals, and between Olszewski’s plates and the diagrams of Atlas and Ingram (1937), there is a difference of about 4 millimeters. Limiting to between 2.5 and 4.5 kg the weight of the monkeys used, diminishes but does not avoid the problem of anatomical variability (Snider and Lee, 1961). Another fact, well-known to the neurosurgeon, is often overlooked by the neurophysiologist: When the cerebral cortex is exposed after a craniotomy, the surgeon does not trust his own visual identification of sulci and gyri, but prefers to use electrical stimulation to identify functionally the cortical representation. 2. Physiological Variability

The fact that individuals look and behave differently depends not only on anatomical differences, but also on physiological variations. Even if the cerebral structures, of two animals were located at exactly the same coordinates, their neuronal connections probably u-ould not be identical. Genetic factors and experience which intervene in the organization of behavioral reactions and performance cannot be identical in different individuals, e.g., the hands of a surgeon may look like the hands of a musician, but the training of each has developed very different skills and potentialities. An auditory cue may be neutral for a naive animal, but after repeated pairing with pleasant or unpleasant stimuli, the sane auditory cue initiates motor activity, such as pressing a lever; it is conceivable that electrical stimulation of auditory cortex could induce a motor response in a trained animal but not in a naive one. The existence of both anatomical and physiologicai variability fortunately does not present unsurmountable obstacles for a systematic investigation of cerebral functions, because variability has a limit. The problem is similar to the study of the multiform manifestations of spontaneous

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behavior, in which we may determine experimentally the range of variability, after which we can predict results with statistical reliability. Functional variability has some relationship with the complexity of the response. Simple effects such as the movement of one eye, flexion of the arm, or chewing are more uniform in pattern of performance and in cerebral localization than expression of rage, sexual activity or sequential behavior. In summary, anatomical variability and the less easily recognized physiological variability complicate the analysis of brain stimulation, but they have a limited range, which may be determined experimentally. Simple effects have less variability than complex patterns. V. Types of Behavior Evoked in Free Situations

Until recently the lack of suitable methods has handicapped the investigation of evoked behavior in free situations. Thirty-five years ago, a few scientists initiated acute experiments in unanesthetized animals, chronic preparations have been employed during the last decade; and the study of completely free animals stimulated by remote control is barely starting at the present time. With the development of new techniques, there are now many areas of animal behavior open to researchers. For example, the electrophysiology of arousal has been extensively studied, while its behavioral aspects are much less clear; no information exists about long-term arousal, or its possible repercussions on social relations. Vocalization has been evoked under anesthesia and as a manifestation of rage, but the "speech" areas of the animal brain remain unknown. The effects of cerebral stimulation on social hierarchy, maternal relations, grooming, courtship, daily movements, night behavior and many other interesting subjects remain to be explored. However, a few other manifestations, including motor responses, emotional reactions, and behavioral inhibition have already attracted considerable investigation and are discussed in this section. In general, the following types of evoked reactions may be considered: a. Tonic actiuity, producing the contraction of a group of muscles and resulting in a determined posture, for example, one limb in a flexed position; b. Phasic activity, which requires the organized contraction and

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relaxation of antagonistic groups of muscles in a determined sequence, as in chewing, licking, lashing the tail, or pawing the air; c. Drirjing uctiuity, characterized by the aim to reach an objective. The motor pattern may vary depending on the relations between experimental subject and the purpose. For example, performance of grooming is related to the respective position and individual characteristics of the animals; d. Inhibition of spontaneous or evoked behavior, which may affect all motility such as in the arrest reaction, or may be specific for a determined behavioral category such as inhibition of food intake; e. Intrinsic effects without motor manifestations cannot be detected unless special methods are used. These include a wide range of physical, chemical, and psychological phenomena such as variations in blood pressure, in gastric acidity, and in moods. Following the concept of autonomic tuning proposed by Gellhorn (1957), we may consider that there is a behavioral tuning which may be modified hy brain stimulation. Behavioral tuning often determines the pattern of response to normal sensory stimulation. Usually a cat responds to petting bv rubbing its head against the experimenter’s hand; however, threshold stimulation of some amygdaloid areas which does not produce any observable effect if the animal is isolated, does modify the behavioral tuning, and then the response to petting or to the presence of another animal is aggressive behavior. f. Abnnmlal t#ects, such as tremors, seizures, and disturbances in equilibrium may also be evoked by cerebral stimiilation, but these effects will not be discussed in the present review. Since behavior is expressed mainly by muscular contractions, it is convenient to consider first the range of motor effects that can be evoked by stimulation of the brain. A. MOTOHRESPOXSES

Identification of behavioral units is made by the observer, analvzing postures, movements and social relations. In spite of extensive investigation, the role of cerebral structures in motor performance is still poorly understood, and it is significant that in the recent symposium devoted to electrical stimulation of the brain (Sheer, 1961a), motor responses are barely mentioned. The classic concept stated in several textbooks (Best and Taylor, 1961; Lovatt

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Evans, 1956) of a homunculus-or simiusculus-lying upside down in the motor cortex and in charge of motor performance, has proven too schematic. Motor behavior may be evoked not only from the secondary and supplementary motor areas, but also from the caudate nucleus, pallidum, amygdala, rhinal fissure, thalamus, central gray, cerebellum and many other areas. As results of new investigations have become known, the original homunculus has been fragmented, repeated, and scattered throughout the brain (Hughes, 1959; Hughes and Mazurowsky, 1962; Lilly, 195%; Woolsey et al., 1950). The participation of so many areas of the brain in motor performance should not be too surprising, considering not only the need for constant correlations, adjustment and integration of a large amount of information, but also the tremendous variety of behavioral manifestations, expressed mainly by motor activity of the organism, which requires complex and extensive guidance systems. The classical question of whether individual muscles or movements are represented in the brain (Chang et d., 1947; Fulton, 1949; Liddell and Phillips, 1950, 1952; Walshe, 1943, 1951), may be debated in experiments performed with the subject under anesthesia, but is less important in awake animals, in which, according to Denny-Brown (1960), it seems likely that the nervous system as a whole contributes to each motor act, and it is difficult to distinguish between mechanisms for posture and for movements. The concept of circular organization of the nervous system (Paillard, 1960) is a useful scheme to explain the complexity of spontaneous motility, and also to clarify some results of brain stimulation. In agreement with original ideas of Exner ( 1894), revitalized by Gooddy (1949), Paillard conceived of the motor organization as a dynamic patterning of nervous command, which is carried along a variety of circular paths that form closed loops at various anatomical levels. The system has working units with its own functional balancing and self-regulating mechanisms, and each unit contributes to the effectiveness of the organism as a whole. It is possible that the brain may be electrically stimulated at different points of this circular path, and at different anatomical levels, activating one or several of the working units. We should therefore expect, depending on the stimulated level, to evoke localized motor effects, fragments of behavior, or complete behavioral patterns. The simplest pattern of motor response is the contraction of a

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small group of muscles producing, for example, flexion of one finger, or estension of one hand. These effects have been extensively described in the literature (Bucy, 1944; Hines, 1929; Penfield and Rasmussen, 1950; Terzuolo and Adey, 1960; Woolsey, 1952). The well-known maps of cortical motor representation express graphically the fact that localized motor responses can be obtained from some areas of the brain. It should be emphasized that deep anesthesia is often mentioned as a requirement for the mapping. Even in unanesthetized animals evoked effects are usually described as clumsy, crude, unnatural, and never skillful or elegant. The lack of finesse in evoked movements is usually attributed to “the inability of artificial stimulation to simulate the natural patterns of cortical stimulation” ( Terzuolo and Adey, 1960). Physiological mechanisms have a temporal and spatial complexity which many authors doubt can be reproduced by electrical stimulation. Are these facts and opinions also true in free situations? The pioneer studies of Hess and his school (1932, 1954, 1957) g’ive some important clues in answer to this question. According to Hess, “our voluntary movements are directed towards the attainment of certain objectives.” For each purpose, it is necessary to have a definite starting position which affects the eye, head, body, and extremities. The postural adaptation of the animal, “which provides the basic conditions for every accurately aimed motion,” is called ereismtic phase ( ereisma = support). The voluntary aimed movements are superimposed upon the postural adaptation, and constitute the teleokinctic phase (telos = purpose). This concept has been developed further by Jung and Hassler (1960). Hess classified the results observed in unrestrained cats into two groups: ( I ) change in the posture of head and body, and ( 2 ) movement of head and extremities and turning of the body. Lowering of the head was obtained by stimulation of the region of the posterior commissure; elevation of the head and sometimes of the entire front part of the body were obtained by stimulation of a zone around the posterior hypothalamus and prerubral region. Rotation of the head and body was obtained by stimulation of the subthalamic part of the brachium conjunctivum. Ipsilateral turning of the head and of the body, resulting in a tight circular movement, developed following excitation of the centrum medianum. The motor responses of the extremities included a “drawing up” of the contralateral forelimb, which

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was kept “hanging” for several seconds after the end of the stimulation. This and other patterns of movement were obtained by stimulation of the thalamic radiations. The motor responses in the face were observed frequently by electrical stimulation of the same area, and included the ear, eye, eyelid, and vibrissae. In these responses, there was a harmonious relationship between their ereismatic and teleokinetic phases, similar to that existing in spontaneous movement. The concept of a play of forces between the applied stimulation, postural reflexes, and the will of the animal, has been emphasized and repeated in the Hess papers. The pattern of the evoked effect in some cases was different (see Figs. 35 and 37 or Hess, 1957), but in other cases, it was similar to spontaneous behavior (see Figs. 34 and 36 of Hess, 1957). The above-mentioned studies were concerned mainly with the motor function of diencephalic structures. Hassler ( 1956), using the Hess technique, demonstrated that low frequency stimulation of the head of the caudate nucleus in the cat produces purposeful turning of the head and body and the animal often circles to the side opposite the stimulation. Our experiments on the hidden motor cortex of the cat showed the existence of three types of response: ( I ) movements which never appear normally in the animal, for example, stimulation of the inferior part of the sulcus presilvius produced displacement of the ocular globe accompanied by elevation of the eyelid, or rotation of one eye in the anterior posterior axis; ( 2 ) movements of the body without postural adaptation, producing in general a loss of equilibrium, for example, stimulation of a few cortical points in the posterior part of the sulcus cruciatus evoked contralateral rotation of the head and extension of the fore limb, and the cats fell to the floor without attempting to recover their balance. They seemed to have lost their spatial orientation, but as soon as the stimulation ended, they recovered their normal position instantaneously. ( 3 ) The majority of evoked responses showed excellent postural adaptation of the whole body, and developed with the smoothness and coordination of spontaneous activities. The following examples illustrate this type of effect. In the cat, excitation of the cortex hidden in the cruciate sulcus evoked flexion of the contralateral hind limb as shown in Fig. 1A. The cat raised its pelvis and tail and lowered the anterior part of the body, properly balanc-

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FIG. 1. A. Electrical excitntion of the right hidden cortex of the sulcus cruciatus is performed with the programmed collar stimulator. Lifting of the

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ing its weight, supported by only three legs. The evoked movement did not produce emotional disturbance or inhibition of spontaneous behavior, for when the experimenter reached out his hand, the cat lowered its head, and rubbed against the hand, seeking to be petted, as shown in Fig. 133. In this case the evoked flexion of the hind limb was increased, probably because of reflex correlations determined by the posture of the animal. In contrast, if the head of the cat was extended at the moment of stimulation, the hind limb was lifted in an extended position, as appeared in Fig. 1C. Postural adaptation, correlation between spontaneous and evoked movements, variability of the response through variation in the initial posture, lack of emotional disturbance, and development of spontaneous behavior during cerebral stimulation were thus demonstrated in these experiments. Similar effects have been recorded in more than twenty-five other cats. Another example of voluntary postural adaptation preceding an evoked movement was observed in a different cat. This animal was also stimulated in the cortex of the cruciate sulcus, but as it was caught in a moment of precarious balance with all paws close to each other, the initial part of its motor response was to spread the fore limbs to secure better equilibrium, shifting the body weight to the right. These effects did not appear if the posture of the cat was appropriate for the initiation of the evoked movement. When crus I1 of the cerebellum was stimulated in the same animal, lifting of the ipsilateral fore limb was evoked with good coordination, without tremor, discomfort or disturbance of equilibrium, proving that cerebellar stimulation may evoke smooth responses without rebound or abnormal manifestations, a fact which has been questioned in the literature (Chambers, 1947; Clark, 1939, 1952; Clark and Ward, 1949; Dow and Moruzzi, 1958; Koella, 1955; Lewandowsky, 1903; McDonald, 1953). Simultaneous stimulation of both motor cortex and cerebellum in this cat produced an alternate lifting of the left fore and hind limbs without loss of equilibrium, representing probcontralateral hind limb was evoked with perfect postural adaptation, without emotional disturbance. B. Spontaneous behavior was not inhibited by motor cortex stimulation. The animal shows friendliness, rubbing against the experimenter’s hand. Observe also that the evoked lifting of the leg is increased. C. Lifting of the cat’s head by the experimenter modified the pattern of the evoked response. The hind limb is raised but in extended position.

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ably an algebraic summation of evoked responses and volitional and postural adaptation of the animal ( Schoolman and Delgado, 1958). ir, variety of effects, such as turning of the head, yawning, chewing, licking, walking, climbing, flexing or extending of the limbs, and so on, have been e.croked by stimulation of the motor cortex (Clark and IVard, 1937; Delgado, 1952a, b; Delgado and Livingston, 1955-1956; Delgado, unpublished observations). Often the cats resemble electric toys, with a predictable series of well-performed movements controled by push-button radio stimulation. Strong intensities produced a violent response, but near-threshold stimulations (about 0 . 2 1 mA) evoked effects very similar to the spontaneous activities observed in the animals. In monkeys completely free within a colony, electrical stimulation of the motor cortex permitted observation of the development of evoked reactions and their relation to spontaneous activities. In general, the evoked effects were simple, such as flexion, extension, or elevation of the contralateral arm, turning of the head, and wrinkling of the face. These effects were stereotyped, reliable, aimless, did not produce emotional disturbance, and lacked positive or negative reinforcing properties. Time-lapse recording of the daily behavior of animals, in which motor effects were evoked repeatedly-up to 60 times in one hourrevealed no modification in quality (with the exception of the evoked responses), or in quantity of behavioral categories compared with control days. Spontaneous activities such as grooming, walking, picking, or eating were not inhibited. The motor cortex of a female monkey was excited while it was grooming the boss of the colony, and the evoked turning of its head did not interrupt grooming, alarm the animals, or disturb their social relations. The other monkeys of the colony watched the evoked movements of the stimulated animal without signs of fear or hostility. Emergency situations may block the effect of cortical excitation. In one experiment, a monkey was stimulated in area 4 when an external threat in the form of a net used to catch the animals was presented to the colony. The monkey’s ensuing off ensive-defensive reactions completely inhibited the previously evoked head turning. This was one more example of the fact that evoked and spontaneous movements often influence each other and the final motor pattern seemed to depend on their algebraic summation.

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Electrical stimulation of the rhinal fissure produced a similar pattern in 4 different monkeys. The head turned to the contralateral side, the mouth opened without showing the teeth, and the ipsilateral side of the face was scratched with the ipsilateral hand, This effect was accompanied by a partial inhibition of spontaneous activity. If the animal was walking, it would hesitate and stop; if it was eating, chewing was interrupted but food was not ejected. Spontaneous behavior halted in a natural way, as if the attention of the animal had been incidentally diverted. In one monkey this effect was evoked 120 times in one hour without habituation. More details may be found elsewhere (Delgado, 1963a). Movements evoked by stimulation of the limbic system, thalamus, and other areas of the monkey brain were usually more complex and purposeful, and with less fatigability than movements produced by motor cortex stimulation. Some other examples are described in the following sections. Interesting data have also been obtained from human patients. In one of our cases, intracerebral electrodes had been implanted for several days, with some contacts located in area 4. Voluntary movements of the contralateral hand produced a desynchronization of the electrical activity localized on one of the contacts (Delgado and Hamlin, 1958). Electrical stimulation of this point evoked forced extension of the fingers of the contralateral hand, abnormal in character. The patient expressed surprise, considered the evoked movement strange and involuntary, and tried to stop it by holding one hand with the other, a phenomenon which we have also observed in monkeys. Movements evoked in a free situation by electrical stimulation of the motor cortex seem to be simpler in the monkey and in man than in cats, suggesting that, with the evolution of the brain, the functions of the so-called motor cortex in areas 4 and 6 have become more closely related to the final output of movements already patterned than to their initiation and spatial-temporal development. In summary: Some localized motor effects evoked by cerebral stimulation are clumsy and look abnormal, but many of them are well organized, have a proper postural adaptation, and look similar to normal activities. They do not produce emotional disturbances and usually do not inhibit spontaneous behavior. Cerebellar stimulation also may evoke spontaneous-looking movements. Motor cor-

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tex stimulation produces simpler effects than those evoked by excitation of the limbic system and thalamiis.

B. SE~IJENTIAL BEHAVIOR Stereotyped responses with a sequence of behavioral categories riot necessarily related to each other have been evoked by electrical stimulation of the brain. The effect is reminiscent of the automatisms observed in some epileptics, but in our experience it was not related to the presence of after-discharges, in spite of the fact that sequential behavior often outlasts a 5-second stimulation by 5-10 seconds. In general, the animal shows a motor effect during the cerebral excitation and then walks on a determined path, avoiding obstacles, and reacting normally to noises or to visual stimulation, and returning to its starting place. The following example is typical: Female Monkey no. 10, under restraint in a chair, was stimulated in the nucleus medialis dorsalis of the thalamus with 1.5 mA, evoking a dorsal extension of the head and restlessness with low-toned vocalization. \\'hen the same stimulation was repeated by radio control with the monkey free inside the colony, a typical pattern was observed: the animal moved its head backward and walked a few steps, avoiding the proximity of the other monkeys, jumped with precise coordinatioii of movement to the back wall of the cage, where it hung for a few seconds, jumped down to the floor, and then walked back to the starting point, resuming the type of behavior which was interrupted by the stimulation. At the end of this sequence, if male monkey no. 4,which was the boss of the colony was present, he usually mounted monkey no. 10. This effect was so reliable that stimulations applied for 5 seconds once every minute during a 90-minute period, produced a total of 81 mountings. No sexual relations were recorded before or after the stimulation period during the entire day. It was interesting to note that 5 of the stimulations were applied while monkey no. 4 was on top of monkey no. 10 without interrupting their relation. However, as soon as the mounting was over, monkey no. 10 performed the stereotyped response of walking, jumping to the back wall of the cage, hanging there for a few seconds, and coming back to the starting place where she would again be mounted. These experiments demonstrated that the evoked behavioral sequence could be momentarily inhibited by a spontaneous activity taking place after the stimula-

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tion was over. A similar sequential response of walking, jumping, hanging, and walking has been evoked in cats by electrical stimulation of the nucleus ventralis of the thalamus. The main difference in this experiment was a fear-like component which was present in the cat, but not in the monkey. In sumnay: A determined sequence of different behavioral categories may be elicited by cerebral stimulation. Similar patterns may be observed in cats and monkeys. If a female monkey was stimulated in the nucleus medialis dorsalis of the thalamus, a great number of mountings (81 in 90 minutes) were induced in the male partner.

C. OFFENSIVE-DEFENSIVE BEHAVIOR Aggressive behavior in the cat with unsheathed claws and wellaimed blows acting as “if threatened by a dog,” has been evoked by electrical stimulation of the periventricular gray matter (Hess, 1928, 1957). “The animal spits, snorts or growls. At the same time the hair on its back stands on end, and its tail becomes bushy. Its pupils widen sometimes to their maximum, and its ears lie back, or move back and forth “to frighten the nonexistent enemy” (Hess, 1928). This first description of rage produced by intracerebral stimulation in awake animals opened an era of extensive studies on emotional behavior by the Hess group and many subsequent investigators (see reviews by Akert, 1959, 1961; Hess, 1957; Ingram, 1952). The wellknown studies in lightly anesthetized preparations of Magoun et ul. ( 1937), and Ranson and Magoun ( 1939), gave evidence of rage but behavioral reactions could be analyzed only to a limited extent. The comprehensive study by Leyhausen (1960) of spontaneous predatory behavior in caged and free-ranging domestic cats and other felines is of great interest because of its detailed analysis in photographs and sketches of the components of aggressive behavior in this species. In the cat, most of the experimental work has been done with single animals, in acute experiments, under some restraint ( Akert, 1961), and, as the results are often generalized, there is perhaps some distortion about patterns and concepts of evoked aggressiveness. For example, threatening gestures (biting its own hand), or submissive acts to appease the aggressor (presenting the genitalia) are typical monkey behavior, without parallel in the cat.

‘TABLE: V OFFENSIVE-DEFENSIVE REACTIONS Source

Animal and brain structure

Nomenclature

Display

Attack

Escape

Observation

CAT

Hess, 1W28; Hess, 1957

Brain stem periventricular gray.

Midriasis, piloerec- Striking welltion flat ears. aimed blows with paw See also Iless and Anterior hypoTrue rage Spitting, snorting, thltlamus, periaggressive growling, defecaRrugger, 1943 fornical and drive, flight tion, urinating drive. in spurts preoptic area. i~iid ]less el al., 1!J45 13tts:d septum Affective de(around anterior fmse reaction commissure). Posterior hypothalamus. Substantia grisea of midbrain Masserman, 1941

Hypothalamus

Gastaut el al., 1852 Habenula, amygdala, hippocampus

YW

Looking for antagonist. Aggression may change to flight

Pseudoaffec- Midriasis, piloerec- Biting and Blind attempts No change in tive reaction tion, flat ears, striking, not to escape spontaneous salivation, liydirected behavior per p nea, responds to crouches, growls, petting lashes tail Fear

Midriasis, piloerec- Not directed, Flight hiding tion, tremor, but may bite arrest respiration followed by hy-

Adversive movements

x

See also Naquet, 1953

Rage (higher voltage)

Spiegel et al., 1954 Periaqueductal gray. Superior and inferior colliculus

Reaction sug- Midnasis, crying, gesting pain struggling, hyperpnea, micturition, defecation

perpnea, flat ears, decreased reactivity

Midrimis, piloerec- Welldirected tion, flat ears, biting and show of teeth, striking hissing, spitting, defecation, urination

Delgado et al., 1954 Tectal area, lateral Fear-like response thalamus, posterior hippocampus

Flight or defense

Turning head, deviation of eyeballs

Well-oriented Trial-anderror escape learning, conditioning

8

Midrimis, piloerec- Seizing and Big jumps, tion, typical biting all protective facial expression, within reach. gestures, high-pitch vocalescape ization, defensive-offensive movements, urination, defeca-

Medial nucleus of Fear-like reamygdala, pensponse. aquaductal central gray, posteroventral n. of thalamus, posterior hippocampus, lateral tegmentum

E i !i

CAT AND MONKEY

Delgado, 1955b; Delgado et al., 1956

2

tion, tachycaria, tachypnea

Social fighting, conditioning

U

9

s5

2i

__

Anand and Dua, 1956

Anteromedial, basal n. amygdala, pyriform cortex, hippocampus

Fear rage reaction

Eating automatisms and autonomic responses, frightened or agitated or quiet and goes to sleep

Runs away w

co

4

Y

g.

CAT

Huiisperyer, 1956. l’reoptic area, hy- Affcvtivc. (I(,(See also Ferpothalamiis, mid- fence, thrwt , Hat ( w s , ini(1ri:t:tttsak iiandez de Molina twain, central attwk, sis, piloc~rt*rtioti, and Hunsperger, gray, ttmygdala, hu11chrcl h:wk stria termindis 1959, 1962 IJunsperger el al., Peripheral to :~l)ovc~ l’hght, e w q w Hissing 1962 t irhavit )r - - ___ - _ _ _ ___ __ Shealy and Peek, Basal and I a t t d Cowering Srisrling, salivaticin, Hitirig and 1957 amygdala. (display) piloerectioii , wr:i trhing baring I -ririatio~~, dc.tcwhledial aiid central Rage areas of amvgdal:t tion

o r fligtii

E:scapc.

E

lo

Roberts, 1958a,b

Posterior hypothalamus

Flight

Medial lemniscus, Alarm ventralis posterior, habenula, posterior hypothalamus

Stereotyped locomotor activity, searching as if looking for escape Rage, fear-like and pain-like

Escape learning without avoidance learning Avoidance

2

F! _____

Nakao, 1958

2

Middle hypothala- Aggressive re- Midriasis, retracViolent dimus sponse tion of ears, rected attack arching back, extension of legs, stops spontaneous behavior Rostra1 hypothala- Flight reaction Midriasis, snarling, Withdraws Well-oriented mus, preoptic (at times hissing from experiescape, leaparea mixed) menter ing t o top of cage

g

i? m

!a

% z

8 ---

Ursin and Kaada, Lateral and central Fear response Searching, scared, amygdala, venanxious glances, 1960. See also midriasis, cringKaada et al., 1954 tral part internal ing, restless capsule Ventromedial and Anger response Growling, hissing, caudal parts crouching, flat ears, pawing lateral nucleus, basal nucleus -__--

_ _ _ _ _ _______

._

Jumps 08,runs away, hides

m#

____

:

2

Seldom direct attack - - _ _ _ _ ~ ~ ____

8 co

8 0 Wasman and Flynn, 1903

1,nteral hypotliala- .kffcctive nius, supr:ioptir attack I i 11clens

I'eriphery of 1atcr:Ll Stalking hypothalamus attllck

illert, mydriasis, Striking rat piloerection, hisses, growls, arches back, urination, h r c s clavv3, salivation, d w p brcriths 1)iscrc~tealerting Vicious biting mydriasiu, walking signs of ininor displity

'rested against rat

tl

CHICKEN

von IIolst and von Saint Paul, 106'2

I3rain sten1

Attack beAlertness, visual Well-directed Urgc to flre havior, urge fixation, apattack with t o flee proach, attitude spurs against of rage animals and humans, pecking, plucking of feathers, triumphant call

Social fighting

P

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One of the difficulties in interpreting the literature is that offensive and defensive behavior are closely related to each other, and there are a variety of terms, sometimes ill-defined, which have been applied to one or both of them, as shown in Table V. Some of these terms mix experimental data with interpretation. The words “anger” and “fear” attribute to the animals emotional reactions that are unverifiable psychological evaluations. We do not know if a cat is afraid: we only observe a display of symptomatology. It would be desirable to have a set of characteristics ascribed to a precise term, but unfortunately, the criteria of different authors is not uniform. It should be emphasized that, in general, a small number of manifestations are selected to characterize offensive-defensive behavior and this selection, again, is not uniform. The classical maps of Hess (1957) of “affective defense reaction” are based on only four reactions : namely, spitting, growling, piloerection, and retraction of the ears. The data of Wasman and Flynn (1962) are based mainly on the direct attack of a cat on a rat. The study of Miller (1961) and his group is based mainly on conditioning. It is therefore necessary to know the meaning of the terms used by each author in order to understand the experimental results. The manifestations which may be observed in aggressive-defensive reactions may be classified in four groups: autonomic, somatic, vocal, and dynamic behavior; the more usual manifestations in the cat are shown in Table VI. Aggressive behavior has two types of essential elements: ( 1 ) Aggressive display, consisting of a variable amount of the manifestations listed above, but without directed attack. A typical example is the false rage shown in Fig. 3. Many of the display manifestations are not specific of aggression, because they may appear in other behavioral patterns. (2) Attack with harmful contactual activities, using nails or teeth, with the definite purpose of hurting the adversary (Fig. 2 ) . There is experimental evidence that these two elements are represented in different areas of the brain and may be evoked in a pure form. Deadly attacks with minimal display have been described by Wasman and Flynn (1962). The combination of display and attack constitutes the complete behavioral pattern of aggressive behavior, as originally observed by Hess and later confirmed by many other authors. I n defensive behavior there is offensive-defensive display plus

402

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JOS6 M. H. DELGADO

TABLE V1 (.)FFENSIVE-DEFENSIVE MANIFESTATIONS I N THE CAT __

.\I-*~OXOMIC

VOCAL

Pupillttry dilatation Eaophtslmus Retraction nictitating meml)rrtiic. Salivation Pilorret4ori Saeatiug I‘rination Defecation Respiratory acceleration (‘ardiar accelemtion

(howling Hissing Spitting Snorting High-pitched screaming

S O M \TI(’

.ilc*rtirig Ketrac*tingwrb I~nshcnthingtin\\ s .\rchiiig Ixic-k Lashing tail Showing teeth Opening morith Snarling Pawing Hoor

._ -.-

DYNAMIC BEHAVIUR Leaping to feet Circling Prowliit g Stalking Poiincing (jumping 011 i Striking ~ i t hpaws Biting Chasing Fighting (reciprocal) Cringing Hiding Escaping Fleeing

the attempt of the animal to withdraw from the experimental situa:ion, as revealed in such actions as hiding, escaping, or fleeing. Manifestations of aggression or defense may appear alone, mixed, or may alterate with each other. 1. Anutoniicul Loccctioii The affective defensive reaction identified by piloerection, spitting, growling, and retraction of the ears is located in Hess’ maps around, but excluding the fornix (Hess and Briigger, 1943). Rostrally, the areas extend up to the intermediate preoptic area and ventral septum ( Hess et at., 1945), and caudally, continue through the posterior hypothalamus down to the periaqueductal central gray (Delgado, 1955b; Glusman and Roizin, 1960; Hunsperger, 1956; Miller, 1961; Nakao, 1958; Roberts, 1958a, b; Spiegel et al., 1954; Wasman and Flynn, 1962, in the cat, and Delgado, 1955b; Segundo et al., 1955; Sheer and Kroeger, 1956, in the monkey). In spite of the general agreement on hypothalamic representa-

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tion of aggressive behavior, anatomical differentiation of effects has been described. According to Hunsperger et al. ( 1962), it is possible to distinguish threat-attack behavior from escape. The first is elicited from a central part of the active area of the hypothalamus, and the second only from the border of this area. According to Wasman and Flynn (1962), affective attacks were elicited in the lateral portion of the hypothalamus from points medial to those from which pure stalking attacks were evoked. Electrical stimulation of several other areas of the brain also is able to produce offensive-defensive responses. Increase of aggressiveness evoked by amygdaloid stimulation has been described by Anand and Dua (1956), Delgado et aZ. (1956); Fernandez de Molina and Hunsperger (1959, 1962), Gastaut (1952), Gastaut et al. ( 1951, 1952), Kaada et d. (1954), MacLean and Delgado (1953), Magnus and Lammers (1956), Naquet (1953), Sano ( 1958), Ursin and Kaada ( 1960). However, excitation of the lateral nucleus of the amygdala is able to inhibit the rage induced by hypothalamic excitation (Egger and Flynn, 1962). This apparent contradiction of previous studies may be explained by accepting the existence of inhibitory points in the amygdala in addition to the rage-provoking areas which already have been described by MacLean and Delgado (1953). In one of our cats, combined stimulation of the lateral nucleus of the amygdala and pyriform cortex produced arrest reaction, while stimulation of the rostral amygdala in the same animal produced aggressive behavior. Fernandez de Molina and Hunsperger (1962) believe that defensive behavior is organized at three levels of progressively increasing importance: amygdala, hypothalamus, and midbrain central gray. The stria terminalis would establish the functional connection for aggressiveness between amygdala and hypothalamus, but the direction of the conduction is controversial (Zbrozany, 1960). According to Bard and Macht (1958), “fragments of what definitely appeared to be a rage reaction” may appear in low mesencephalic cats with the brain sectioned at the level of the rostral part of the superior colliculi. A strong nociceptive stimulation of the tail produced hissing, growling, tail lashing, and attempts to escape, demonstrating that the amygdala and hypothalamus are not the only structures which play a role in the display of rage. Rage and flight have been evoked in the cat in self-stimulation experiments of the median forebrain

404

JOSS M. R. DELG.4DO

bundle, periaqueductal gray and lateral hypothalamus ( Doty, 1961). In the chicken with implanted electrodes and equipped with tiny radio receivers (von Holst and von Saint Paul, 1962) brainstem stimulation was able to produce “disgust,” “urge to flee,” and “attack” on stuffed animals, real chickens, and caretakers. Analyzing the experimental data of different authors (see also Table V ) , it is clear that there is not a single “rage center” but that there are many points concentrated in the amygdala, hypothalamus, diencephalon, tectum, and reticular region. These structures probably have a close relationship to each other and perhaps a hierarchical dependence ( Fernandez de Molina and Hunsperger, 1962), or a functional specialization. The clarification of these possibilities requires further investigation. 2. Functioiml Characteristics Some of. the offensive-defensive manifcstations evoked by electrical stimulation of the brain are similar to the normal reaction evoked by noxious stimulation, such as tail pinching, which in the cat may induce a directed attack. It is possible that stimulation of the central gray or of the posteroventral nucleus of the thalamus may activate pain pathways, and therefore the aggressive reaction could be a secondary effect to painful and unpleasant sensations. The hypothalamus and the amygdala, however, do not seem to be involved in pain perception, and their roles are prabably more directly related to the central organization of offensive-defensive mechanisms. The study of fatigability of evoked aggressiveness is of considerable interest, but the experiments are difficult to perfom for ethical reasons. In three monkeys mesencephalic stimulation was applied, using the lowest possible intensity and limiting its duration to 5 minutcs to minimize possible discomfort. The high-pitch vocalization, pupillary dilation, and aggressive movements observed in these thxperimcnts continued during the whole stimulation period without m y signs of fatigue (Delgado, 1955b).In another study on cats, the cimygdala was continuously stimulated for 20 minutes, and during all this time, the animal hissed and threatened when we tried to pet him, and would growl even when left alone, reverting to peaceful and affectionate behavior when stimulation was discontinued. The lack of fatigability of this effect contrasted with the known

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quick fatigability of some other areas of the brain, such as the motor cortex. Direct electrical stimulation of the thalamus, tectum, and other cerebral structures in the cat and monkey can be used to establish conditioned responses, to motivate trial-and-error learning, and to induce the performance of instrumental responses, demonstrating that the offensive-defensive effects elicited from these areas have the drive properties of true motions (Delgado, 1955a; Delgado et al., 1954, 1956). However, as demonstrated by Roberts (1958a, b, 1962; see also Miller, 1961), stimulation of the posterior hypothalamic nucleus produces a ‘‘ilight” response which is able to induce escape learning but not avoidance learning, The cat is able to run in a T maze to stop the stimulation but cannot be conditioned to give a response in order to avoid the start of the stimulation.

3. True and False Rage Pseudo-affective responses with considerable display without directed attack, known as false or sham rage, have been observed in decorticated animals (Bard, 1928; Bard and Rioch, 1937; Cannon and Britton, 1925), and also have been evoked in intact cats by hypothalamic stimulation ( Masserman, 1941). False rage cannot be conditioned and, therefore, is considered a motor display without awareness, lacking the motivational properties of true emotion. The presence or absence of directed attack is the differential factor between true and false rage. An object of attack is therefore essential for this type of experiment. Gloves, sticks, a live rat or guinea pig, or even the experimenter himself have been used as targets for feline aggression. Probably a preferable subject would be other members of the same species. In the excellent studies of von Holst and von Saint Paul (1962), the aggressive behavior evoked in roosters by brainstem stimulation in the presence of a hen produced a full display and attack similar to normal aggressiveness, while if the rooster was alone, a good part of the display was absent and the evoked effect appeared mainly as motor restlessness. In our experiments with cat colonies, the difference between true and false rage could be clearly demonstrated, as seen in Figs. 2 and 3. Stimulation of the lateral hypothalamus produced the usual aggressive display with mydriasis, piloerection, hissing, showing of the teeth, snarling expression, and other manifestations. The display

406

JOSk S l . H. DELGADO

FIG. 2. True rage. Evoked by stimulation of the lateral hypothalamus. Characterized b) : A. Aggressive di\play oriented towards another cat. B. Attack with well-oriented blows directed against other cats. C. Attack against friendly experimenter.;. D. Learning of instrumental response to stop brain stimulation.

was directed towards a control animal which reacted properlyfacing the threat ( Fig. 2A ). The stimulated animal then advanced, directing well-aimed blows with unsheathed claws against other cats which fought back or withdrew (Fig. 2B). Attempts by the

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Fig. 2 C and D. See facing page for legend.

investigator to protect the other colony animals diverted the attack of the stimulated cat, which bit and clawed the gloved hand ferociously (Fig. 2C). The aggressive behavior usually started and

FIG. 3. False rage. A. Control: two friendly cats together in the colony. B. Stimulation of anterior hypothalamus evoked aggressive display, not directed against the other cat, which, however, reacts with defensive pattern. C and D. The control cat attacks the stimulated animal, which, in spite of its apparently aggressive display, lowers its head, flattens its ears, withdraws, and does not retaliate. 408

FReE BEHAVIOR AND BRAIN STIMULATION

409

Fig. 3 C and D. See facing page for legend.

ended with the stimulation, but a state of mistrust was created in the cat by the experiments. Attacks and fights were elicited as many times as fhe hypothalamus was excited, even if the stimulated cat

410

JOSh M. R. DELCADO

was small and was struck and clawed by the others (see Delgado, 1955b), proving that a painful experience did not inhibit evoked aggressiveness. Instrumental responses to stop the cerebral stimulation were easily learned during hypothalamic stimulation. Fig. 2D shows the cat, in whom aggressive display was evoked in the preceding figures, turning a wheel while being stimulated, indicating that memory orientation and coordination are intact in spite of the autonomic and somatic changes. In contrast, one example of false rage is seen in Fig. 3. A control picture (Fig. 3A) shows the peaceful relations between two cats. Stimulation of the anterior hypothalamus produced a typical aggressive display with hissing and growling, which was not directed against other animals. The blue-gray cat, which was close by, withdrew defensively (Fig. 3B), and then responded to the aggressive display by launching a series of blows against the stimulated animal, which lowered its head and flattened its ears (Fig. 3C). After being struck and bitten several times (Fig. 3D), tlie stimulated cat retreated without any sign of retaliation. After repeated stimulations, the cat moved around the colony cage, showing aggressive display, but avoiding the proximity of the blue-gray cat, which was the most belligerent of the group. This study does not clarify the question of whether the stimulated animal had only external manifestations of rage without emotional awareness. However, it demonsbates that consciousness was not impaired, and that some learning and behavioral adaptation may exist in cases of false rage. In the same cat, true and false rage were evoked by stimulation of the lateral or anterior hypothalamus, respectively, proving that the effects depend on the stimulated area rather than on individual animal's reactions. The main element lacking in false rage is the directed attack. In our experiments, pinching the tail during anterior hypothalamic stimulation increased the aggressive display, but there was no threatening of the experimenter, and even the attempts to escape were mild. Tlie animal did not orient itself against the nociceptive stimuli. False rage could be explained as a lack of one important aggressive element, the attack, which may not be represented in the anterior hypothalamus. Another explanation, which seems more logical is that stimulation which evokes false rage induces aggressive display but at the same time has an inhibitory effect upon directed attack. The fact that the animals failed to re-

F%EE BEHAVIOR AND BRAIN STIMULATIOiV

411

spond aggressively to painful sensory stimulation supports this explanation. It is known that conflicting drives may be induced by brain stimulation, and the final result depends on the integration of all factors (Delgado, 1952b; von Holst and von Saint Paul, 1962). It should be emphasized that adynamia has been evoked by stimulation of the anterior hypothalamus in the cat ( Hess, 1944a), and in the monkey (Sheer, 1961b). It is not clear which precise points may evoke rage (true or false), or adynamia, but all these effects seem to be represented in the anterior hypothalamic area. 4. Selectivity and Spread

of Evoked Aggressivsness

If there is a choice among several targets, purposeful aggression is usually preferentially directed against one of them. This fact has been observed in groups of cats and monkeys, and one example is presented here. A monkey colony was formed of two males and four females. The boss was a big male, no. 8, which usually was avoided by the other animals with the exception of a small female monkey (no. 7), with which he had friendly and intimate relations. Electrical stimulation of the central gray in the boss monkey produced an immediate increase of motor activity and aggressiveness (Fig. 4) , with the animal running around the cage and attacking the other members of the group, chasing them, and even biting them if they were unsuccessful in eluding him. The pattern of this aggressive behavior, which was distinguishabIe from spontaneous attacks only by its violence and persistence, lasted as long as the stimulation was applied. The attacks were well-aimed and well-organized and showed social selectivity, because they were directed especially towards monkey no. 4, the other male of the colony, against which spontaneous attacks had previously been recorded. The aggressiveness was only exceptionally directed against monkey no, 7, the female friend of the stimulated animal. The increase of evoked aggressiveness in the boss was also manifested sometimes by biting the swing, shaking the cage, and attacking its mirrored image in the glass front of the cage. After the experiment was repeated 20 times, once every minute, there was a social spread of aggressiveness, and each time that monkey no. 8 was stimulated, monkey no. 7 joined him in attacking the rest of the colony. In a few cases, the attacks were even launched by monkey no. 7, in spite of the fact that she was much smaller than the other animals, as shown in Fig. 4C. In summary: Offensive-defensive behavior is characterized by a

412 J O d M. R. DELGADO

FIG. 4. A. Stimulation of the central gray in the boss of the colony evokes an aggressive display and he advances threatening the rest of the group. A female tries to pacify him by presenting. Other monkeys retreat to a corner. B. The evoked aggression of the boss is selectively directed against the other male of the colony, which grimaces, showing fear and submissiveness. C. Backed and excited by the boss’ increased aggressiveness, his tiny consort launches attacks against all the other monkeys, u-hich are twice her size. This experiment shows the social spread of evoked aggressiveness.

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display and by attack or escape. Stimulation of hypothalamus, amygdala, thalamus, tectum, central gray, and other structures may evoke this type of behavior. These areas have low fatigability. The presence or absence of attack differentiates true from false rage. Conditioning is not possible with false rage but some awareness and adaptation seem to exist. Evoked offensive-defensive reactions have the drive properties of true emotions and can induce learning and conditioning. The behavioral performance is similar to spontaneous rage including the selective direction of the attack against natural enemies and the social spread of aggressiveness.

D. BEHAVIORAL INHIBITION In 1875, Danilewsky demonstrated the cessation of spontaneous movements in the dog, produced by stimulation of the caudate nucleus. Since then, inhibitory effects have been repeatedly demonstrated. Recent symposia have summarized the present basic knowledge about neuronal inhibitory mechanisms (Florey, 1961; Roberts, 1960; Wolstenholme and OConnor, 1962), which will not be reviewed here. Extensive literature exists on the inhibition of spontaneous behavior evoked by brain stimulation, and this subject is summarized below. Three types of evoked inhibition may be considered: ( I ) Induced sleep, which has a slow onset, and affects all behavior. This is easily interrupted by sensory stimulation. (2) General inhibition, which starts immediately, affects all behavior and persists in spite of sensory stimulation. ( 3 ) Specific inhibition, which starts immediately, affects only determined behavioral patterns, and may or may not be modified by sensory stimulation. 1. Induced Sleep

It is well known that repeated monotonous sensory stimulations, such as the noise of a motor, the roaring of the sea or the rocking of a cradle have hypnogenic effects. The internal inhibition and the sleep induced by rhythmic sensory input may be interpreted as a protective function (Pavlov, 1957) against signals which have no meaning, while several other authors consider it a form of habituation. The cortex is necessary for this phenomenon because in the decorticated cat there is no habituation to visual or auditory stimuli; habituation depends on some inhibitory influence from the cortex

414

JOS$ M. R. DELGADO

upon the reticular activating system (Jouvet, 19el). It seems reasonable to expect that rhythmic stimuli applied to the sensory areas of the brain would induce sleep. However, experience has shown that sleep-like responses do not appear following cortical stimulation, but instead may be induced by activation of thalamus, caudate nucleus, and septum. The classical experiments of Hess (1944b) demonstrated that in the cat, electrical stimulation of the internal medullary lamina of the thalamus produced a sleep-like pattern. Several minutes after a 30to 60-second excitation, repeated two or three times at intervals of 2-5 minutes, animals previously alert paced slowly around looking for a place to lie down, curled up, and closed their eyes, with a spatial and temporal pattern of behavior similar to spontaneous sleep. Autonomic phenomena, such as pupillary constriction, relaxation of the nictitating membrane, and slowing down of respiration, as well as electroencephalographic signs of slower activity in the thalamus and cortex were similar to those present in natural sleep. The cats were less reactive to exteroceptive stimulation, but they could be easily aroused. These experimental results have been reviewed by Hess ( 1954, 1957); conErmed by Akert et al. (1952), Hess et al. (1950, 1953), Hunter and Jasper (1949), but could not be duplicated by Hamson (1940). The experimental conditions under which sleep can be evoked seem to be rather critical. Recently Akert (1961) emphasized the need for using special parameters of stimulation with the Hess waveform of dampened DC pulses, frequency below 8 cps, and a very low voltage. It is also necessary to give freedom of movement to the cats, to have quiet surroundings, to avoid sensory stimulation from the connecting wires or from the surgical wound, and to eliminate the interference of drugs. Some criticism has been expressed that the hypnogenic effect in Hess’ experiments was due to electrolysis rather than to electrical stimulation (Ranson and Magoun, 1939; Harrison, 1940), and Akert (1961) has answered some of these questions. The importance of the parameters of stimulation in the production of inhibitory effects was emphasized by Akimoto et al. (1956), who, working with dogs, demonstrated that stimulation of the diffuse thalamic projection system induced sleep with 5 cps, but the animals woke up if 30-90 cps were used. These experiments suggest that sleep was induced when the thalamic reticular

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system was driven at a slow rate similar to that of spontaneous EEG sleep activity. The mechanism of sleep induction by thalamic stimulation is not well understood, and no efforts have been made to learn whether it depends on stimulation of fiber tracts or of neurons in the intralaminar nuclei or in adjacent structures. Akert (1961) and Grastyh et al. ( 1955) mentioned anatomical and physiological similarities between recruiting responses and inhibitory effects, but they are not necessarily interdependent, and Morison and Dempsey (1942) did not find electrical-behavior relations between them. Electrical stimulation of the caudate nucleus also seemed able to produce a sleep-like effect (Akert and Anderson, 1951; Hess et al., 1953). The electroencephalographic activity is similar to that of spontaneous sleep, but the behavioral pattern is different and the animals are in an akinetic state with sensory functions little affected, and with awkward paw and ankle movements suggesting disturbed proprioceptive mechanisms. Heath and Hodes ( 1952) produced sleep-like behavior in monkeys, and also in one schizophrenic patient by stimulation of the head of the caudate nucleus. Stevens et al. (1961) also observed in the cat quietude, failure to respond to conditioned avoidance, and drowsiness when the caudate nucleus was stimulated electrically (8-10 cps), or chemically, with carbachol. Similar inhibitory effects are also reported by Buchwald et al. (1961a), using low frequency stimulation, but arousal was obtained by high frequency or by using strong stimulation (Buchwald and Ervin, 1957). Some authors (see discussion by Jasper, Akert, and Mettler in Heath and Hodes, 1952) doubt that the inhibitory effects induced from the caudate nucleus should be considered as true sleep, and Forman and Ward (1957) deny that inhibitory phenomena could be observed in the unanesthetized cat under caudate stimulation. The earlier reports are contradictory, and Danilewski (1875) described inhibitory effects while Ferrier ( 1873) and fachon and Delmas-Marsalet (1924) described activation and motor effects when the caudate nucleus was stimulated in dogs. Electrophysiological studies indicate that the caudate nucIeus has inhibitory functions upon cortically induced movements (Mettler et al., 1939); upon cortical activity (Gerebtzoff, 1941); upon the knee-jerk reflex Hodes et al. (1951), and upon seizure a&-vity in

41 6

J O d hf. R. DELGADO

other structures (Umbach, 1959). Spindles have been induced in the gyms cruciatus by low frequency stimulation and even by single shocks applied to the caudate (Buchwald et al., 1961a; Stoupel and Terzuolo, 1953). Cortical recruiting responses elicited from the caudate have been described by several authors (Buchwald et al., 1961a), but could not be detected by others (Laursen, 1961). The functional similarity between the caudate nucleus and the reticular system has been emphasized by several authors. Stimulation of both structures modifies monosynaptic spinal reflexes ( Peacock and Hodes, 1951),and may have facilitatory or inhibitory effects on the cortes (Fox and OBrien, 1962). In both barbiturate-induced and natural sleep the caudate shows the earliest electroencephalographic changes. The fact has been demonstrated in patients (Knott et al., 1950; Meyers et al., 19!j4), and in monkeys (Hodes et al., 1952), with implanted electrodes, indicating the importance of the caudate nucleus in the mechanism of sleep and in the inactivation of the cortes (Jung and Hassler, 1960). A problem in the interpretation of the results of stimulation in the caudate nucleus is the lack of precision in anatomical data of some published papers. The caudate nucleus, from its head to its tail, occupies a long stretch of brain, and even if most of the results are probably related to the stimulation of the head of the caudate nucleus, it is doubtful that this structure has a functional unity, or that stimulation of its medial part, bordering on the septum, should be considered identical with its lateral part, bordering on the internal capsule and putamen. In addition, changes in experimental conditions may explain some of the contradictory findings of the literature. Species differences may also be significant, and, as shown by Harman and Carpenter (cited by Mettler, 1959), the proportion of striatal tissue in the caudate is 83%in the fox, 81%in felines, 56% in lower primates, and 36% in higher primates. Stimulation parameters are also important. Stimulation of the thalamus with low frequency (5 cps) induced sleep, while higher frequencies ( 15 to 90 cps) awakened the animals (Hess, 1957; Akimoto & al., 1956). Caudate stimulation below 10 cps causes inhibition; between 20 and 30 eps produces “arrest-like” reaction; between 30 and 300 cps causes arousal (Buchwald et al., 1961b; see also Stevens et al., 1961). There is a close anatomical and functional correlation be-

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tween the intralaminar nuclei of the thalamus and the caudate area ( Cowan and Powell, 1955). According to Buchwald et al. ( 1961b), perhaps there is a caudate loop which receives input from the diffuse thalamic system, feeding back modified impulses to the nucleus ventralis anterior and then to the cortex. This system usually would be antagonistic to the reticular arousal system, but in some conditions it also could be synergetic. In addition to the thalamic and caudate systems, the septum should also be included among the sleep-inducing areas of the brain. As demonstrated by Rosvold and Delgado (1956) in free moving monkeys, intermittent stimulation of the septum with 60 cps for 2 seconds once every 3 seconds for about 5 minutes decreased spontaneous activity of the animals, which started dozing and finally closed their eyes, assuming a sleeping posture. The animals could be easily aroused, but immediately dozed again without further stimulation, with the effect lasting for about 15 minutes. In these experiments, three different effects were evoked from three different structures: the septum produced sleep; the head of the caudate nucleus, decreased activity and, in addition, the animal failed to give correct delayed-alternation responses; and the putamen produced circling and increasing activity. All these effects have been described by other authors as results of caudate stimulation. Perhaps some of them depend on spread of current to nearby structures. Stimulation of the septa1 region and the anterior hypothalamus in the monkey produced quiescence and dozing, but there was also a generalized loss of muscular tonus (see Fig. 5D), and this effect simulates the adynamia described by Hess (1944a) rather than natural sleep. It is surprising that sleep has not been induced by direct excitation of the main region which regulates the wakefulness-sleep rhythm: the reticular system (Jasper et al., 1958; Wolstenholme and O’Connor, 1962). As this system has arousal and also inhibitory properties, blocking of the first or excitation of the second should produce dozing. Its destruction produces a sleep-like state which is analogous but not identical with normal sleep. Quietude and a drop in authority have been observed during reticular stimulation in free-moving monkeys (Delgado, 1963a), but not sleep; the thalamus, caudate and septum are still the only areas in which direct

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electrical stimulation induces sleep. We should, however, keep in mind that a close correlation must exist between the “waking brain of Magoun and the “sleeping” brain of Hess. In szcrnnzury: Electrical stimulation of thalamus, caudate nucleus and septum in cats and monkeys may induce a pattern similar to spontaneous sleep. Mechanism of action and correlation among these areas remain to be elucidated. 2. General Inhibition

A variety of reactions have been described in the literature, having as their common denominator a cessation of spontaneous motor activity. Cortical inhibition of motor functions in anesthetized animals was demonstrated long ago by Bubnoff and Heidenhain ( l881), and by Hering and Sherrington (1897). Many years later, Tower ( 1936) observed that inhibition could be obtained from many cerebral areas, concluding that it was a general cortical function. Hypothalamic inhibition of motor effects evoked by cortical stimulation has been demonstrated by several authors in animals under anesthesia (see discussion and bibliography in Grastyhn et al., 1953). h4ore specific effects were described by the authors mentioned in Table VII, who worked with unanesthetized animals. The fact that motor inhibition was obtained by stimulation of so many different cerebral structures supports Towers’ conclusions. The effect described by Hunter and Jasper (1949) and subsequently observed by many other authors, may be defined as follows: sudden cessation of spontaneous motor activity, the animal remaining motionless with decreased responsiveness to sensory stimuli, but with maintanance of postural tonus and reflexes, without autonomic changes, and resuming spontaneous activity as soon as cerebral stimulation ceases. In this case, the animals are “frozen” in the position in which they were caught at the onset of the stimulation, as if a moving picture had been halted, for example, surprising a cat with its tongue out of its mouth or immobilized between two steps of a stair (Delgado, 1952b). It is confusing to notice in the literature the application of a variety of terms to similar reactions. The studies numbered 1 to 10 in Table VII should probably be included in the same group in spite of Goldzbands reservation that his dogs were easily distracted while Hunter and Jasper‘s cats even

TABLE VII INHIBITION OF BEHAVIOR GENEEAL Name of effect

Author

Year

Animal

Brain structure

ARREST REACTIONa ~

1. Arrest reaction

2. 3. 4. 5.

Suppression Cessation of walking Inactivation Syndrome Arrest reaction

6. Arrest reaction (searching, attention) 7. Arrest reaction

8. Cessation of walking 9. Freezing 10. Arrest reaction

Hunter and Jasper Ward and LeQuire Goldzband et al. Akert and Andersson Vigouroux et al. Gastaut Kaada Sloan and Kaada Delgado MacLean and Delgado Forman and Ward Buchwald and Ervin Bursten and Delgado

1949 1950 1951 1951 1951 1952 1951 1953 1952a 1953 1957 1957 1958

cat cat dog cat cat cat cat cat cat cat cat cat monkey

~~

Intralaminar nucleus of the thalamus Anterior cingulate gyrus Cortical and subcortical points of forebrain Caudate nucleus Amygdala Orbito frontal cortex-cingulate g.yrus Hidden motor cortex Pyriform cortex and lateral amygdala Putamen and internal capsule Pallidum, caudate nucleus, basolateral amygdala Septum

ADYNAMIAb 1. Adynamia 2. Hypotonic reaction

Hess Delgado

1944a 1960b

3. Quiescent behavior

Sheer

1961b

~__ - __ cat Lateral hypothalamus, preoptic area monkey Caudate nucleus, anterior cingulate g y r ~ s ,reticular system monkey Septum, anterior hypothalamus

a Arrest reaction is characterized by sudden cessation of spontaneous activities with preservation of suitable posture and muscular tonus. Adynamia is characterized by cessation of spontaneous activities and loss of muscular tonus without sleep.

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stopped while chasing mice. In all the above-mentioned experiments, the reaction affected the whole animal, and therefore the term “cessation of walking” seems too restricted. The word “supression” has connotations of electrical activity which may be confusing, and the writer thinks that the phrase “arrest reaction” of Hunter and Jasper is descriptive and suitable, as well as being the original term. Kaada ( 1960) grouped together the terms “arrest reaction,” “searching,” “attention,” and “arousal,” describing an initial cessation of all spontaneous activities followed by changes in facial expression, to attention or arousal, and then raising of the head, dilatation of the pupils, glancing movements, and sometimes circling. It is true, as Kaada claims, that searching may have an initial phase of cessation of all movements, but in the writer‘s opinion “arrest reaction” should be clearly differentiated from searching and arousal. In arrest reaction the animal is inhibited as long as stimulation is applied (one minute or longer in our experiments), resuming its interrupted activity as soon as stimulation is over. In searching, the animal often starts moving at the onset of stimulation without any period of arrest. Reactivity to sensory stimuli is decreased in one case and increased in the other. The pattern of responses is different, and, as stated by Jasper (1961), arrest reaction is not the same as aggressive arousal: “. . . neither is adequately described as simple awakening.” It is possible, however, that in some cases two different reactions (“arrest” and “arousal”) may be evoked from the same point or may appear mixed with each other, if the physical characteristics of stimulation were changed. It should be remembered that Buchwald and Ervin (1957) have claimed that from one single site in the basolateral amygdala of the cat, by varying the parameters of stimulation, it was possible to evoke “the whole gamut of behavioral responses reported by workers such as Kaada . . . to be functions of specific subdivisions of the amygdaloid.” The mechanism of arrest reaction is not well understood. Jasper (1961) suggests that perhaps it is related to interference in the integrated normal functioning of the thalamic reticular system, but the effect may be obtained also from some other areas of the brain. Hess (1957) believes that caudate stimulation inhibits the whoIe sensorimotor system and activates part of the integrated mechanisms

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for sleep. Mettler et d.(1939) has described in cats and monkeys a “melting away” of cortically induced movements if the caudate nucleus was stimulated. These results were not confirmed by Forman and Ward (1957). The decrease in activity and in readiness evoked by caudate excitation is perhaps different from the one elicited by thalamic stimulation, because the exteroceptive and vegetative mechanisms are more normal in the first case than in the second. There is probably a close functional interdependence among several of the cerebral structures from which arrest reaction may be evoked, and stimulation of one of them may activate another. The author has experimental evidence suggesting that stimulation of different points within the limbic system activates the areas with greater excitability. The type of response may thus depend more upon a low threshold area than upon the stimulated point (Delgado and Hamlin, 1960). In monkeys septal stimulation may evoke an afterdischarge in the hippocampus with minor electrical disturbances of the stimulated point, and it is conceivable also, that in the absence of an after-discharge, stimulation of one point may activate the function of distant structures, Part of the behavioral inhibition evoked by septal and caudate stimulation perhaps should be related to amygdala-hippocampal activation (Gloor, 1955); or it may depend on the intralaminar nucleus of the thalamus with which a close anatomical relationship exists (Cowan and Powell, 1955). When electrical afterdischarges are evoked, it would be necessary to undertake a thorough exploration of the brain in order to estimate the anatomical correlates of the evoked effects. The behavioral inhibition observed during petit ma1 activity (Ingvar, 1955), should not be identified with arrest reaction because it involves autonomic disturbances and automatisms not present in arrest reaction. However, the inhibition observed in both may have similar mechanisms (Hunter and Jasper, 1949). Arrest reaction and inhibition of instrumental responses in the monkey have been demonstrated by electrical stimulation of the septal area (Bursten and Delgado, 1958; Delgado, 1957). Monkeys were trained to approach a light within 2 seconds to avoid an electric shock to the feet, and the animals performed with reliability above 90%.During septal stimulation, the monkeys kept their eyes

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open, and maintained a good postural tonus, but did not respond to the light or even to the shock, remaining motionless until the septal excitation was over. Arrest and bewilderment of the animal during hippocampal after-discharges propagated to the limbic circuit have been described bv several authors (Andy and Akert, 1955; Delgado and Sevillano,’1961, and others). Injection of chemicals into the hippocampus (XIacLean, 1957) or of alumina cream into the caudate nucleus (Spiegel and Szekely, 196l), produced catatonic effects and decreased sensory reactivity. But these effects related to epileptic or abnormal activity probably involve large areas of the brain and are less specific than the arrest reactions. Another type of general motor inhibition has been described by Hess (1944a) as “adynamia,” which is characterized by the animals lying down on the floor with decreased muscular tonus, abnormal posture, diminished spontaneous activity, but without being asleep. The main difference between arrest reaction and adynamia is the existence of normal muscluar tonus in the first, and considerable hypotonia in the second. The distinction is clear in typical cases, but may be difficult to ascertain precisely when the hypotonia is only moderate. Adynamia has been evoked in the cat by stimulation of the lateral preoptic area, and lateral anterior hypothalamus (Hess, 1944b, 1957). It should be emphasized that Grasty6n et al. (1953, 1955); Grossman and Wang (1956), and Grossman (1958), in cats under anesthesia, have described a somatomotor inhibitory system in the thalamic reticular area and in the ventral part of the hypothalamus, which can be traced up to the septal nuclei and the lateral part of the head of the caudate nucleus. Inhibitory properties of all these structures should have in this way a functional correlation. In the monkey, stimulation of the septal region and of the anterior hypothalamus between the optic chiasma and the anterior commissure produced a diminution of spontaneous activity, less reactivity to external stimuli, quiescence, dozing and generalized loss of muscular tone “resembling the state described by Hess as adynamia,” (Sheer, 1961b). A similar hypotonic reaction has been observed in the monkey by excitation of the anterior cingulate gyms and of some points of the reticular formation (Delgado, 1960b).

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A summay of anatomical representation and functional characteristics of arrest reaction and adynamia may be seen in Table VII. 3. Specific Inhibition Some inhibitory effects may be localized to a few or even to only one behavioral manifestation, and may pass unnoticed unless the observation was directed towards the specific effect and the experimental conditions were appropriate. For example, inhibition of the use of one limb would not be detected if the animal was lying down; inhibition of appetite could not be demonstrated unless the animaI was hungry and food was offered. Probably many behavioral categories may be inhibited by cerebral stimulation, but the availabIe information is related mainly to inhibition of motor, alimentary, and aggressive reactions. a. Motor Znhibitwn. Electrical stimulation of some areas of the motor cortex may produce a localized motor deficit. For example, in the cat, mild stimulation of the cortical area of the right foreleg did not stop spontaneous walking, but inhibited the use of the limb and made the animal limp (Delgado, 1952b). In the monkey, cortical stimulation of area 4 evoked flexion of both arms, putting them out of voluntary control. Peanuts were then offered to the animal, which promptly took the food with its feet, showing that the purposeful organization of movement had not been disrupted by the cortical stimulation. In monkeys free in the colony, diminution or lack of use of one limb in spontaneous activities such as grooming or feeding themselves, has been evoked by electrical stimulation of area 6. Localized motor inhibition has a great variety of manifestations. This type of inhibition in general may be determined by two kinds of mechanisms: ( a ) direct neuronal blocking, which impedes the transmission or the organization of the impulses necessary for the movement at any synaptic level; and ( b ) indirect inhibition depending on reciprocal innervation or on the predominance of one group of muscles over the agonistic. Localized motor inhibition also has been produced in man. As reported by Penfield and Rasmussen (1950), and commented on by Feindel (1961) voluntary apposition of the thumb to the little finger may be stopped by stimulation of the precentral hand area, and arrest of different types of repetitive movement have also been

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observed by electrical excitation of the primary, secondary, and supplementary motor areas. In some cases, during stimulation, patients expressed the desire to move an extremity, but were unable to do so. b. Alimentary Inhibition. In the M a c a m rheas, as demonstrated in a film2 (see also Delgado, 1957), and illustrated in part in Fig. 5, stimulation of the head of the caudate nucleus produced ( a ) loss of interest in food, dropping it if it was already being held, or rejecting the food with the paws if it was forced close to the mouth; ( b ) immediate cessation of eating if the animal was already biting into a banana; ( c ) spitting out of any food in the mouth, and if the animal had filled its pouches, active movements with the paws trying to empty them; ( d ) grimaces of distaste; ( e ) movement of the whole body away from the proximity of food. In these experiments there was a specific inhibition of a behavioral category-food intake-without generalized inhibition of the animal, which, on the contrary, showed active motor performance of acts related to the rejection of food. In the same animal we have seen two types of effects: pure inhibition of alimentary reactions as described above, and general inhibition of the animal with decreased aggressiveness and diminution in postural tonus, as shown in Fig. 5D.Both effects were obtained by stimulation of the medial part of the head of the caudate nucleus from two different points 3 mm apart. Cessation of chewing and ejection of food evoked by electrical stimulation of septum, amygdala and other structures in monkeys have also been obtained by Robinson and Mishkin ( 1962), who emphasized that brain stimulation modified only alimentary reactions without producing observable effects in the absence of food. In a systematic exploration of the anatomical location of food inhibitory areas ( Rubinstein and Delgado, 1963), three monkeys were prepared with up to 100 needle guides spaced 2 mm apart on the skull over the septal-caudate-cingulate-amygdalaregion for the exploration of cerebral tracts at l-mm steps. Several other monkeys were studied with permanently implanted electrodes in these areas. Food inhibition was obtained mainly from the head of the caudate

’ “Brain Stimulation in the Monkey: Technique and Results,” distributed by the Psychological Cinema Register, Pennsylvania State University, University Park, Pennsylvania.

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nucleus and the septum, but not from the rest of the explored structures. In most cases, there was a combined inhibition of food intake and of aggressiveness, but some points gave predominantly one or the other effect. Radio stimulation of the monkeys free in the colony demonstrated that caudate and septa1 excitation abruptly stopped food intake, and also any movement being made towards the food. This inhibition lasted up to 150 seconds without fatigue. Some of our results agree with the experiments of Knott and Ingram (196l),in which cats were trained to press a bar to obtain food. Stimulation of the septum and also of the anterior and posterior hypothalamus inhibited the bar-pressing response. Working with cats, some of them stimulated by radio control, Fonberg and Delgado (1961) demonstrated that excitation of the basolateral and anterior part of the amygdala inhibits spontaneous food intake and also conditioned alimentary reactions in hungry animals without signs of fear or anxiety. In these experiments, the inhibition of food intake was immediate and chewing stopped at the onset of the stimulus without any other observable symptoms. The inhibition of food intake usually outlasted the five-second stimulation by several minutes, and in one case lasted for three days. In some experiments the animal moved to the corner of the cage opposite the food tray, and refused to eat even when food was put into its mouth. If the cat was playing with the investigator, or resting, stimulation of the amygdala at the low intensities used in these experiments (0.1-0.6 mA) did not disturb the play or rest. Stimulation of other areas, such as cingulate gyrus, motor cortex, gyrus olfactorium, and corpus callosum did not produce inhibitory effects below 0.6 mA and with higher intensities-up to 2.5 mA, the inhibition of food intake was transient, with adaptation if the stimulations were repeated. An inhibitory effect on food intake has been obtained by stimulation of the medial hypothalamus in the rat (Smith, 1961), but could not be demonstrated in the cat (Delgado and Anand, 1953). Working with unanesthetized squirrel monkeys, MacLean and Delgado (1953) showed that stimulation of the pyriform cortex inhibited the eating of a banana and the animal spit the fruit into the palm of its hand, peered at it with grimaces of distaste and tried to escape from the testing stage. In our opinion, food intake is regulated by two systems which

FIG.5 . Control: monkey tries anxiously to grab a banana offered to him. B. As soon as the head of the caudate nucleus is stimulated, interest in food is lost. C. Monkey turns away from the banana, showing that motility is not arrested, that muscular tonus is well kept, and that awareness is preserved. D. Stimulation of head of caudate nucleus, but of a different point-3 mm away from the area stimulated in B and &produced also a loss of interest in food plus a generalized hypotonicity. T h i s reaction is similar to the adynamia described by Hess. 426

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Fig. 5 C and D. See facing page for legend.

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have a functional relationship, but are not necessarily dependent on one another; the ventral-lateral hypothalamic area (“satiation” and “feeding” centers) (Anand and Brobeck, 1951; Anand and Dua, 19,S; Brobeck, 1957; Wheatley, 1944; and Wyrwicka et al., 1959), and the amygdaloid-septal-caudate system ( Brutkowski et al., 1960; Delgado, 1957, 196Ob; Gloor, 1955; Green et aZ., 1957; and Morgane and Kosman, 1960). Inhibitov effects are easier to obtain by stimulation of the latter system than of the former (Knott and Ingram, 1961; Robinson and Mishkin, 1962). c. Inhibition of Aggressiueness. Arrest reaction and adynamia, as mentioned earlier, produced a general inhibition of activity which implied the absence of aggressive acts normally present in ferocious animals such as the rhesus monkey. In other experiments, however. it was possible to evoke a specific inhibition of aggressiveness without changes in the general behavior of the animal. If a glove, ii stick, or the experimenter’s hand suddenly approached a monkey under restraint on a chair, the animal usually opened its mouth, showing teeth, moved its head forward, vocalized with a characteristic low-toned sound, and tried to grab, to scratch, and to bite. When the monkey was free in a colony, the reaction against a threat was similar, with the difference that attempts to escape alternated with pawing the floor, shaking the cage, and launching sudden attacks. At any moment before or during threatening situations, stimulation of some points of the caudate nucleus made the animal close its mouth and lose its aggressive attitude; it was then safe to touch the animal, and even to put a finger in its mouth (Fig. 6 ) . The animals, however, did not seem to be depressed, arrested, adynamic or stuporous. They were well oriented, alert, able to follow moving objects with their eyes, and to use their paws to push away a glove or a stick close to their faces. The monkeys had a well-preserved muscular tonus, and good coordination of spontaneous movements, without biting, threatening attitudes or destructive acts. As soon as the caudate stimulation stopped, the animals reverted to their usual ferocious behavior. The fact that inhibition of aggressiveness was obtained without changes in other behavioral manifestations indicates that this effect had a specific cerebral representation. This effect, however, appeared in its pure form in only a few cases, and more often inhibi-

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FIG.6. A. Monkeys usually are aggressive and try to scratch and bite anything within reach. B. Stimulation of some points of the caudate nucleus inhibits aggressiveness and it is safe to touch the monkey with bare hands. Observe that monkey is not depressed and is aware of the approaching hand.

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tion of aggressiveness was combined with inhibition of food intake, and in several experiments some degree of hypotonia, diminished sensory reactivity and inhibition of instrumental performance indicated a generalization of the inhibitory effect. VI. Fragmental Organization of Behavior

A. EXPERIMENTAL DATAAKD THEORY

The performance of behavior requires a sequence of motor acts typical for each category within a range of variability. Suppose, for example, that we offer an orange to a monkey. First there is an orienting reaction, looking at and recognizing the quality of the object offered. Then, if the animal is interested and is not afraid, there is an approaching response, and the monkey walks a few steps forward and extends a paw, reaching for the food, which then may be taken between both paws, cleaned, licked, bitten, chewed and swallowed in the consumatory response. The whole sequence of acts constitutes the complete behavioral pattern of food intake. This pattern may be analyzed in each of its several components as a series of behavioral fragments involving different parts of the body, with temporal programming towards the achievement of a specific aim. Each fragment may be studied in greater detail with respect to its motor characteristics and with respect to the areas of the brain involved in its performance. Analysis of these responses introduces a basic question. H o w is behavior organized?-as a homogeneous motor performance, or as fragments with considerable independence which may be assembled together for a purpose? The author’s thesis is that behavioral fragmcnts hate amtomicd and functional realit9 inside of the brain. In the above-mentioned example of food intake, none of its successive acts can be considered as specific of alimentary reaction, because the animal may orient itself towards many different stimuli, may use its limbs for many different purposes, may swallow because it is anxious, and may bite in a fight. It is the successive acts taken together which constitute a specific pattern of alimentary reaction. A more detailed analysis of the act of licking will clarify the concept of the fragmental organization of behavior. Licking may be a spontaneous act, or may be evoked, for example, in the cat by electrical stimulation of the suicus presilvius. In spite of its simple appearance,

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it is an exceedingly complex reaction, with intervention, among other organs, of the tongue, lips, jaw, facial and neck muscles, plus a complicated postural adjustment of the head, trunk, and limbs. The majority of the muscles of the body participate directly or indirectly in the act of licking. In a detailed analysis, licking may be subdivided into simpler elements, such as motility of the tongue, of the lips, jaw, and other organs; tonus of the head, of the limbs, of the trunk, and so forth. Each element may be analyzed further: for instance, it may be determined how much the tongue protrudes from the mouth, the speed of movement, the number of lickings per unit of time, and the shape of the tongue, whether flat, pointed, or spatulate. Among all the combinations of movements and postures, some elements are indispensible, while others are accessary. Without the tongue, without the hypoglossus nerve, there is no licking. However, the animal may lick in different postures, whether blind or deaf, or even under anesthesia, if the corresponding cerebral point is stimulated. The anatomical representation of licking is extensive and includes the orbital cortex, amygdala, pyriform cortex, hypothalamus, preoptic area, and ventral nucleus of the thalamus ( Akert, 1961; Andersson, 1951; Baldwin et al., 1954; Delgado, 1952b; Gastaut, 1952; Hess, 1954; Kaada, 1951; Larsson, 1954; MacLean and Delgado, 1953; Rioch and Brenner, 1938; Takahashi, 1951) but does not appear when many other areas of the brain are stimulated. The so-called motor areas are not essential, because the decorticated animal is still able to lick. During the spontaneous activities of the animal, licking usually has a purpose which depends on experience, memory, conditioning, learning, drive, and other more or less obscure psychological entities. During brain stimulation, evoked licking may also have a purpose as directed by the free will of the animal, which may lick the floor, our hands, or its own body (Delgado, 1952b). From the functional point of view, licking may be part of one of the following behavioral patterns: ( I ) Alimentary reactionAmong several subcategories, we should mention: cleaning of food before eating, tasting of the food, taking semiliquids and drinking. ( 2 ) Exploration of the surroundings-licking the sides or the floor of the cage, or various objects such as sticks, balls, wire mesh, and so forth. (3) Body cleaning-in the individual category of selfgrooming, and also in the social category of grooming others. ( 4 )

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Maternal behavior-for the grooming and care of the offspring. ( 5 ) Playing-which may be part of exploration, but at other times is typical play, as in the case of monkeys licking the glass front of the cage, and smearing the saliva with a paw. (6) Cooling-In the opossum, licking is used as a cooling device. ( 7 ) Submissive behavior-as when a dog licks the hand of its master. (8) Sexual behavior-licking their own or their partner’s genitalia. ( 9 ) Accessory motor manifestations-such as licking movements during the performance of a difficult task by children, The act of licking, therefore, should not be necessarily related to feeding mechanisms, as some authors assert. Licking is a phasic activity involving the precise timing of several motor activities, such as opening of the mouth, protrusion of the tongue, extension of the neck muscles, withdrawal of the tongue and closing of the mouth, It would be difficult to explain how a monotonous excitation of one point of the brain could produce such a well-organized and well-timed sequence, unless we accept the existence of a specific neuronal organization which has been triggered by an unspecific stimulus (electrical in this case). It is possible that matemal behavior, alimentary reactions, submissiveness, sex, and the other forementioned categories have a determined and distinct anatomical representation in the brain. It is not probable, however, that the motor pattern of licking would have a separate cerebral representation for each behavioral category. Rather, we may suppose that the act of licking has a single neuronal organization. It may be considered a fragment of behavior, which may be used for different purposes. This does not imply the existence of one “center” of licking in the old sense of a single and determined structure, but we may accept the theory that licking has a central organization with one or more areas responsible for the initiation of the act and for the functional coordination of several other cerebral structures. These areas are perhaps located in different parts of the motor cortex, hypothalamus, cerebellum and other regions, and may be modified by different types of sensory and central influences, which also can modify each level of the efferent circuits. In this way, the central organization is dynamic and flexible, but does not involve the whole brain, and probably we can exclude from the basic motor organization of licking, areas from the frontal, temporal, and occipital lobes and many other regions of

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the brain. At the same time, many of these areas could be associated with licking and might even evoke this act by being electrically stimulated, if, through experience, learning and training, a functional association had previously been established. Fragments of many different behavioral categories have been described in the literature by several authors. Recent studies have shown that penile erection without a complete sexual pattern may be evoked in the squirrel monkey by stimulation of the septum, anterior and midline thalamic nuclei, hypothalamus, cingulate gyrus, and other structures ( MacLean and Ploog, 1962). In the rat, ejaculation has been elicited by hypothalamic stimulation without observation of mounting or attempts to copulate, suggesting that “. . . we may be dealing with a network of nuclei rather than with any single center” (Miller, 1961). In roosters, the behavior evoked by cerebral stimulation “consists of a chain of different individual movements, that are all related to a single goal.” Each of these effects can be evoked individually from different cerebral foci, but all of them are subordinated to the evoked drive. When individual movements are evoked, the electrical stimulation “merely furnished an impulse for a particular movement.” In the case of evoked drives, “the stimulus sets up a goal that can be attained in different ways, depending on external circumstances” (von Holst and von Saint Paul, 1962). There is also abundant clinical data in favor of the anatomical and functional existence of behavioral fragments. Kliiver mentioned that “affectively charged experiences are not necessary to produce the appearance of spatiotemporal fragments and the splitting up into isolated and very often relatively meaningless details. In fragmentary eidetic imagery the reproduction of words or figures may even start with such elements or fragments as a part of a ‘u’, the dot of an ‘i’, the upper part of an ‘8’, or the lower part of a ‘6”’ (Kluver, 1958; see also 1933). Other examples may be found in the extensive investigations of different types of aphasia. Speech has four basic elements: namely, phonation, respiration, articulation and resonation, but each of these elements is not necessarily related to speech, as is evident especially in the case of respiration, and each has independent anatomical representation in the brain, as demonstrated by the fact that cerebral stimulation may evoke changes in respiration, sounds without any meaning, or the articulation of

words in a completely normal manner. The above-mentioncd four fragments must be precisely coordinated in the elaboration of speech (Garde, 1953: Husson, 1952), but they are only a pattern of movement, which must be related with other ps~7chological mechanisms with symbolic basis for the expression of thought ( Walshe, 1947). Disturbances may occur independently in the articulation of words, in their recollection, identification or expression, in their emotional quality, or in other aspects of speech, indicating the existence of different fragments, which are usually precisely coordinated, but not necessarily related to each other. la sirnznury: Behavioral categories are conceived of as an assembly of motor patterns, each one having individuality and specialized organization. -411 these patterns are combined in time and space under the supervision of a special set of cerebral structures which is responsible for the behavioral category as a whole, but not for the specific motor details.

H.

POSTULL\TES

The theory of fragmental organization of behavior has not been formally stated in the literature, but its spirit may be recognized in many publications. In animals trained to open a problem box, bilateral partial destruction of the motor cortex does not prevent the animal from performing the learned response, and if there is a motor deficit, a different pattern of movement is used. This motor equivalence (Lashley, 1924) and the principle of stimulus equivalence (Kliiver, 1933; Lashley, 1948) demonstrate the relative independence of a central organization of motor patterns which may be chosen for the behavioral performance according to the requirements of the experimcntal situation. In general, the theory of the fragmental organization of behavior may be formulated as follows: 1. Each behavioral category is formed by a series of motor fragments organized in space and in time. 2. Single fragments of behavior may form part of different behavioral categories, and therefore have a different functional meaning. For example, (as stated earlier) licking may be part of alimentary, exploratory, sexual or other reactions. 3. Fragments of behavior may have a functional affinity and form

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a sequence, like notes of a melody, This linkage is reinforced by usage. 4. Fragments of behavior may be linked together by learning. This occurs during conditioning, and may help to explain individual variability of functional representation. 5. Fragments of behavior are not rigidly organized, but may be modified by external and internal stimulation, including proprioceptive information. 6. Motor performance of each fragment of behavior depends on a set of cerebral structures which is modified and simplified by usage. Temporal-spatial coordination of fragments which constitute a behavioral category depend on a different set of cerebral structures. Deficits may appear in the performance of a fragment of behavior (local trauma of the hypoglossus nerve may handicap tongue movements), or the deficit may be in the coordination of the fragments of behavior and the activity may appear to lack logical content. For example, stereotyped movements during psychomotor epileptic attack, false rage in decorcitated animals, or nail-biting in anxiety. 7. The cerebral structures which initiate and coordinate the behavioral fragments are mainly responsible for the choice of independently organized motor patterns, which also may have a mutual influence (food may be taken by lowering the head or grasping with either hand, and monkeys can even use their feet). Perhaps in some cases this initiation and coordination of behavior may be identified with a central motive state (Beach, 1958; Morgan, 1959). Initiation and termination of each type of behavior may depend on different cerebral structures, as has been demonstrated experimentally by Deutsch (1960) in the case of drinking. 8. The organization of these general mechanisms of behavior, as well as the organization of each behavioral fragment, depends to a great extent on the previous history of the individual. For example, an enraged person may display aggressive behavior, but his motor performance will be very different according to whether he is accustomed to a sedentary life or has been trained in boxing or jujitsu. Possibly one of the merits of the theory of fragmental organization of behavior is that it may be useful as a working hypothesis to analyze the elements which form each behavioral category; to investigate the cerebral representation of each of these elements, and

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to study the areas of the brain which coordinate and integrate these different elements. VII. Summary

The study of the total spontaneous behavior of groups of animals (cats and monkeys) provides a base line with which effects evokcd by cerebral stimulation may be compared. Complete freedom of experimental subjects requires the use of special techniques. Objective, continuous day and night recording of behavior is accomplished by telemetry and by time-lapse photography. Spontaneous behavior has been classified quantified and analyzed in time and in space, establishing individual and social correlations. Spontaneous behavior is unpredictable in short samples of several minutes, but has a limited and predictable range of variability in samples obtained over several hours, Methods have been developed for wireless electrical and chemical stimulation of the brain of free animals. These methods are based on the reception of a radio signal which closes a circuit in order to apply a predetermined amount of stimulation. Electrical stimulation of the brain is a rather crude procedure, and to explain the finesse, coordination and drive of many of the evoked reactions, it is necessary to assume the activation of physiological mechanisms. This assumption is supported by several facts. Many of the evoked effects have physiological qualities and may interact algebraically with voluntary activity. Adaptation to unforeseen circumstances, which is characteristic of spontaneous behavior, a150 exists in evoked reactions. Depending on the stimulated ccrebra1 area, the evoked effects may fatigue in seconds, in minutes, or mav not fatigue at all. In general behavioral aftereffects have not been observed, but in some cases a few seconds stimulation may modify a specific type of conduct for days. Under the same experimental conditions, brain stimulation is reliable, and the principle of stability of cortical points should be accepted. Comparing results of cerebral excitation among different animals is handicapped by the existence of anatomical variability and also by the less known physiological variability. The range of variability, however, has limits, which may be determined experimentally. In general, brain stimulation may evoke: ( a ) stereotyped tonic

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or phasic activity without any emotional disturbance; ( b ) driving activity characterized by the aim to reach an objective, with a motor performance adapted to the relations between subject and purpose; ( c ) change in behavioral tuning which may not be detected in isolated animals because of the lack of manifestations, but may modify decisively the character of response to normal stimuli; ( d ) inhibition of spontaneous or evoked behavior; ( e ) abnormal effects such as tremor or seizures. A summary of specific types of evoked effects (motor, sequential, offensive-defensive sleep, arrest, inhibition of motor, alimentary and aggressive reactions) may be found in the corresponding sections of this paper. To explain performance of spontaneous behavior and results of cerebral stimulation, a working hypothesis of fragmental organization of behavior is proposed. Behavioral categories are formed by a series of fragments which have anatomical and functional bases inside the cerebral organization. Single behavioral fragments form part of different behavioral categories and may have a different functional meaning. The act of licking is a good example because it forms part of alimentary, exploratory, maternal, submissive, sexual, and other types of behavior. Motor performance depends on a set of cerebral structures. Choice of the motor patterns and their temporospatial organization depends on a different set of cerebral structures. Behavioral deficits may be related to a disturbance in the motor performance, or in the general coordination of behavioral fragments. In the latter case the activity may lack logical content. Fragments of behavior may have a functional affinity and form a sequence like the notes of a melody. This linkage may be established by learning and reinforced by usage. ACKNOWLEDGMENTS The collaboration of Mrs. Caroline S. Delgado, who reviewed behavioral literature and wrote part of Section I11 is warmly acknowledged. The collaboration of Dr. Rafael Rodriguez Delgado in part of the theoretical and experimental studies on social behavior is also gratefully acknowledged, Our investigations have been supported by grants from the United States Public Health Service, the Office of Naval Research and the Neuro-Research Foundation. The following photographs are reproduced with the kind permission of their publishers: Fig. 5, Life Magazine; Fig. 6, Yale University Press Bureau

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AUTHOR INDEX Numbers in italic show the page on which the complete reference is listed. Amassian, V. E., 169, 184, 192, 213, 251 Abe, K., 414,416,438 Amini, F. B., 309, 346 Aboul-Nour, B., 54, 83 Anand, B. K., 397, 398,403,425,428, Abraham, D., 18, 93 438, 440 Acs, G., 43, 44, 51, 52, 53, 83, 91, 98 P., 108, 112, 120, 141, 144, Andersen, Adams, C. W. M., 25, 64,65, 66, 79, 399, 403,444 83, 96 Anderson, E., 75, 97 Addison, W., 283,293 Ades, H. W., 173, 177, 178, 180, 187, Anderson, F. D., 167, 184 Andersson, B., 374, 415, 419, 431, 438 415, 421, 445 Andersson, E., 267, 293 Adey, W. R., 310, 316, 345, 346, 375, Andy, 0. J., 422, 438 388, 448 Anfinsen, C. B., 48, 63, 66, 69,93, 96, Aelony, Y., 17, 83 97 Agassiz, L., 107, 141 P. J., 80, 91 Angeletti, Aguilar, V., 11, 84 Anker, H. S., 46, 93 Ainsworth, S., 21, 93 Ansell, G. B., 61, 63, 64,65, 83 Aitken, R. C. B., 307, 311, 347 Ajmone-Marsan, C., 154, 162, 173, Anthony, J., 128, 145 178, 180, 184, 186, 189, 198, 213, Apelbaum, J., 363, 440 Apgar, J., 53, 89 214, 215, 217, 251, 255 Akert, K., 395, 414, 415, 419, 422, Appel, E., 5, 83 Appel, K. R., 5, 83 431, 438, 443 Appelmans, F., 62, 86 Akimoto, A., 414, 416, 438 Apter, J. T., 155, 160, 163, 184 Albert, S., 23, 89 Araki, T., 230, 235, 251, 255 Alcarez, M., 169, 172, 186 Arana, R., 402,447 Allee, W. C., 358, 438 Arat, F., 22, 96 Allen, E. H., 45, 52, 53, 83, 95, 283, Arcuate, L. R., 289, 298 293 Arden, G. B., 172, 184, 209, 211, 251 h e y , L. B., 153, 159, 184, 185 Allen, M. L., 4, 88 Arloing, S., 107, 124, 125, 141 Allen, W. F., 113, 141 Aronson, L. R., 356, 439, 448 Allfrey, V. G., 51, 83, 87 Arrheniuq E., 59, 89 Alonso de Florida, F., 358, 374, 381, Arteta, J. L., 288, 293 438 Aschheim, E., 72, 96 Altman, J., 28, 83, 155, 156, 157, 161, Ashworth, M. E., 71,86 162, 163, 165, 167, 169, 170, Ask, F., 267, 293 173, 174, 175, 178, 184, 283, 293 Askonas, B. A., 2, 46, 48, 83, 84 Alvord, E. C., 23, 89 Atlas, D., 384, 395, 438, 445 Amador, L. V., 383, 384, 438 Attardi, D. G., 267, 293 Amaducci, L., 24, 25, 83 Atwood, J. M., 309,347 451

A

452

AUTHOR INDEX

Austin, G . M., 292,297 Austin, J . L., 102, 141 Ayers, H., 109, I41

B Babson, A. L., 49, 84 Bach, L. X1. N.,361, 447 Badier, M.. 403, 419, 441, 448 Bard, P., 404, 438 Balfoni, G. M., 271, 293 Bagdenamian, U., 68, 96 Bagel, S., 307, 347 Bailey, B. F. S., 20, 21, 24, 84 Bailey, P., 384, 446 Bainbridge, R., 105, 134, 141 Bak, A. F., 242, 254 Bakty, L., 4, 84 Raker, C. l'., 289, 291, 297, 299 Bakker, C. B., 309, 346 Balado, M., 159, 184 Baldwin, H. A., 361, 447 Baldwin, hi., 431, 438 Bales, R. F., 366, 438 Ballou, J. E., 40, 41, 96 Ban, T. A., 308, 313, 346 Banister, J., 75, 84 Bard, P., 403, 405, 438 Barets, A., 99, 108, 117, 121, 125, 129, 130, 131, 132, 136, 138, 141, 141, 142 Barfurth, D., 274, 293 Barker, D., 104, 105, 117, 142 Barkulius, S. S., 11, 84 Barlow, H. B., 208, 209, 251. 254 Barnard, J. W., 286,293 Barris, R. W., 157, 158, 161, 162, 163, 184, 165, 166, 167, 173, 174, 175, 176, 177, 178, 180, 182, 184, 189 Barron, K . D., 21,84 Bart, B., 17, 83 Bartorelli, C.,357, 438 Basilio, C., 57, 95 Bassett, C. A. L., 287, 290, 293, 294, 298 Bassham, J. A,, 13, 92 Bates, H. M., 51, 89

Bauer, H., 20, 84 Baum, J., 115, 133, 142 Baust, W., 361, 438 Baxter, C. C.,6, 94 Bayliss, 0. B., 65, 79, 83 Beach, F. A., 435,438 Beams, H . W., 262, 298 Beaufay, H., 62,84 Becker, M. C., 206, 230, 232, 239, 240, 253 Beech, 13. R., 308, 313, 314, 331, 343, 347 Beevor, C. E., 182, 184 Beidler, L. M., 171, 188 Belik, Y.V., 25,42, 74, 93 Beljanski, M., 60, 84 Bell, E. T., 270, 293 Bellegie, N. J., 292, 295 Belo€€-Chain, A,, 11, 84 Bellrose, F. C., 361, 444 Bennett, M. G. L., 231, 232, 251 Benzer, S., 53, 84 Beresford, W. A., 167, 173, 178, 180, 184

Berg, P., 2, 52, 53, 54, 57, 84, 85, 94, 9s

Bergmann, F. H., 52, 53, 84, 94 Berkowitz, E. C., 138, 142 Berl, S., 5, 11, 13, 14, 15, 17, 31, 32, 84, 90, 94 Berleur, A. M., 62, 84 Berman, A. L., 205, 254 Berman, hl., 75, 92 Bernsohn, J. I., 21, 84 Berry, C. M., 157, 167, 173, 175, 177, 184, 186 Berry, J . F., 75, 84 Beskow, G., 59, 89 Bessman, S. P., 4 9 5 Best, C. H., 386, 438 Bianchetti, G., 357, 438 Bickford, R. G., 354, 438 Bidwell, R. G. S., 13, 96 Bielschowsky, M., 282, 292, 293 Biermann, L., 13, 97 Birch, H. G., 353, 439 Bishop, G. W., 157, 158, 162, 165,

AUTHOR INDEX

174, 178, 180,184,185, 192, 196, 198, 235, 251 Bishop, J. E., 54, 57, 84 Bishop, P. O., 153, 159, 184, 192, 195, 196, 197, 198, 199, 200, 201, 203, 204, 205, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 241, 242, 243, 245, 251, 252, 254 Blatt, N., 267, 293 Blech, W., 62, 88 Blinn, K. A., 360, 446 Block, M. A,, 17, 87 Block, R. J., 26, 84 Bliimcke, S., 155, 184 Blum, B., 361,441 Blum, J. S., 159, 185 Blum, R. A., 159,185 Bock, F., 16, 89 Boddeke, R., 124, 127, 132, 134, 142 Bodian, D., 163, 166, 170, 184 Boeke, J., 109, 110, 142 Bohner, B., 304, 310, 314, 316, 333, 334, 344, 347 Bolling, D., 26,84 B o h a n , J. L., 17, 87 Boman, H. B., 52, 91 Bone, Q., 109, 112, 113, 115, 117,142 Bonner, J., 57, 84 Booij, J., 24, 84 Booker, B., 47, 80, 81, 91 Borek, E., 28, 71, 84 Born, G., 270,293 Bornschein, H., 209, 252 Borenstein, P., 162, 173, 175, 180, 181, 185 Borsook, H., 42, 45, 50, 84, 85 BouIanger, G. A., 127, 142 Bourne, G. H., 22, 44, 96 Bovis, M., 17, 83 Boyarsky, L. K., 74, 75, 95 Boyd, I. A., 103, 104, 142 Brachet, J., 50, 57, 85

453

Brady, J. V., 383,439 Braekkan, 0. R., 117, 132, 142 Brandt, I. K., 51, 91 Brattgard, S.-O., 71, 73, 79, 85 Braus, H., 259, 293 Brazier, M. A. B., 350, 439 Breakell, C. C., 360, 439 Bremer, F., 172, 184 Brenner, C., 431, 446 Bridgman, C. F., 104, 142 Brindley, G. S., 159, 170, 184 Brinley, F. J., Jr., 229, 254 Britten, R. J., 13, 85 Britton, S. W., 405, 439 Brobeck, J. R., 428, 438, 439 Brock, L. G., 192, 219, 228, 229, 239, 241,252 Brodal, A., 167, 184 Brodmann, K., 152, 177, 180, 184 Brody, E. B., 351, 439 Brouwer, B., 159, 163, 184 Brown, A. W., 78,86 Brown, B. J., 54, 87 Brown, J. L., 398,403,443 Brown-Sequard, C. E., 279, 280, 293 Briigger, M., 396,402,443 Bruesch, S. R., 153, 159, 185 Bruner, J., 162, 173, 175, 180, 181,

185 Brunk, H. J., 383, 384, 438 Brust, B., 74, 75, 95 Brutkowski, S., 428, 439 Bubnoff, N., 418, 439 Bucciante, G., 92 Buchanan, D. L., 40,85 Bucher, V. M., 156, 165, 166, 167, 169, 173, 178,185,187,396,402, 443 Buchholz, Dr., 283, 293 Buchwald, N. A., 382, 415, 416, 417, 419, 420, 439 Bucy, P. C., 388, 439 Bueker, E. D., 81, 95, 259, 261, 291, 293 Buell, M. V., 25, 85 Biirgi, S. M., 156, 165, 166, 167, 169, 185

354

AUTHOR INDEX

ChafFee, E. L., 362, 439 Chaikoff, I. L., 4, 87 Chain, E. B., 11, 84 203. Chamberlin, M., 57, 85 214, Chambers, W. W., 286, 287, 288, 290, 292, 294, 297, 301, 391, 439 223, 231, Chance, hl. R. A., 365, 439 239, Chance, R. R., 275, 300 Chang, H. T., 153, 185, 387, 439 Chang, M. W., 25, 85 Chang, T., 341, 347 181. Chantrenne, H., 2, 57, 85 Chapman, L. F., 72, 85 Chatschaturian, A,, 177, 186 C Chavaz, M., 171, 185 Calvert, F., 23, 92 Chen, G., 304, 309, 310, 314, 316, Calvin, hl., 13, 92 333, 334, 344, 347 Cameron, D E., 307, 311, 3-17 Chevrel, R., 105, 142 Campbell, A. W., 152, 177, 185 Chiaverini, P., 20, 25, 85 Campbell, B., 140, 142, 192, 254 Chin, L. E., 49, 85 Campbell, j. B., 287, 290, 293, 294, Chipman, L. M., 25, 85 298 Chirigos, M. A., 4, 6, 8, 9, 85 Campbell, P. N., 2, 46, 47, 83, 85 Chiron, A. E., 304, 307. 347 Canellakis, E. S., 53, 88 Chou, S. N., 218, 254 Cannon, W.B., 405, 439 Chow, K. L., 159, 185 Cantmi, G . L., 53, 95 Christensen, N. N., 8, 85 Caporaso, L., 274, 294 Christopherson, F., 360, 439 Capriata, A., 274, 300 Ciaccio, G. V., 107, 116, 126, 133, Caravaglios, R., 20, 25, 85 142 Caimichael, M. W., 5, 94 Clarc, hl. € I . , 153, 157, 158, 162, 165, Carneiro, J., 44, 85 174, 178, 180, 184, 185 Carpenter, C. R., 350, 353, 365, 366, Clark, D. D., 11, 14, 17, 84, 94 439 Clark, E., 356, 439 Carpenter, hl. B., 156, 161, 165, 167, Clark, G., 353, 419, 439, 442 169, 170, 175, 177, 184 Clark, j. R., 25, 91 Clark, S. L., 362, 383, 391, 392, 439 Carpenter, W., 286, 29.3 Cam, S., 22, 24, 87 Clark, W. E. Le G., 150, 155, 158, Caspersson, T. O., 50, 85 159, 161, 164, 165, 166, 174, 175, Castelfranco, P., 52, 92 185, 186, 285, 289, 294 Castelis, C., 344, 347 Clarke, R. H., 384, 439, 440 Castillo, J. del, 235, 252 Clausen, J., 20, 85 Catanzaro, R., 11, 81 Clearwaters, K. P., 278, 294 Cattaneo, D., 280, 289, ,294 Clemente, C. D., 287, 288, 290, 294, CavaliB, $I., 116, 125, 142 298, 299, 301, 428, 442 Cavanaugh, M. W., 74, 97 Clemente, L. D., 4, 87 Cerletti, A., 338, 346 Clouet, D. H., 17, 23, 27, 32, 39, 42, Ccrvi, M., 274, 300 43, 51, 76, 78, 85

Buller, A. J., 101, 142 Burgen, A, S., 25, 85 Burger, E., 75, 93 Burke, W., 100, 142, 196, 197, 205, 207, 210, 212, 213, 215, 216, 217, 219, 222, 225, 227, 228, 229, 230, 232, 234, 235, 237, 238, 242, 243, 245, 251, 252 Burr, H. S., 269, 293 Bursten, B., 354, 419, 421, 439 Huqer, P., 162, 173, 175, 180, 185

455

AUTHOR INDEX

Clow, J. E., 25, 91 Cobb, S., 350, 374, 440 Cochran, W. W., 361, 444 Cohen, A., 278, 300 Cohen, B. D*,304, 306, 307, 30%309, 343, 347 Cohen, L., 18, 93 Cohen-Bazire, G., 47, 92 Cohen, S.9 80, 85, 91, 277, 291,294 Cohn, G. L., 51, 91 Cohn, M., 28,89 Cohn, P., 32, 37, 60, 85, 94 Cohn, R., 158, 185 Cole, F. J., 110, 111, 112, 142 Cole, J., 361, 440 Cole, R. D., 52, 85 Coleman, D. L., 71, 86 Collin, R., 197, 251 Colucci, V. S., 274, 294 Comly, L. T., 57, 88 Cook, L., 311,347 Coombs, J. S., 192, 206, 219, 228, 229, 241y 252 230’ 231’ 232’ 239’ Cooper, S., 104, 139, 142 Coote, J., 52, 85 Cordeau, J. P., 353, 440 Corner, E. 1). S., 106, 142 Corriol, J., 403, 441 Costello, R. T., 350, 417, 444 Couch, J., 110, 143 Couteaux, R., 99, 117, 121, 128, 143 Cowan, W. M., 417, 421, 440 Cowie, D. B., 13, 86, 88 Cragg, B. G., 156, 170, 185 Craighead, F. C., 354, 361, 440 Craighead, J. H., 354, 361, 440 Crain, S. M., 231, 232, 251 Cravioto, R. O., 16, 86 Crawford, E. J., 25, 91 Crosby, E. C., 166, 167, 186 Csillik, B., 101, 143 Culler, E. A,, 415, 421, 445 Curti, B., 65, 93 Curtis, D. R., 206, 216, 218, 230, 231, 232, 238, 239, 240, 242, 243, 244, 245, 246, 247, 248, 252 Cuypers, A., 117, 143

D Daigle, H. J., 155, 187 Dale, H. H., 76, 86 Dalrymple, D., 75, 93 Daniel, J. F.,107, 118, 143 Daniel, P. M., 139, 142 Danilewski, B., 413,415,440 Darian-Smith, I., 213, 252 Davey, w. L, M., 362, 445 David, G. B., 78, 86 David, M., 383, 448 Davidson, H. M., 23, 95 Davie, E. E., 52, 86 Davies, B. M., 308, 313, 314, 331, 343, 347 Davies, J. W., 68, 86 Davies, P. W., 205, 254 Davies, R. K., 20, 86 Davis, G. D., 361, 440 Davis, R., 159, 184, 196, 197, 200, 203, 204, 205, 207, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 225, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 239, 241, 242, 243, 244, 245, 246, 247, 248, 251, 252 Davison, A. N., 29, 40, 86 Dawson, R. M. C., 16, 17, 86, 94 De Ajuriague*ay ’*’ 383’ 448 Deasy, G. L., 42, 50, 84 de Beer, E. J., 366, 373, 445 De Duve, C., 62, 86, 97 Defalco, A., 12, 96 de Groot, J., 428, 442 de la Haba, G. L., 59, 98 Delgado, J. M. R., 350, 354, 358, 360, 361, 362, 363, 366, 367, 373, 374, 376, 377, 378, 379, 381, 382, 383, 392, 393, 397, 402, 403, 404, 405, 410, 411, 417, 418, 419, 421, 422, 423, 424, 425, 428, 431, 438, 439, 440, 441, 443, 445, 446, 447 Dell, P., 172, 185 DeJmas-Marsalet, P., 415, 446

456

A U T H O R LWEX

Demling, L., 20, 86 DeMoss, J. A., 52, 86 Dempsey, E. W., 415, 445 Denber, H, C. B., 342, 347 Denny-Brown, D. E., 101, 243, 387, 440 Dentan, E. J., 106, 142 Denton, P., 281, 291 Denz, F. A , , 102, 143 De Risio, C., 73, 92 De Robertis, E., 62, 86 DeRopp, R. S., 16, 17, 86 Desmedt, J. E., 170, 185 Detwiler, S. R., 259, 260, 269, 294, 295 Deuticke, H. J., 25, 73,86 Deutsch, J. A., 435, 441 DeVault, i l . , 304, 306, 313, 315, 333, 334, 347 de Vito, R. V., 169, 184, 213, 251 De-Waart, C., 17, 86 Dews, P. B., 361, 441 Dieckmann, M., 52, 53, 84, 94 Dingman, W., 4, 12, 20, 86, 96 Dinhis, H. hl., 54, 57, 86 Dobkin, J., 87 Dobrzecka, C., 428, 448 Doctor, B. P., 53, 89 Dodge, H. W., Jr., 354, 438 Dodt, E., 171,185 Dogiel, A. S., 103, 116, 143 Domino, E. F., 317, 318, 323, 343, 347 Doty, R. W., 155, 158, 173, 174, 177, 180, l8S, 404, 441 Dow, R. S., 391, 441 Doyen, A., 62, 84 Dreyfus, J., 71, 90 Droz, B., 43, 70, 77, 86, 97 Dua, S., 398, 403, 428, 438 Duesberg, J., 275, 295 Dumont, S., 172, 185 Dunbar, H. R., 360, 441 Duncan, D., 292, 295 310, 316, 345, 346 Dunlop, C. W., Dunn, D. B., 53, 86 Dustin, A. P., 262, 295

E Eagle, H., 4, 29, 86 Eccles, J. C., 101, 142, 143, 192, 203, 206, 218, 219, 221, 228, 229, 230, 231, 232, 233, 238, 239, 240, 241, 252, 253, 292, 295 Eccles, R. M., 101, 142, 143, 218, 221.' 238, 243, 247, 253, 292, 295 Ed&, M. V., 292, 295 Edstrom, J. E., 79, 85 Edwards, C., 219, 230, 232, 253 Egger, M. D., 403,341 Eggleston, L. V., 6, 7, 96 Eichhorst, H., 281, 295 Eidelberg, E., 16,93 Eldred, B., 104, 142 Eldred, E., 104, 142 Eliassen, E., 361, 441 Elinson, 170, 185 Elliot, K. A. C., 7, 86 England, S. J. M., 361, 441 Ensor, C., 304, 309, 310, 314, 316, 333, 334, 344, 347 Erikson, L. B., 290, 295 Erickson, R. P., 241, 253 Emsting, M. J. E., 17, 86 Errera, M., 51, 91 Erulkar, S. D., 213, 253 Erulkar, S. C., 159, 185 Ervin, F. R., 382, 419, 420, 439 Evans, V., 307,347 Evans, W. A., 198, 199, 236, 237, 238, 251 Evarts, E. V., 242, 243,253,254, 342, 347 Exner, S., 387, 441 Eyzaguine, C., 103, 143, 217, 219, 253 F

Fatt, P., 218, 226, 230, 235, 239, 252, 253 Feigen, I., 286, 295 Feindel, W., 423, 441 Feldberg, W., 25, 75, 86, 363, 441 Fender, F. A,, 362, 441

457

AUTHOR INDEX

Ferguson, T., 270, 295 229, 230, 232, 233, 238, 239, Feringa, E. R., 287, 294 240, 241, 253 Fernandez de Molina, A. F., 398,403, Franke, E., 159, 184 404, 441 Frankel, S., 18, 94 FernQndez-Guardiola, A., 169, 172, Frantz, I. D., Jr., 50, 98 186 Franz, V., 110, 143 Fernandez-Moran, H., 73, 86 Freeman, G. L., 166,188 Ferrer, S., 344, 347 Freeman, L. W., 286, 290, 295, 296, Ferrier, D., 415, 441 300 Fessard, A., 117, 125, 131, 142, 143 Frei, E. H., 361, 441 Fickler, A., 282, 295 French, E. F., 13,85 Ficq, H., 51, 91 French, J. D., 169, 185, 362, 402, 447, Field, G., 242, 251 448 Filimonov, V. G., 361, 441 Frenster, J. H., 51, 87 Fillenz, M., 158, 159, 170, 185, 213, Frey, E., 155, 185 253 Freygang, W. H., Jr., 196, 205, 206, Findlay, M., 23, 86, 87 207, 213, 232, 233, 242, 253 Finneran, J. C., 286, 295 Freidberg, F., 4, 30, 33, 50, 87, 98 Fischer, E. H., 45, 85 Friede, R. L., 76, 78, 87 Fischer, H., 8, 85 Freis, B. A., 4, 87 Fischer, J., 37, 79, 87 Frost, L. L., 431, 438 Fischler, H., 361, 441 Fugita, H., 268, 295 FitzHugh, R., 209, 254 Fukada, T., 76,87 Flanzbaum, S., 365, 445 Fuortes, M. G. F., 206, 210, 213, 219, Fleischman, R., 29, 86 221, 229, 230, 232, 238, 239, 240, Flexner, J. B., 11, 32, 34, 46, 47, 71, 241, 253 87, 94 Fukai, N., 15, 48, 87, 93, 95 Flexner, L. B., 11, 32, 34, 46, 47, 71, Fuller, J. L., 361, 441 87, 94 Fulton, J. F., 387, 441 Flock, E. V., 17, 87 Furst, S., 5, 15, 29, 30, 31, 32, 33, 36, Flood, P. R., 110, 143 39, 41, 42, 43, 51, 77, 87, 90 Florey, E., 413, 441 Furusawa, S., 74, 78, 79, 96 Flynn, J. P., 400, 401, 402, 403, 441, Fusari, R., 114, 115, 143 448

G

Folch, J., 19, 20, 21, 22, 24, 27, 87, 90, 93

Fonberg, E., 381, 425, 428, 439, 441 Forbes, A,, 230,253 Forman, D., 415, 419, 421, 441 Forssman, J., 261, 295 Forster, G. R., 106, 142 Foster, L., 280, 295 Fowler, I., 278, 300 Fox, S. S., 172, 181, 185, 416, 441 Fraisse, P., 274, 277, 295 Francis, M. D., 48, 87 Frank, K., 206, 207, 210, 213, ,221,

Gaddum, J. H., 363,441 Gadsden, C.P., 361, 447 Gaitonde, M. K., 6, 11, 17, 22, 23, 27, 29, 30, 32, 33, 37, 40, 85, 87, 94 Gajewski, J. E., 304, 315, 333, 334, 347

Gall, F. G., 150, 185 Galt, W., 365, 448 Ganser, S., 156, 185 Garde, E., 434, 441 Garfinkel, D.. 14, 87 Garol, H.'W.; 177, 185

458

AUTHOR

Gastaut, H., 344, 347, 396, 403, 419, 431, 441, 448 Gasteiger, E. L., 102, 145 Gastineau, D. C., 286, 300 Gaze, R., 275, 295 Geddes, L. A,, 361, 442 Gegenbaur, C., 276, 295 Geiger, A., 11, 84, 87 Celler, E. H., 286, 295 Gellhorn, E., 382, 386, 442 Gengerelli, J. A,, 362, 442 Genuth, S. M., 52,86 Gerard, R. W., 74, 75, 95, 155, 173, 185, 285, 286, 295, 300 Gerebtzoff, hl. A., 110, 114, 115, 143, 415, 442 Geren, B. B., 73, 95 Gemstein, G. L., 211, 253 Gershon, S., 307, 347 (:er5tein, A,, 5, 30, 32, 39, 90 C;estll, A,, 357, 442 (;ewll, R., 230, 253 Gestring, G . F., 171, 186 Gey, G. O., Jr., 71, 90 Ciacobini, E., 101, 102, 143 Giacomini, E., 107. 116, 120, 128, 133, 143 Gimetto, R., 62, 86 Giarman, K.J., 7, 8, 9, 95 Gillespie, J. S., 206, 253 Gillilan, L. A., 164, 185 Cinsborg, B. L., 100, 101, 103, 142, 143 Giolli, R. A., 164, 185 Girado, J. M., 17, 84, 287, 290, 294, 298 Giuditta, A,, 42, 93 Glassman, E., 52, 83 GIazko, A. J., 341,347 Glees, P., 157, 158, 161, 167, 170, 173, 185, 186, 188, 225, 253, 290, 295, 361, 440 Clenner, G. G., 64, 66, 83 Gley, H. E., 280, 295 Gloor, P., 421, 428, 442 Glusman, Sf., 402, 442 Gobbel, W. G., Jr., 167, 186

INDEX Codin, C., 46, 48, 83 Gokay, H., 286, 295 Goldberg, S. E., 419, 442 Caldfarb, A. J., 274, 295 Goldstein, A., 54, 87 Goldzband, M. G., 419, 442 Colgi, C., 105, 143 Goddy, W., 387,442 Goodrich, E. S., 138, 143 Gordon, J. J., 25, 87 Gordon, M., 356, 439 Gore, M., 153, 184 Goslar, H. B., 37, 87 Gotovtseva, C. P., 93 Gottlieb, J. S., 304, 306, 307, 308, 309, 343, 347 Granit, R., 101, 103, 104, 143, 205, 206, 209, 219, 235, 254 Granit, R. E., 171, 186 Grastyin, E., 376, 415, 418, 422, 442 Gray, E. G., 99, 100, 143, 144 Gray, J., 105, 135, 136, 137, 140, 144 Grazer, F. M., 4, 87 Green, J. D., 219, 255, 428, 442 Green, L., 287, 294 Greenberg, D. M., 4, 30, 33, 46, 50, 87, 94 Greene, C. W., 132, 144 Creengard, P., 4, 6, 8, 9, 85 Greer, M. A., 362, 442 Greifenstein, F. E., 304, 306, 313, 315, 333, 334, 347 Crenacher, H., 107, 108, 110, 144 Griffin, D. R., 361,442 Grimm, F. R., 177,185 Grinker, R. R., 286, 295 Grindlay, J. H., 17, 87 Grontoft, O., 4, 87 Gros, F., 54, 96 Gross, C. S., 71, 95 Gross, J, 50, 54, 91 Grossack, M., 353, 442 Grossman, R, G., 422, 442 Grover, J. W., 67, 91 Griisser, 0. J., 159, 186, 196, 200, 203, 228, 237, 238, 254

AUTHOR INDEX

Griisser-Cornehls, U., 196, 200, 203, 228, 237, 238, 254 Grundfest, H., 192, 197, 231, 232, 251, 254 Grunert, F., 279, 295 Gudden, B., 151, 163, 186 Curry, R. W., 197, 254 Gurewitsch, M., 177, 186 Guroff, G., 6, 7, 9, 87 Cumin-Flores, C., 169, 172, 186 Gwynn, G. W., 341,347

H Haagen-Smit, A. J., 42, 50, 84 Haggqvist, G., 101, 104, 144 Hakkinen, H.-M., 17, 87 Haley, T. J., 363, 442 Halverson, H. M., 357, 442 Halvorson, H. O., 13, 28, 29, 47, 71, 88

Hamasaki, D. I., 170, 184 Hamburger, J., 80, 91 Hamburger, V., 259, 260, 277, 291, 295, 296, 297 Hamlin, H., 350, 354, 393, 421, 440, 443 Handler, P., 4, 89 Hannett, F. I., 162, 186, 384, 443 Hansen, R. G., 68, 98 Hanson, H., 62, 63, 88 Hanzawa, S., 265, 300 Hare, W. K., 165, 186 Harlow, H. F., 365, 442 Harman, P. J., 157, 173, 175, 177, 186 Harris, A., 13, 48, 49, 91 Harris, G., 68, 86, 88 Harris, G. W., 362, 442 Harris, H., 29, 88 Harrison, F., 414, 442 Harrison, R. G., 258, 262, 269, 270, 296 Harrison-Matthews, L., 106, 144 Hartelius, H., 293, 296 Hartley, R. W., Jr., 73, 92 Hartmann, G., 52, 91 Haselkom, R., 57, 93 Hashimoto, Y., 206, 255

459

Hassler, R., 388, 389, 416, 442, 444 Hasznos, T., 418, 422, 442 Hayashi, M., 57, 88 Hayhow, W. R., 155, 157, 158, 161, 162, 163, 164, 175, 186, 194, 196, 213, 215, 216, 242, 251, 254 Hayes, J. S., 366, 446 Hayne, R. A., 416, 444 Heald, P. J,, 20, 21, 22, 23, 24, 84, 88 Healey, E. G., 139, 144 Heath, R. G., 354, 415, 442, 443 Hebb, C. O., 75, 88, 247, 254 Hebb, D. O., 365,442 Hecht, L. I., 53, 54, 88, 89 Heerowski, M., 53, 98 Heidenhain, R., 418,439 Heinz, E., 8, 9, 88 Heisey, S. R., 9, 93 Hellner, K. A., 361, 438 Helrich, M., 309,347 Hemmer, R., 17, 88 Hems, R., 6, 7, 96 Henatsch, H. D., 101, 103, 143 Henderson, E. E., 384, 439, 440 Henderson, N., 23, 73, 94 Hendler, R. W., 70, 88 Hennessy, B. L., 242, 251 Henning, N., 20, 86 Hensen, V., 259, 296 Herbert, C. C., 350, 447 Herbert, E., 53, 88 Hering, H. E., 418, 442 Hermann, P., 62, 88 Hernindez-P&n, R., 169, 172,186 Hess, A. R., 21, 84, 104, 144, 170, 186 Hess, R., Jr., 414, 415, 438, 443 Hess, W. R., 354, 357, 362, 368, 374, 376, 388, 389, 395, 396, 401, 402, 411, 414, 416, 419, 420, 422,431,442,443 Heuser, G., 415, 416, 417, 439 Heymans, J. F., 109,144 Hibbard, E., 264, 296 Higgins, J. W., 350, 443

480

AUTEIOR INDEX

LIild, W., W6, 209, 254 Hill, A. V., 137, 144 Himwich, H. E., 317, 326, 347 Himwich, W.A., 4, 16, 86 Hinde, R. A., 365,366,443 Hines, M., 366, 388,443 Hinsey, J. C., 99, 120, 144 Hirano, S . , 6, 7, 96 fh,W.,Sr., 262, 296 Hiscoe, H. B., 74, 75, 97 Hixon, W.S., 23, 91 Iloagland, H., 140, 144 Hoadley, L., 278, 296 Hoagland, ill. B., 2, 52, 53, 54, 5 i , 68,88, 89 Hodes, R., 415, 416, 442, 443, 446 Hoebel, B. G., 363, 443 Hoessly, G. E., 160, 163, 186 Hofman, G., 20, 89 Hogeboom, G. H., 13, 95 Hogness, D. S., 28,89 Holley, R. W.,53, 89 Hollinshead, M. B., 269, 296 Holmes, M'., 109, 144 Holm-Hansen, O., 13, 92 Holmstedt, B., 102, 143, 144 Holter, N. J., 361, 443 Holzer, I-I., 269, 296 Hooker, D., 265, 269, 271, 272, 275, 284, 283, 296, 298 51, 57, 83, 96 Hopkins, J. W., Horak, K., 16, 90 Horsley, V., 182, 184 Horvath, N., 11, 87 Nouwer, A. W., 163, 184 Hovels, O., 25, 73, 86 Howard, S. Y., 218, 254 Hoyle, G., 140, 144 Hubbard, J. I., 240, 253 Hubel, D. H., 153, 159, 172, 177, 197, 207, 210, 213, 214, 216, 254 Huber, G. C., 166, 167, 186 Hudson, R. D., 343,347 Hughes, A., 277, 296 76, 91 Hughes, A. F. W., Hughes, J. R., 242, 253,254,381, 387, 443

Hullin, R. Y.,61, 94 Hunter, J., 173, 175, 176, 186, 284, 296 tFulSmdl1, w. c., 52, 91 Hultin, T., 42, 50, 54, 59, 60, 89, 94, 97 Hunsperger, R. W., 398, 402, 403, 404, 441, 443 Hunt, C. C., 103, 144, 213, 214, 218, 238, 254 Hunter, J., 414, 418, 419, 421, 443 Husby, J., 287, 290, 293, 294, 298 Husson, R., 434, 443 Huxley, A. F., 100, 145 Huxley, H. E., 54, 89 Hydkn, €I., 3, 71, 73, 79, 85, 89, 293, 296 I

Iggo, .4., 218, 253 Imanishi, K., 365, 366, 443 Imber, R., 416, 445 Ingersoll, E. H., 395, 445 Ingram, W. R., 157, 161, 162, 164, 165, 167, 173, 174, 178, 184, 186, 384, 395, 428, 438, 443, 444 Ingvar, D. H., 173, 175, 176, 421, 443 Ingvar, S., 262, 296 Inoma, S., 197, 255 Ivanova, T. N., 22, 97 Izquierdo, J. J., 16, 86

163, 175, 425, 186,

J

Jacob, F., 57, 89, 94 Jacobson, J. H., 171, 186 Jakoby, R. K., 290, 296 Jansen, J., Jr,, 104, 108, 112, 120, 141, 144, 167, 184, 399, 403, 444 Janssen, P., 17, 95 Jarman, G. M., 106,144 Jasper, H. H., 154, 162, 186, 212, 254, 350, 414, 417, 418, 419, 420, 421, 443, 444 Jeffay, H., 34, 35,89

AUTHOR INDEX

Jefferson, G., 167, 186 Jefferson, J. M., 155, 186 Jensen, G. D., 361,444 Jent, M., 292, 296 Jeremy, D., 153, 157, 159, 184, 212, 215, 216, 241, 251 Jervie, A., 164, 186 Jewell, P. A., 102, 144, 374, 438 Jimenez-Castellanos, J., 162, 180, 181, 182, 186,187 Jinnai, D., 16, 98 Jiracek, V., 16, 90 Joan, H. M., 75,92 Johnson, R. M., 23, 89 Johnston, J. B., 115, 144 Johnstone, M., 307, 347 Johnstone, R. M., 9, 89 Jones, 0. W., 54, 57, 92 Jordan, E. F., 9,93 Jordan, M., 272,275, 296 Joshi, S., 60,96 Jouvet, M., 414,444 Jung, R., 172, 186, 187, 209, 254, 388,416,444

K Kaada, B. R., 399,403,419,420, 431, 444, 447, 448 Kabak, K. S . , 73, 93 Kahler, O., 282, 296 Kallen, B., 277, 278, 296 Kafoe, W. F., 17, 86 Kalf, G. F., 51, 89 Kallejian, V., 362, 442 Kamin, H., 4,89 Kamrin, A., 16, 89 Kamrin, A. P., 16, 89 Kandel, E. R., 213,218, 220, 229,254 Kappers, C. U. A., 261, 262, 296 Kapphan, J. I., 25,85 Kaps, G., 20,89 Karcher, D., 20, 24, 89 Katz, B., 125, 144, 235, 252 Katz, D., 353, 444 Kaufman, I. C., 365,444 Kawakami, M., 102,144

461

Kawakita, Y., 11, 84, 87 Keefe, E. L., 259, 296 Keighley, G., 42, 50, 84 Keighley, J. J., 45, 85 Keil, A. W., 25, 73, 89 Keil, J. H., 265, 296 Kbkesi, F., 376, 442 Keller, E. B., 42, 50, 51, 52, 54, 88, 89, 91, 98 Kelley, B., 23, 89 Kelley, R., 304, 306, 307, 308, 343, 347 Kennedy, J. L., 150,187 Keup, W., 20,89 Kiang, N. Y. S., 211, 253 Kies, M. W., 23, 61, 73, 89, 94 Kim, C., 415,416,448 King, R. L., 241, 253 King, W., 6, 7, 9, 87 Kinnaman, A. J., 353, 444 Kinzelmeier, H., 20, 86 Kirsche, W., 128, 144, 265, 266, 274, 296 Kishinouye, K., 137,144 Kiyota, K., 20, 21, 24, 32, 89 Kletzkin, M., 397, 402, 448 Kleine, R., 62, 88 Kliiver, H., 351, 433, 434, 444 Knauff, H. G., 16, 26, 89 Knighton, R. S., 350, 417, 444 Knoche, H., 155, 187 Knott, J. R., 416, 425, 428, 444, 445 Koechlin, B. A,, 73, 89, 292, 296, 297 Koella, W. P., 391, 414, 415, 438, 443, 444 Koelle, G. B., 76, 78, 87, 90 Koenig, E., 76,78,90 Koenig, H., 38, 75, 77, 90 Kogan, A. B., 363, 444 Koizumi, K., 243, 245, 252 Koketsu, K., 218, 252 Kolmer, W., 267,297 Kolousek, J., 16, 37, 79, 87, 90 Konig, M. P., 292, 297 Konigsberger, J. J., 52, 86 Koppanyi, T., 264, 265, 267, 272, 285, 295, 297, 298

462

AUTHOR INDEX

Korey, S. R., 6, 7, 90 Korner, A., 51,96 Kosciuszko, H., 273, 297 Kosman, A. J,, 428, 445 Kosugi, Y., 197, 255 Kozak, W., 196, 200, 208,252 Krachko, L. S., 25, 42, 74, 93 Krause, M., 75, 90 Kravchinski, E., 39, 90 Krebs, H. A,, 6, 7, 96 Kroeger, D. C., 402, 447 Kruger, P., 101, 104, 144, 145 Kruger, L., 288, 289, 291, 297, 299 Kruh, J., 71, 90 Krushinskii, L. V., 353, 444 Kuffler, S. W., 100, 103, 144, 145, 208, 209, 214, 217, 219, 253, 254 Kulonen, E., 17, 87 Kuno, hi., 213, 214, 218, 238, 254 Kupfer, C., 103, 104, 145 Kurland, C. G., 54, 90 Kutchin, fi. L., 109, Id5 Kuypers, H. G. J. hl., 167, 187 Kwaitkow\ki, C., 273, 297

L Lance, J. W., 288, 297 I.iinsim&i, T. A,, 107, 145 L a j h i , A., 2, 3, 4, 5, 6, 8, 9, 10, 13, 14, 15, 18, 19, 25, 29, 30, 31, 32, 33, 36, 39, 41, 42, 43, 44, 51, 61, 62, 63, 64,65,67, 69, 70, 73, 74, 77, 84, 87, 90, 97 Lambertini, G . , 104, 145 Lamborg, M. R., 59, 90 Lamfrom, H., 45, 59, 90, 95 Lnmmers, H. J., 403, 445 Lampard, D. G., 209, 211, 254 T.ance, J. W., 153, 157, 184, 241, 251 L'mdau, W., 242, 253 Lange, P., 89 Lankester, E. R., 124, 145 Laporte, Y., 100, 145 Lashley, K. S., 159, 161, 162, 163, 164, 174, 187 l,arsson, S., 374, 431, 438, 444

Lashley, K. S., 434,444 Lauenstein, K., 25, 73, 86 Laupheimer, R., 356, 448 Lauprecht, C. W., 416, 417, 439 Laursen, A. M., 416, 444 Lavocat, A., 107, 124, 125, 141 Leahy, J., 57, 84 Lear, E., 304, 307, 347 LeBaron, F. N.,19, 20, 22, 87, 90 Leblond, C. P., 44, 85 Le Danoh, Y., 132, 145 Lee, J. C., 384, 447 Lees, H., 341, 347 Lees, M., 22, 24, 27, 87 Lehmann, H. E., 308, 313, 346 Le Mare, D., 135, 145 Lengyel, P., 57, 91, 95 Lennox, M. A,, 153, 187 Leon, H. A., 59,89 Le Quire, V., 419, 448 Lesevre, N., 344, 347 Lessel, I. M.,71, 95 Le Toud, S., 131, 142 Levick, W. R., 196, 197, 200, 207, 209, 210, 211, 215, 252, 254 Levi-Montalcini, R., 47, 80, 81, 91, 260, 291,294,297 Levy, L., 307, 311,347 Levy, M., 4, 86 Lewandowsky, M., 391, 444 I,ewis, P. R., 76, 91 Lewis, W. H., 269, 270, 297 Leyhausen, P., 395, 444 Li. C. L., 212, 218, 254 Li, T.-P., 73, 91 Libet, B., 74, 75, 95 Liddell, E. G. T., 387, 444 Liebl, G. J., 32, 94 Light, R. V., 362, 439 Lilcs, G . W., 167, 186 Lilly, J. C., 354, 387, 444 Lindsley, D. B., 172, 187, 360, 444 Lingrel, 3. B., 52, 97 Lipman, E., 415, 421, 415 Lipmann, F., 52, 53, 54, 59, 86, 91, 92, 97, 98 Liss,L., 170, 189

AUTHOR INDEX

LissAk, K., 376,415, 418,422, 442 Lissmann, H. W., 105, 139, 140, 144, 145 Littlefield, J. W., 50, 51, 54, 91 Liu, C. N., 287, 292, 297 Liu, C. Y., 292, 297 Livingston, R. B., 170, 187, 392, 440 Lloyd, D. P. C., 192, 203, 254 Locate&, P., 275, 297 Lodin, Z., 37, 79, 87 Loevinger, R., 288, 297 Lflyning, Y., 108, 112, 120, 141 Loftfield, R. B., 2, 13, 42, 45, 46, 48, 49, 50, 67, 89, 91, 98 Logan, R., 51,91 Logothetis, J., 17,83 Lohrenz, J. J., 308, 313, 346 Looney, E., 397, 403, 405, 440 Lord, R. D., 361, 444 Lorente de N6, R., 200, 254, 272, 297 Lorenz, K. Z., 353,444 Lorenzini, S., 107, 145 Loucks, R. B., 362,445 Lovatt Evans, Sir C., 382, 387, 445 Low, G., 107, 145 Lowenstein, O., 126, 145 Lowenthal, A., 20, 24, 89 Lowry, 0. H., 25, 85, 91 Lowy, P. H., 42, 50, 84 Lu, E. S., 213, 214, 255 Lubinska, L., 75, 76, 91 Luby, E. D., 304, 306, 307, 308, 309, 315, 343, 347 Lumsden, C. E., 75, 91 Lundberg, A., 101, 143, 218, 221, 238, 239,252,253 Lushnat, K., 344, 347 Lyons, R., 87

M McCaman, R. E., 25, 65, 80, 91 Mecarthy, D. A., 341, 347 McClure, F. T., 13, 86 McCormick, W. G., 363, 442 McCouch, G. P., 292, 297 McCreight, J., 275, 297

463

McDonald, J. V., 391, 445 McDowell, A. A., 351, 445 McIlroy, C. A., 8, 9,95 McIntyre, A. K., 192, 203, 213, 215, 217, 218, 254 Mackay, B., 103, 107, 108, 110, 112, 114,143,145,146 MacKay, R. S., 361,445 McKean, C. M., 95 McLean, J. R., 51, 91 MacLean, P. D., 353, 403, 415, 416, 419, 422, 425,431, 433,445, 448 McLeod, J. G., 159, 184, 196, 198, 201, 204, 212, 215, 216, 226, 251 McPherson, C. F., 75, 84 Macht, M. B., 404,438 Magasanik, A. K., 59, 91 Magasanik, B., 59, 91 Magee, W. L., 23, 86 Magladery, J. W., 292, 300 Magnes, J., 87 Magni, F., 203, 221, 253 Magnus, O., 403, 445 Magoun, H. W., 156, 165, 166, 169, 186, 187, 188, 395, 414, 445, 446 Mahl, G. F., 350,443 Mahler, H. R., 49, 97 Malis, L. I., 157, 163, 184, 288, 289, 291, 297, 299 Mandeles, S., 46, 93 Mandelstam, J., 28, 71, 92 Mann, F. C., 17,87 Manocchio, I., 65, 79, 93 Mantegazzini, F., 219, 253 Marburg, O., 167, 169, 187 Marinesco, G., 275, 282, 297 Mark, R. F., 213, 215, 217, 218, 254 Marks, N., 61, 62, 63, 64, 65, 92 Mar&, K., 264, 297 Marotto, M., 276, 298 Marsh, G., 262, 298 Marshak, A., 23, 92 Marshall, W. H., 155, 180, 173, 177, 178, 180, 185, 187, 188, 192, 242, 253, 254

464

AUTHOR INDEX

Martin, R. G., 57, 92 Martini, V., 292, 298 Marx, D,, 26, 89 Masahashi, K., 414, 416, 438 Mase, K., 43, 51, 92, 95 Masi, I., 11, 84 Masius, V., 275, 298 hlaslow, A. €I., 353, 363, 445 Mason, J. W., 354, 445 Masserman, J. H., 396, 405, 445 Massidda, R., 273, 300 hlassieu, G. H., 16, 86, 92 Massopust, L. C., Jr., 155, 187 hlatthaei, J. II., 54, 57, 92 hlatthew, P. B. C., 104, 144 hlatthewb, S. A., 267, 298 hlatthey, R., 275, 298 Matzelt, D., 20, 84 Maurer, F., 106, 107, 110, 145 bfaurer, W., 5, 30, 37, 43, 83, 92, 95 hlauro, A., 362, 445 Xlaxfield, M., 73, 92 Slajer, G., 26, 89 hlayer, L. C., 165, 187 Maynard, E. A,, 285, 298, 299 Mazurowski, J. A., 387, 44.3 Mead, A. P., 366, 446 hleister, A,, 52, 92, 97 hlela, P., 8, 9, 15, 90 \[elchoir, J. B., 50, 92 hlempel, E., 428, 439 hiettler, F. A., 167, 178, 180, 181, 187, 415,416, 421, 445 hleudt, R., 51, 83 Meyer, D. R., 387, 448 Meyer, € I . , 30, 92, 291, 297 Lleyers, R., 416, 444, 445 hfeyer, J. S., 306, 313, 315, 347 Miale, I., 283, 298, 299 hliani, N., 75, 92 hlihailovii., L., 382, 445 \filler, N. E., 397, 402, 405, 433, 440, 445 hiillo, A., 65, 79, 92, 93 blillot, J., 128, 145 hfinea, J., 282, 297 Itfinko\$\ki, hl., 152, 157, 158, 159,

161, 164, 163, 164, 173, 174, 171, 187 Xiirsky, A. E., 51, 83, 87 Mirsky, A. F., 351, 356, 365, 445, 446 Mishkin, M., 424, 428, 436 blissere, G., 73, 92 hlitchell, R., 6, 7, 90 Mitchell, S. Q., 287, 294 hlizutani, K., 100, 146 hloldave, K., 49, 52, 53, 85, 92, 98 Moldave, hi., 10, 32, 97, 98 hlollica, A., 169, 188 XfolnC, L., 418, 422, 442 Monod, J., 28, 47, 57, 89, 92, 94 \loore, S., 18, 96 \lorgan, C. T., 435, 445 Itlorgan, T. H., 265, 298 Slorgane, P. J., 428, 445 Lforgenstern, F. S., 314, 331, 343, 347 Mori, A., 16, 98 hlorillo, A., 180, 184, 213, 215, 251 Morison, R. S., 415, 445 Morin, F., 167, 187 hlorlock, N. L., 242, 254 hlorrell, F., 17, 83 Xlorrell, R. M., 361, 445 Xlorris, A. J., 59, 92 hloruzzi, G., 169, 188, 391, 441 hioses, V., 13, 92 Mountcastle, V. B., 192, 203, 205, 213, 214, 215, 216, 217, 254, 255 Miiller, H., 272, 276, 277, 298 ?rlUller-Limmroth,H. W., 171, 187 bfunk, H., 151, 187 Munzer, E., 164, 187 Llurakami, M., 206, 233, 254, 2-55 Murray, J. G., 292, 298 h4urray, P. D. F., 117, 1-45 Slybridge, E., 357, 445

N Nachmawsohn, D., 75, 92 Xagata, Y., 6, 7, 96 Nageotte, J., 282, 298 Saka, K., 197, 255 Nakagawa, T., 414, 416, 438

AUTHOR INDEX

465

Nakao, H., 399,402, 445 Okabe, K., 414, 416, 438 Naquet, R., 396, 397, 403, 441, 445 Okamoto, T., 54, 96 Nathaniel, E. J. H., 290, 298 Okuma, T., 415,416,439 Nathans, D., 54, 92 Okumura, N., 15, 93 Naunyn, B., 281, 295 Olariu, I., 307, 347 Nauta, W. J. H., 162, 163, 164, 167, Olds, J., 354, 362, 363, 380, 383, 446 173, 178,187 Olds, M. E., 363, 446 Nauta, W. Th., 17, 86 O’Leary, J. L., 157, 158, 159, 161, Neal, G. E., 68, 86 167, 173, 175, 176, 177, 178, Neame, K. D., 7, 8, 92 179, 187, 192, 196, 198, 251 Negishi, K., 176, 187, 213,214, 255 Olson, M. E., 46, 94 Neidhardt, F. C., 59, 91 Olszewski, J., 384, 446 Neidle, A,, 43, 44, 51, 52, 83 Oosterhuis, H. K., 17, 86 New, D., 277,296 Orkand, R. K., 100, 145 Nicholas, J. S., 260, 265, 285, 296, Orrego, F., 196, 201, 213, 235, 255 298 Ortega, B. G., 16, 92 Niemer, W. T., 180, 181, 182, 187 Ortin, L., 289, 298 Niemierko, S., 76, 91 Osawa, S., 51, 83 Niklas, A., 30, 43, 92 Oscarsson, O., 218, 240, 253 Nirenberg, M. W., 54, 57, 92 Otani, T., 230, 251 Nishioka, H., 17, 93 Otsuki, S., 15, 93 Nissen, H. W., 353, 364, 445 Ottoson, D., 219, 230, 232, 253 Noback, C. R., 287, 290, 294, 298 Overbosch, J. F. A,, 153, 157, 159, Nohara, H., 53, 93 160, 187 Nomura, M., 74, 78, 79, 96 Owens, H. F., 317, 326, 347 Norris, F. H., 102,145 Oyama, V. I., 29, 86 Norton, S., 366, 373, 445 Noshay, W. C., 350, 417, 444 P Novelli, G. D., 52,86 Pachon, V., 415, 446 Nowlis, V., 366, 446 Packer, A. D., 161, 164, 188 Nursall, J. R., 106, 145 Paillard, J., 378, 387, 446 Palade, G. E., 54, 70, 76, 93, 95 0 Palatine, I. M., 8, 85 Palay, S. L., 76, 93 Ober, R. E., 341, 347 OBrien, J. H., 172, 181, 185, 416, 441 Palladin, A. V., 21, 25, 26, 33, 37, 42, 73, 74, 93 Oehlert, W., 37, 43, 95 Pallin, I. M., 304, 307, 347 Ochoa, S., 53, 57, 60, 84, 91, 94, 95 Panizza, B., 150, 188 Ochs, S., 74, 75,93 Pansini, S., 116, 120, 145 OConnor, C. M., 350, 413, 417, 448 Pantchenko, L. F., 33, 37, 93 Oderfeld, B., 76, 91 Papez, J. W., 166, 188 ODoheay, D. S., 350, 446 Pappenheimer, A. M., 47, 92 Oettinger, W. H., 197,254 Pappenheimer, J. R., 9, 93 Ofengand, E. J., 52, 53, 94 Pardee, A. B., 57, 94 Ofengand, J., 57, 93 Parish, H. D., 73, 89 Ogata, G., 93 Parker, C. S., 360, 439 Ogata, K., 43, 51, 53, 92, 93, 95

436

AUTHOR I N D E X

Parker, H. W., 106, 144 Pasamanick, B., 361, 441 Pattay, J., 292, 298 Pavlov, I. P., 355, 413, 426 Peachep, L. D., 100, 103, 109, 145 Peacock, S . XI., Jr., 415, 416, 443, 446 Pearce, G. W., 167, 188 Pearcy, J. F., 264, 298 Pearson, H. E., 10, 32, 94, 95, 98 Pease, D. C., 285, 298, 299 Pecot-Dechavassine, M., 102, 108, 131, 141, 142, 146 Peelr, ?’. L., 398, 447 Pellegrino de Iraldi, A., 62, 86 Penfield, W., 350, 382, 388, 423, 446 Penn, N. W., 46, 64, 93 Pennycuick, C. J., 123, 124, 135, 146 Perroncito, A., 117, 146 Peters, A,, 107, 108, 110, 112, 114, 145, 146 Petersen, J. C., 4, 16, 88 Petrushka, E., 48, 93 Pfaffmann, C., 241, 253 Phillips, C. G., 101, 143, 205, 206, 219, 229, 235, 240, 254, 255, 387, 444 Phillis, J. W., 243, 245, 252 Piatt, J., 259, 261, 269, 271, 273, 275, 298 Piatt, M., 275, 298 Picard, D., 43, 93 Pick, A., 151, 188 Piez, K. A., 4, 29, 86 Pigon, A., 89 Pinto Hamuy, T., 387, 448 Pisano, J. J., 18, 93 Ploog, D. W., 433, 445 Pocchiari, F., 11, 84 Poddie, hl., 273, 300 Poliakova, N. M., 25, 33, 37, 73, 93 Polley, E. H., 196, 201, 213, 235, 255 Poloumordwinoff, D., 116, 125, 139, 146 Polyak, S., 150, 155, 159, 161, 162, 165, 167, 170, 173, 174, 175, 177, 178, 180, 181,188 Ponticorvo, L., 28, 71, 84

Pope, A., 25, 63,93 Porcellati, G., 65, 79, 92, 93 Porter, H., 21, 93 Porter, K. R., 76, 94 Potter, V. R., 32, 94 Powell, T. P. S., 417, 421, 440 Prast, J. W., 360, 446 Pravdina, N. I., 22, 97 Preiss, J., 52, 94 Pressman, B. C., 62, 86 Pribram, K. H., S l , 356, 365, 445, 446 Price, S. A. P., 15, 94 Probst, hl., 151, 164, 167, 188 Proctor, L. D., 350, 417, 444 Prosser, C. L., 136, 146 Przibram, H., 267, 298 Psychoyos, S., 72, 96 Purpura, D. P., 5, 11, 17, 84, 94 Purvis, G. C., 126, 146

Q Quastel, J. H., 9, 89, 94 Quinke, E., 30, 92

R Habinovitz, M.,46, 94 Racah, hl., 128, 146 Rademaker, W., 62, 94 Rafelson, M. E., Jr., 10, 91 Rafelson, N. E., 10, 32, 98 Rall, W., 218, 255 Ramey, E. €I., 292, 300 Ramey, E. R., 350,446 Ram& y Cajal, S., 104, 146, 159, 161, 170, 177, 178, 188, 261, 282, 283, 298 Ransmeier, R. E., 100, 145 Ranson, M., 156, 165, 187 Ranson, S. W., 157, 161, 162, 163, 164, 165, 166, 167, 173, 174, 175, 178, 184, 186, 187, 384, 395,414,443,445 Ranvier, L., 101, 104, 107, 117, 124, 146

467

AUTHOR INDEX

Rashevsky, N., 388, 446 Rasmussen, A. T., 107, 109, 188 Rasmussen, T., 423, 446 Rasquin, P., 268, 298 Ratner, S., 50, 95 Razzauti, A., 116, 146 Reger, G. F., 75, 97 Reis, P. J., 51, 94 Rendi, R., 53, 60,94 Renshaw, B., 214, 255 Renson, J., 94 Retzius, G., 107, 109, 111, 116, 146 Rhines, R., 278, 298 Rhode, E., 109, 146 Richards, G., 75,93 Richter, D., 3, 8, 11, 17, 30, 32, 33, 37, 39,42,43, 51, 61, 03, 64, 65, 83, 85, 87, 94 Riggle, G. C., 302, 442 Riggs, T. R., 8, 85 Riley, M., 57, 94 Rinkel, M., 342, 347 Rioch, D. Mck., 162, 167, 188, 405, 431, 438, 446 Rittenberg, D., 28, 50, 71, 84, 94, 95 Rizzoli, A,, 92 Roberts, E., 6, 16, 18, 94, 413, 446 Roberts, N. R., 25, 85, 91 Roberts, R. B., 11, 13, 32, 34, 46, 47, 71, 85, 87, 94 Roberts, W. W., 397, 399, 402, 405, 440, 446 Robertson, D. M., 20, 22, 24, 94 Robins, E., 25, 65, 80, 91, 94 Robinson, B. W., 424, 428, 446 Roboz, E., 23, 73,89, 94 Rodin, E. A., 315, 347 Rodriquez de Lores Amais, G., 02, 86 Rodriquez, C., 361,447 Rodriquez Delgado, R., 305,374,446 Rodriquez Perez, A. P., 146 Roe, A., 350, 446 Roizin, L., 402, 442 Romano, D., 72,96 Roodyn, D. B., 51,94 Rose, A. S., 24, 27, 98

Rose, J. E., 180, 188, 192, 203, 213, 214, 215, 216, 217, 255, 288, 289,291,297,299 Rose, S. P. R., 23, 24, 25, 94 Rosen, H., 22, 27, 96 Rosenbaum, G., 304, 306, 307, 308, 309,343, 347 Rosenblatt, J. S., 356, 447 Rosenblum, L., 365, 444 Rossi, C. A., 11, 94 Rossi, J. P., 287, 294 Rossi, O., 276, 299 Rossiter, R. J., 23, 75, 84, 86, 87 Rosvold, H. E., 351, 356, 365, 397, 398, 403, 405, 417, 439, 440, 443,445,446 Rothleder, E. E., 22, 90 Rothstein, M., 0, 94 Rotman, B., 47,94 Royle, N. D., 284, 296 Rowell, T. E., 365, 366, 443 Rubin, R., 270, 299 Rubinstein, E. H., 424, 446 Ruch, T. C., 378, 387, 439, 446 Rudnick, D., 32, 94 Ruffini, A., 104, 146 Ruguski, H., 275, 298 Rumley, M. K., 25, 96 Rumley, M. L., 63, 64,96 Russell, D., 304, 310, 314, 316, 333, 334, 344,347 Russell, W. M. S., 366, 446

5 Saeki, R., 48, 95 Salganicoff, L., 62, 86 Samuels, A. J., 74, 75,95 Sand, A., 125, 135, 136, 143, 144 Sano, T., 403,446 Santa, B., 273, 299 Sasaki, Y., 206, 255 Satake, M., 43, 51, 95 Sato, J,, 48, 95 Saul, L. J., 155, 173, 185 Saw, G., 159,186 Sawyer, C. H., 75, 95

468

AUTHOR INDFX

Schanberg, S., 7, 8, 9, 95 Schaper, A,, 270, 299 Schapira, G., 71, 90 Schaltenbrand, G., 384, 446 Schatzmann, hl., 357, 446 Scheibel, A. B., 169, 171, 188 Scheibel, XI. E., 169, 171, 188 Schenkein, I., 81, 95 Schepartz, B., 12, 95 Scher, A. M., 362, 445 Scherrer, H., 172, 186 Schiefferdecker, P., 282, 299 Schinko, H., 20, 69 Schlegel, 0. M.,286, 295 Schlessinger, D., 54, 96 Schmidt, G., 23, 95 Schmitt, F. O., 73, 95 Schneidcr, W. C., 13, 32, 94, 95 Schneiderman, N.,52, 83 Schneirla, T. C., 356, 447, 448 Schoenheimer, R., 50, 95 Scholl, D. A,, 178, I88 Schoolman, A., 392, 447 Scott, D., Jr., 287, 288, 291, 297, 299, 301 Schotti., O., 275, 299 Schulman, M. P., 33, 87 Schulz, D. w., 25, 91 Schultz, R. L., 285, 298, 299 Schultze, B., 37, 43, 87, 95 Schwartz, 11. G., 167, 187 Schwarzacher, H. G., 110, 146 Schwarze, I., 20, 84 Schwerin, P., 4, 95 Schwimmer, S., 61, 89 Sclavunos, G., 283, 299 Scott, J. F., 54, 89 Scott, J. P., 351, 353, 366, 447 Scrase, N., 75, 84 Scott, D., Jr., 288, 299 Sefton, A., 162, 186 Seite, R., 43, 93 Schweclt, R. S., 45, 52, 53, 54, 57, 59, 83, 84, 92, 95 Sefton, A,, 161, 163, 186 Segundo, J. P., 402, 447 Sem-Jacobsen, C. W., 354, 447

Sencer, W., 387,448 Sengstaken, R. W., 197, 254 Settlage, P. H., 387, 448 Sevillano, M., 422, 440 Seymour, R. J., 287, 294 Sgobbo, F., 274, 275, 280, 299 Shamarina, N. M., 100, 146 Shealy, C. N., 292, 295, 398, 447 Sheatz, G. C., 362,447 Sheer, D. E., 350, 354, 383, 386, 402, 411, 419,422,447 Shemin, D., 71,94 Sheng, P., 20, 95 Sheng, P. K., 73, 91 Sherrington, C. S., 104, 146, 350, 382, 418, 442, 447 Sherwood, S. L., 354, 363, 441, 447 Shimura, K., 48, 95 Shipton, H. W., 361, 447 Shorey, hl. L., 259, 299 Sibbing, W., 269, 299 Sidman, R. L., 283, 298, 299 Siekevitz, P., 54, 70, 93, 95 Silitch, T. P., 33, 37, 39, 73, 90, 93 Silva, P. S., 158, 161, 188 Simhadri, P., 363, 440 Simkin, J. L., 2, 59, 84, 95 Simoes-Raposo, L. R., 275, 299 Simon, A., 350, 447 Simon, E. J., 71, 95 Simpson, G. G., 350, 446 Simpson, M. V., 2, 38, 41, 47, 51, 66, 89, 91, 95 Singer, A., 361, 447 Singer, M. F., 53, 95 Siou, G. B., 131, 146 Sirlin, J. L., 2, 95 Skinner, B. F., 374, 447 Skoglund, C. R., 101, 143 Skultety, F. M., 416, 415 Sky-Peck, H. H., 10, 95 Slater, L., 361, 447 Slijper, E. J., 124, 127, 132, 134, 142 Sloan, N., 419, 447 Sloane-Stanley, G. H., 22, 87 Smimov, G. D., 350, 443 Smith, J. D., 53, 86

469

AUTHOR INDEX

Smith, J. R., 242, 251 Smith, K. U., 174, 188 Smith, 0. A., 425, 447 Smith, V. R., 361, 447 Snedeker, E. H., 16, 17,86 Snider, R., 167, 188 Snider, R. S., 384, 447 Soderberg, U., 172, 184, 209, 211, 251 Soep, H., 17,95 Soons, J. B. J., 62, 94 Spahr, P. F., 53, 86 Speidel, C. C., 262, 299 Spemann, H., 271,299 Spencer, W. A., 213, 218, 220, 229, 2S4 Sperry, C. J., 361, 447 Sperry, R. W., 266, 267, 275, 293,299 Sperry, W. M., 41, 97 Speyer, J. F., 57, 91, 95 Spiegel, E. A,, 171, 185, 354, 383, 397,402,422, 447,448 Spiegelman, S., 47, 57, 88, 94 Spirito, A., 270, 299 Sporn, M. B., 4, 12, 20, 86, 96 Spratt, N. T., Jr., 277, 299 Sproul, E. E., 71, 94 Spurzheim, G., 150,185 Srebro, Z., 272, 273, 299 Stahl, A., 43, 93 Stancer, H. C., 23, 88 Stannius, H., 107, 110, 146 Stary, Z., 22, 96 Starzl, T. E., 169, 188 Stefanelli, A., 115, 146, 271, 273, 274, 276, 299,301) Steg, G., 101, 103, 143, 146 Stein, W. H., 18, 96 Steinberg, D., 48, 66, 69, 96 Steiner, J., 213, 254 Stendel, 100, 146 Stephenson, M. L., 50, 53, 54, 67, 88, 89, 91, 98 Stern, J. R., 6, 7, 96 Stevens, J. R., 415, 416, 448 Stevens, L. B., 271, 300 Steward, F. C., 13, 96 Still, J. W., 29,96

Stirhg, W., 107,146 Stjostrand, J., 71, 73, 79, 85 Stokes, G. E., 286, 300 Stone, D., 60, 96 Stone, L. S., 275, 300 Storm Mathrisen, J., 110, 143 Stoupel, N., 172, 184, 416, 448 StoyanofE, V. A., 41,97 Strasser, H., 262, 300 Straub, F. B., 70, 96 Straus, W. L., Jr., 261, 300 Strauss, R., 350, 447 Streifler, M., 361, 441 Strickland, K. P., 23, 87, 96 Striebich, M. H., 13, 95 Stroebe, H., 282, 300 Sugar, O., 286, 300 Sulkin, N. M., 21,96 Suntay, R., 304, 307, 347 Supino, F., 107, 146 Suttie, J. W., 51, 94 Svaetichin, G., 232, 234, 255 Swick, R. W., 41,96 Syrquin, A., 16, 92 Szab6, J., 415, 422, 442 Szafranski, P., 68, 96 Szekely, E. G., 397, 402, 422, 447, 448 Szware, L., 76, 91

T Takagaki, G., 6, 14, 17, 84, 96 Takahashi, K., 431, 448 Takahashi, Y., 43, 51, 74, 78, 79, 92, 95, 96 Takanami, M., 54,96 Talairach, J., 383, 448 Talbot, S. A., 160, 188 Takeuchi, A., 107, 108, 132, 146 Talbot, S. A,, 173, 177, 178, 180, 187, 188 Taliaferro, W. H., 48, 96 Tallan, H. H., 15, 18, 96 Talmage, D. W., 48,96 Tannenberg, W. J., 49, 85 Tarnecki, R., 428, 448

470

A U T H O R INDEX

l'arver, H., 4, 30, 45, 50, 87, 92, 96 Tasaki, I., 100, 146, 196, 200, 201, 206, 209, 213, 235, 237, 254, 255 Tahaki, N., 200, 255 Taw, L., 206, 207, 220, 230, 232, 234, 235, 255 Taylor, A. L., 263, 291, 300 Taylor, C. W., 169, 188 Taylor, G., 105, 146 Taylor, N. B., 386, 438 Teasdall, R. D., 292, 300 Teitelbaum, P., 363, 443 Tello, F., 200, 255, 289, 300 Ten Cate, J., 110, 147 Tendis, S., 63, 88 Terni, T., 276, 300 Terry, R. J., 270, 300 Termolo, C. A., 230, 235, 255, 375, 388, 416, 448 'Teuber, H.-L., 350, 448 Tewari, H. B., 22, 44, 96 Therman, P. O., 192,255 Thermes, G., 273, 276, 300 Thieulin, G., 150, 188 Thompson, J. W., 292, 298 Thompson, R. C., 40, 41, 96 Thompson, W. R., 366, 448 Thulin, C. A., 287, 288, 290, 294, 299, 300 Thuma, B. D., 154, 158, 168, 176, 179, 188, 194, 196, 255 Tieg4, 0. W., 99, 117, 147 Timiras, P. S., 17, 98 Tissieres, A., 54, 57, 96 Tobach, E., 356,448 Tomita, T., 206, 233, 254, 255 Tong, C., 197,255 Tonini, G., 73, 92 Torii, H., 414, 416, 438 Toth, J., 4, 6, 8, 9, 90 Tower, S. S., 418, 448 Travis, A. M.,387, 448 Trinchese, S., 116, 147 Tretjakoff, D., 115, 147 Truman, D. E. S., 51,96 Tsai, C., 162, 166, 174, 188 Tsang, Y. C., 163, 188

Tsao, T., 20, 95 Tsukada, Y., 7, 96 Tucker, D., 171, 188 Tuena, M., 16, 92 Tuge, H., 265, 300 Tuqan, N. A., 25, 65, 79, 83, 96 Turbes, C. C., 286, 290, 296, 300

U Udenfriend, S., 4, 6, 7, 8, 9, 18, 85, 87, 93, 94 Ueki, S., 317,323,347 Uihlein, A., 354, 438 Umbach, W., 416, 448 Ungar, G., 72, 96 Urba, R. C., 28, 71, 96 Ursin, H., 399, 403, 448 Ussing, H. H., 8, 96 Uzman, L. L., 22, 25, 27, 63, 64,96, 97

V Vakkur, G. J., 196, 200, 208, 252 Van Breemen, N. L., 75, 97 Van den Noort, S., 63, 64,96, 97 van der Stelt, A., 124, 127, 132, 134, 142 van der Stricht, O., 109, 144 Van Gelder, N. M., 7, 86 Vanlair, C., 275, 298 Van Meter, W. G., 317, 326, 347 van Sande, M., 20, 24, 89 Van Straaten, J. J., 162, 163, 164, 187 van Wijhe, J. W., 109, 147 Vastola, E. F., 162, 175, 181, 188, 196, 198, 236, 237, 241, 242, 255 Vaughan, M., 48, 66, 69, 96, 97 Vaughn Williams, E. h4., 100, 145 Velasco, M., 172, 186 Velick, S. F., 38, 41, 47, 95, 97 Vernadakis, A., 17, 98 Verne, J., 43, 70, 77, 86, 97 Verzeano, M., 176, 187, 213, 214, 255, 362, 448

AUTHOR INDEX

471

Warner, J. F., 167, 169, 187 Wasman, M., 400, 401, 402, 403, 448 Watanake, K., 233, 254 Watterson, R. L., 278, 300 Watkins, J. C., 243, 252 Wattiaux, R., 62, 86, 97 Watts, J. W., 29,88 Webb, C., 161, 162, 163, 164, 186 Webster, G. C.,52, 54, 59, 83, 97 Weidley, E., 311,347 Weisberger, A. S., 57, 97 Weisblum, B., 53, 84 Weiss, P., 74, 75, 77, 97, 262, 263, 264, 272, 277, 284, 291, 297, 300 Weissbach, H., 94 Weissfeiler, J., 272, 300 Welch, K., 350, 382, 446 Wender, M., 27, 32, 53, 98 Wenger, E. L., 278, 300 West, G. B., 15, 94 Weston, J. K., 310, 347 Wheatley, L. D., 428,448 W Whitteridge, D., 139, 142 Wid& L., 173, 178, 180, 189, 198, Waddington, C. H., 277, 278, 300 213, 214, 215, 217, 255 Wagner-Jauregg, T., 292, 296 Waelsch, H., 2, 3, 4, 5, 8, 11, 13, 14, Wieman, H. L., 269, 300 15, 17, 18, 19, 29, 30, 31, 32, 33, Wiener, H., 164, 187 36, 39, 41, 42, 43, 44, 51, 52, 73, Wiesel, T. N., 153, 159, 177, 186, 74, 76, 77, 78, 83, 84, 87, 94, 95, 201,213, 216,254 Wilken, D. R., 68, 98 97 Willemse, J. J., 106, 147 Wahren, R., 383, 384, 438 Williams, R. G., 260, 278, 300 Waites, G. M. H., 75, 88 Williams, S. C., 262, 284, 300, 301 Waligora, Z., 27, 32, 53, 98 Williams, W. O., 197, 207, 209, 210, Wall, P. D., 213, 218, 255 211, 215, 254 Wallace, H. W., 97 Willis, W. D., 203, 218, 253 Walls, G. L., 150, 155, 189 Waller, W. H., 174, 176, 177, 178, Wilson, D. P., 132, 147 Wilson, J. D., 18, 93 180, 189 Windle, W. F., 28, 98, 278, 286, 287, Walsh, P. M., 8, 9, 88 288, 290, 293, 294, 298, 301 Walshe, F. M. R., 387, 434, 448 Winnick, T., 33, 48, 49, 50, 84, 87,98 Walter, H., 13, 49, 67, 97 Winzler, A. G., 10, 32, 98 Walton, B. P., 13, 86 Winzler, R. J., 10, 94 Wang, S. C., 422, 442 Wiseman, A., 68, 88 Ward, A. A., Jr., 387, 438 Ward, J. W., 362, 383, 391, 392, 415, Wolf, A., 286, 295 WOW, H. G., 72,85 419, 421, 439, 441, 448 Wolfgram, F., 24,27,98 Warden, C. J., 365, 448

Vigouroux, R., 396, 403,419, 441,448 Vincent, S., 150, 188 Visser, D. W., 10, 95 Vladimirov, G. E., 22, 23, 97 Vogt, M. E., 25, 86 Vogt, O., 107,141 Voit, Dr., 279, 300 von Baumgarten, R., 361, 438 von der Decken, A., 54, 59, 89, 97 von Ehrenstein, G., 59, 97 von Euler, C., 219, 255 von Holst, E., 378, 400, 404, 405, 411, 433, 448 Von Monakow, C., 151, 159, 173,188, 189 von Muralt, A., 292, 296, 297 von Saint Paul, U., 378, 400, 404, 405, 411, 433, 448 Vraa-Jensen, G., 43, 97 Vrba, R., 11, 87 Vulpian, M. A., 270,300

472

AUTHOR INDEX

Wolstenholme, G. E. W., 350, 413, Yamasaki, S., 87 417, 448 Yannolinsky, M. B., 59, 98 Yelen, D., 304, 306, 347 Wolter, J. R., 170, 189 Yemm, E. W., 13, 96 Wong, G. K., 53,98 Yerkes, A. W., 351, 353, 449 Wood, C. D., 431,438 Yerkes, R. M., 351, 353, 449 Wood, W.B., 54, 98 Yoshitake, J., 304, 315, 333, 334, 347 Woodbury, D. XI., 17, 98 Woolsey, C . N., 176, 180, 188, 189, 387, 388, 448 Z Work, T. S., 2, 46, 48, 51, 52, 83, 84, Zachau, H. G., 53, 98 85, 94 Zaimis, E. J., 102, 144 Wu, M., 25,91 Zamecnik, P. C., 2, 42, 50, 51, 52, 53, Wunderer, H., 126, 147 54, 88, 89, 91, 98 Wycis, H. T., 383, 447 Zannone, L., 276, 301 Wyers, E. J., 415, 416, 417, 439 Zecman, W. P. C., 163, 184 Wyrwicka, W., 428, 449 Zbrozany, A. W., 403, 449 Wyss, 0. A. M., 357, 438 Zipper, H., 13, 97 Y Zubny, G., 54, 89 Zuckerman, S., 353, 366, 449 Yamaguchi, N., 414, 416, 438 Zworykin, V. K., 361, 449 Yamamoto, Y., 16, 98

SUBJECT INDEX A Acetylaspartic acid, in brain, 18 Acetylcholine, metabolism of in nerve proteins, 75-76 Acrania, muscle innervation in, 108-110 Aggressiveness, inhibition of, 42-30 Agnatha, muscle innervation in, 110116 Alimentary inhibition, study of, 424428 Amino acid ( s ) , in brain, half-life of, 5 transport of, 5-10 pool, in brain, 3-19 y-Aminobutyric acid, in brain, 18 Amphibians, CNS regeneratian in, 268-275 Anurans, CNS regeneration in, 270-271, 275

B

metabolism in, 10-12 pool, 3-19 transport, 5-10 convulsions, causes of, 1&17 enzymes in, 25-26 proteins, see Brain proteins stimulation, see Brain stimulation Brain proteins, 19-28 amino acid content of, 2&28 catabolism of, 60-72 cerebral proteinases in, 61-66 mechanism of, 6668 relation to formation, 69-72 in development, 31-35 dynamic state of, 28-29 formation of, 45-60 amino acid incorporation systems of, 50-52 mechanism of, 5260 precursors in, 47-50 problems in, 45-47 haJf-life of, 29-31 of individual cerebral areas, 23-26 metabolism of, localization in, 4 2 4 5 of various regions, 3538 purified, 20-23 turnover of, 2 8 4 5 heterogeneity in, 38-42 Brain stimulation, fatigue in, 379-380 free behavior and, 3-49 remote control of, 362-363 variability of, 383-385

Behavior, categories of, 364-367 classification of, 363-374 definition of, 363-374 evoked, flexibility of, 37-79 study of, 374-385 types of, 385-430 free, see Free behavior inhibition of, 413-430 methodology used in study of, 356362 C offensive-defensivereaction in, 395-413 Cat, organization of, 430 lateral geniculate nucleus of, 171units of measurement of, 367-369 172 variability of, 372374 mesencephalic projections of, 165Birds, CNS regeneration in, 277-281 172 Brain, neocortical projections of, 177-182 amino acid(s), 3-19 optic chiasm of, 155 473

474

S u B J E m INDEX

optic nerve of, 15%155 optic tract of, 156158 accessory, 163-164 pretectum of, 162, 165-166 primary optic termination of, 152165 reticular formation in, 169-172 retina of, 170-171 retinohypothalamic tract of, 155156 superior colliculiis of, 163, 166169 supraoptic commissures of, 156 thalamic projections of, 17S177 thalamus of, 158-162 visual cortex of, 172 visual pathways of, 149-189 Cathepsins, role in brain protein cataholkm, 60-72 Central nervous system (vertebrate), regeneration in, 257301 in fishes, 264-268 Chick, CNS regeneration in, 277-279 Chlorpromazine, compared to phencyclidine, 311-313 Chordates, Iower, muscular innervation of, 99-147 Cint.manalysis, of behavior, 357360 Collagenlike protein, in brain, 23 Conditioned reflexes, free behavior and, 351-353 Convulsions, brain, amino acids in, 16 Copper protein, in brain, 21 Cystathionine, in brain, 18

D Depressants, effect on lateral genicd a t e nucleus, 245-246 Ditran, compared to phencyclidine,

307 E Elasmobranchs, muscle innervation in, 116126

Encephalomyelitis, collagenlike protein in, 23 Enzymes, in brain, 25-26 Epilepsy, brain amino acid changes in, 1 6 1 7 Excitants, effect on lateral geniculate nucleus. 246-248

F Fishes, CNS regeneration of, 264-268 dual motor system in, 134-135 muscle innervation in, 105-106 proprioception in, 139-140 swimming muscles of, 105-106 Free behavior, brain stimulation and, 34-49 cinemanalysis of, 357-380 conditioned reflexes and, 351-352 telemetry in study of, 3-2 Frogs, muscular innervation in, 100105

G Glucose, in brain amino acid metabolism, 10-11 Glutamic acid, in brain, 18 Glutamine, in brain, 14 Glycine, metabolism in brain, 14-15

H Haloperidol, effects of, 309 Hepatolenticular degeneration, copper proteins in, 21 Homocamosine, in brain, 18

I Insulin, effect on brain amino acid, 16, 18

L Lateral geniculate nucleus, 194-196 afferent synapses of, 191-255 dendrites of. 23S236

47s neurons, pharmacology of, 242-248 refractory, 23&241 pharmacology of, 242-248 repetitive fuing in, 212-223 responses, waveforms of, 196-209 sensory neurons of, 191-255 spontaneous activity of, 2W212 LSD-25, compared to phencyclidine, mff. effects on body temperature, 338339 effects on photic driving responses, 327

M Mammals, CNS regeneration in, 281293 Mescaline, effects on photic driving responses, 330 Muscle innervation, in frogs, 100-105 in lower chordates, 99-147 in mammals, 1W105 Myotomes, muscle fibers of, 106-108 Myxlne, muscle innervation in, 111-

115

N Nerve( s), growth of, 261-264 orientation of, 261-2434 peripheral, proteinases of, 65-66 Wallerian degeneration of, 78-80 Nerve growth factor, 80-82 Nerve proteins, exoplasmic flow of, 74-78 incorporation of, 73-74 metabolism of, 7 2 8 2 nerve growth factor and, 80-82 peripheral, 72-73 Nervous system, protein metabolism of, 1-98 Neural proteinases, role in brain protein catabolism, 84-65 Neurokeratins, in brain, 22

0 Optic nerve, of cat, 153-155 Osteichthyes, muscular innervation in, 127-128

P Peptidases, role in brain protein catabolism, 63-64 Phencyclidine, cardiovascular action of, 333-337 chemical structure of, 304-305 conditioned reflex and, 310-313 effects of, on animals, 310 on blood pressure, 334-337 on man, 305-310 on sciatic nerve responses, 331 EEG effects of, 315-326 metabolic actions of, 3384.42 neurobiology of, 303347 respiratory action of, 333-337 sensory and motor reflexes and, 313-315 Phosphoproteins, in brain, 22-23, 24 Pretectum, of cat, 162 Proprioception, in fishes, 139-140 Protein( s ) , brain, see u h Brain metabolism, of nervous system, 198 nerve, see Nerve protein Proteinases, role in brain protein catabolism, &72 Proteolipids, in brain, 2 p 2 5

R Reptiles, CNS regeneration in, 276277 Ribonucleic acid, role in brain protein formation, 5WM

s Schizophrenia, phencyclidine effect on, 307

SuSJEcr m Ex

476

Sernyl, see Phencyclidine Sleep, induced, study of, 413-418

T Telemetry, use in behavior studies, 3W62 Teleosts, muscle innervation in, 128-133 optic nerve regeneration in, 266-

288 spinal cord regeneration in, 264266 Thalamus, of cat, 158-162

U Urodeles, CNS regeneration in, 268270, 273-275

V Visual systems pathway, of cat, 149-

189

W Wallerian degeneration, protein metabolism in, 7 8 8 0