International Review of Neurobiology Volume 5

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INTERNATIONAL REVIEW OF

Neurobiology VOLUME 5

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INTERNATIONAL REVIEW OF

Neurobiology Edited by CARL C. PFEIFFER New Jersey Psychiatric Institute Princeton, New Jersey

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

Associate Editors V. Amassian J. A. Bain D. Bovet lord Brain Sir John Eccles

VOLUME

E. V. Evarts H. J. Eysenck F. Georgi G. W. Harris

R.

C. Hebb K. Killam S. Martens

G. Heath

5 1963

ACADEMIC PRESS

0

New York and London

COPYRIGHT 0 1963, BY ACADEMICPRESSINC. ALL RIGIiTS RESERVED.

NO PART OF "€XIS BOOK MAY BE REPRODUCED IN ANY FORM, BY PHOTOSTAT, MICROFILM, OR ANY OTHER MEANS, WITHOUT WRITTEN PERMISSION FROM THE PUBLISHERS.

ACADEMIC PRESS INC. 111 Fifth Avenue, New York 3, New York

United Kingdom Edition published by ACADEMIC PRESS INC. (LONDON) LTD. Berkeley Square House, London W.1

LIBRARY OF CONGRESS CATALOG CARDNUMBER:59-13822

PRINTED IN THE UNITED STATES OF AMERICA

CONTRIBUTORS F. J. BFUNLEY,JR., D e p r r r t n t o f Physiology, The Johns Hopkins School of Medicine, Baltimore, Mayhnd

R. V . COXON,University Labmatoy of Phqsiobgy, Oxford, Enghnd WALTERJ . FREEMAN, Department of Physiology, University of Calif ornia, Berkeley, Califmnia RUTH S. GEIGER,Division of Behuoioral Sciences, Institute for Medical Research, Chicago Medical School, Chicago, I l l i d

L. GIBBONS,Department o f Psychiuty, Institute of Psychiaty, M a d l e y Hospital, London, England

JAMES

GUNNARHOLMBERG, Psychiatric Clinic, Centralhrettet, D a m b y d , Sweden, and Karolinska lnstittitet Medical School, Stockholm, Sw& RICHARD P. MICHAEL, Department of Psychiatry, Institute of Psychiatry, M a d l e y Hospital, London, England

KOITI MOTOKAWA,Department of Physiology, Tohoku Uniumsity School of Medicine, Sendai, J a p m

AND& SOULAIRAC,Laboratoire de Psychophpiologie, Facultby des Sciences, Paris, France

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PREFACE This review has now reached its fifth birthday and this seems a suitable point to discuss its course and progress so far. Our original aim was to present to scientists of many disciplines a wide coverage of neurobiology in the form of essays written by authorities in the various overlapping facets of this rapidly expanding field of study. As each science in neurobiology progresses it relies more and more on those higher in the positivistic hierarchy of science. Anatomists discuss their problems in chemical terms, physiologists deal with theirs by drawing on the concepts of biophysics, psychology shades off into the higher mathematics of information theory. In the first five volumes the essays have been divided among the following main fields (although in some cases there was much overlap with another field) : neuroanatomy ( 6), neurophysiology ( lo), neurochemistry ( 11), neuropsychopharmacology ( 7 ) , clinical psychiatry and neurology ( 6 ) . Of these 8 had some direct bearing, 8 an indirect bearing, and 24 no apparent bearing on clinical neurological and psychiatric problems. So we feel that a fair balance has been achieved between the various disciplines concerned and between basic and more applied research. The diseases that have been concerned (directly and indirectly) are schizophrenia ( 7 ) , epilepsy (4),multiple sclerosis ( 1), alcoholism ( 1) , miscellaneous ( 3 ) . This particular distribution probably reflects the particular interests of the editors but also, in part, the volume of basic neurobiological work under course in these fields.

August 1963 CARLC. PFEIFFER R. SMYTHIES

JOHN

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CONTENTS CONTRIBUTORS

PREFACE

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The Behavior of Adult Mammalian Brain Cells in Culture I. I1 I11. IV V VI VII VIII . IX. X XI XI1 XI11. XIV .

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XVI XVII XVIII

RUTH S . GEIGER Introduction . . . . . . . . Methods . . . . . . . . . Description and Properties of Neurons . . Interactions among Neurons . . . . . Properties of Glial Cells . . . . . Interaction of Neurons and Glial Cells . . Glial Cells and Capillaries . . . . . Effects of 2-hinotricyanopropene . . . Effects of Cellular Narcotics (Sodium Barbital) . Effects of Stimulants (Pentylenetetrazole) . . Effects of Serotonin . . . . . . Effects of Epinephrine . . . . . . Effects of Hallucinogens (LSD-25) . . . Effects of Acetylcholine and Eserine . . . Effects of Brain Extracts . . . . . Enzyme Activity of Brain Cell Cultures . . . . . Some Uses in Neuropathology . Conclusions and Summary . . . . . References . . . . . . . .

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The Electrical Activity of a Primary Sensory Cortex: Analysis of Eeg Waves

I. I1. I11 IV. V. VI VII . VIII IX .

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. . . . . . . . Introduction . . The Functional Subdivision of the Brain . The Spatial Structure of Neuronal Electrical Fields . Distribution of Prepyriform Electrical Activity . . Isolation of the Prepyriform Signal . . . . Comparison of Evoked and Spontaneous Potentials . Correlation of Electrical Activity with Behavior . . Input-Output Relationships for the Prepyriform Cortex Conclusions and Summary . . . . . . References . . . . . . . . . ix

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53 54 59 66 76 79 96 102

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CONTENTS

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Mechanisms for the Transfer of Information Along the Visual Pathways KOITI MOTOKAWA . . I. Introduction . . . . . . . . . . . . . . . . . . . I1 Retina . . I11. Impulse Conduction in the Optic Nerve . . . . . IV. Centrifugal Fibers within the Optic Nerve . . . . . . . . V. Lateral Geniculate Body . . VI . Nonspecific Afferents and Visual Transmission . . . . . . . . . . VII . Visual Cortex . . . VIII Corticopetal and Corticofugal Nonspecific Effects . . IX . Color Vision . . . . . . . . . . . . . . . . . X . Pattern Vision . . . . . . . . . . XI . Summary . . . References . . . . . . . .

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Ion Fluxes in the Central Nervous System F. J . BRINLEY. JR . Theoretical Section . . . . . . . Extracellular Space and the Ionic Composition of Brain Exchangeable and Nonexchangeable Ions . . . Spreading Cortical Depression . . . . . Effects of Drugs on Membrane Permeability of Central Nervous System Cells . . . . . . . Summary . . . . . . . . . . References . . . . . . . .

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Interrelationships between the Endocrine System and Neuropsychiatry RICHARDP. MICHAELAND JAMES L GIBBONS Introduction . . . . . . . . . . Adenohypophysis. the Adrenal Cortex. and Emotion . . The Thyroid and Psychiatry . . . . . . . References . . . . . . . . . .

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121 122 129 134 138

Neurological Factors in the Control of the Appetite AND& SOULAIRAC The Regulating Nervous Structure . . . . . . Nervous Mechanisms Controlling Feeding Behavior . . . Tentative Explanatory Hypothesis Regarding the Control of Appetite . . . . . . . . . . . General Conclusions . . . . . . . . . References . . . . . . . . . . .

304 314 332 339 342

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CONTENTS

Some Biosynthetic Activities of Central Nervous Tissue R . v . COXON I. Introduction . . . . . . . . . . . I1. Methods of Study . . . . . . . . . . I11. Fatty Acid Synthesis in Brain . . . . . . . IV . Protein Synthesis in Brain . . . . . . . . . . . . . . . V Glycogen Turnover in Brain . VI . Conclusion . . . . . . . . . . . References . . . . . . . . . . .

347 349 356 363 371 379 383

Biological Aspects of Electroconvulsive Therapy GUNNARHOLMBERC Elicitation of Therapeutic Convulsions . . . . . Modifications of Treatment . . . . . . . . Physiological Effects of ECT . . . . . . . Endocrine and Biochemical Changes Associated with ECT . Clinical Effects of ECT . . . . . . . . . Psychological Effects of ECT . . . . . . . Physiopathology and Neuropathology of ECT . . . . Complications of ECT . . . . . . . . . Prognostic Test Procedures and ECT . . . . . . Mode of Action of ECT and Its Relation to Other Therapies . References . . . . . . . . . . .

389 390 391 393 395 397 399 400 402 403 406

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THE BEHAVIOR OF ADULT MAMMALIAN BRAIN CELLS IN CULTURE1

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By Ruth S Geiger Division of Bchaviorol Sciences. Institute for Medical Research. Chicago Medical School. Chicago. Illinois

I. Introduction . . . . . . . . . . I1. Methods . . . . . . . . . . . I11. Description and Properties of Neurons . . . IV . Interactions among Neurons . . . . . . V Properties of Glial Cells . . . . . . . VI Interaction of Neurons and Glial Cells . . . VII . Glial Cells and Capillaries . . . . . . VIII Effects of 2-Aminotricyanopropene . . . . IX Effects of Cellular Narcotics (Sodium Barbital) X Effects of Stimulants (Pentylenetetrazole) . . XI Effects of Serotonin . . . . . . . . XI1 Effects of Epinephrine . . . . . . . XI11. Effects of Hallucinogens (LSD-25) . . . . XIV Effects of Acetylcholine and Eserine . . . XV. Effects of Brain Extracts . . . . . . . XVI. Enzyme Activity of Brain Cell Cultures . . . XVII Some Uses in Neuropathology . . . . . XVIII Conclusions and Summary . . . . . . References . . . . . . . . . . .

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I . Introduction

The in vitro cultivation of tissues makes it possible to observe directly in a living state. under standardized conditions. and sometimes for long periods of time. various cells and their metabolic activities. their interactions. and reactions to environmental conditions and to drugs. their regenerative and growth properties. etc It is pos-

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Supported jointly by grants from the Illinois State Department of Public Welfare. the United States Public Health Service. and the Scottish Rite Mason's Committee on Research in Schizophrenia and the Brain Research Foundation .

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sible to maintain cultures and subcultures of adult mammalian brain tissue and to obtain up to sixth-passage subcultures over extended periods of time. This paper deals with the behavior of such adult mammalian brain cells in culture. The history of the cultivation of nervous tissue is coincident with the inception of tissue culture itself. Harrison (1907, 1910) isolated neural tissue from the frog embryo and maintained it in sterile frog lymph. He was able to demonstrate conclusively that axons are generated by the nerve cells themselves; this established an irrefutable basis for the concept of the neuron proposed by Ramon y Cajal ( 1934). Further studies by Levi and Meyer (1936-1937, 1941), Weiss and Wang (1936), Peterson and Murray (1955), and Murray ( 1959), using embryonic chick material, showed the differentiation of neuroblasts into ganglion cells, the development of Nissl substance, the appearance of neurofibrils, the outgrowth of neurites, and the myelinization of axons. Levi-Montalcini et al. (1954) were able selectively to accelerate greatly the growth of processes of sympathetic and spinal ganglia, at first with factors isolated from mouse sarcomas, and then even more effectively with mouse salivary-gland factors. Crain (1958) recorded intracellular resting and action potentials of neurons in embryonic spinal-cord explants maintained in vitro for a month to 6 weeks. Cunningham et al. (1960), Cunningham and Rylander (1961) have recorded spontaneous potentials over a period of 14 days from explants of embryo brain from 8-dayold chickens. Hild et al. (1958) showed that both astrocytes and neurons in explants of newborn kitten cerebellum have a membrane potential of 50 mv. Neurons, when stimulated by extracellular electrodes, showed an action potential of l-msec duration and 40-70 mv amplitude. The astrocyte response was a sudden depolarization followed by a slow return to resting level, the latter about 1000 times longer than that of neurons. Hild and Tasaki (1962) recorded resting and action potentials from neuron somata and dendrites in cultures from newborn rat and kitten cerebella with intracellular microelectrodes. These cultures were maintained in vitro up to 25 days either on a plasma clot or on reconstituted collagen gel (Ehrman and Gey, 1956 ) . With extracellular microelectrodes they also demonstrated spontaneous firing of neurons in these cultures and showed that both neuron somata and dendrites were excitable and capable

MAMMALIAN BRAIN CELLS IN CULTURE

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of producing all-or-none, propagated impulses. They assumed that synaptic structures and presynaptic fibers were not present in these cultures. However, in cultures of adult mammalian brain cortex maintained for long periods of time, structures having all the morphological characteristics of synapses can be demonstrated both within the explant and the migratory zone (Geiger, 1958a, 1962b; Geiger and Stone, 1962a, b ) , as will be discussed below. In the cultures of the embryonic nervous tissue referred to above, the neurons themselves did not migrate out from the explant. Hogue ( 1946), however, observed migration of neurons from explants of cerebral gray matter of human fetuses. In marked contrast to neurons from embryonic tissue, the neurons from adult tissue migrate out from the explant. Murray and Stout (1947) were the first to demonstrate migration of adult neurons in cultures of adult human sympathetic ganglia. They also showed that such neurons may occasionally undergo mitosis. Costero and Pomerat (1951) and Hogue (1953) were successful in maintaining cultures of adult human cerebral and cerebellar cortex and demonstrated that neurons as well as glial elements migrate out from the explants of such tissue. Geiger and Behar (1953) and Geiger (1957a, 1958a) were able to obtain cultures and subcultures from the cerebral and cerebellar cortex of adult humans, monkeys, and rabbits; all these cultures showed migrating neurons and glial elements. Some of these cultures were maintained for as long as 2 years. II. Methods

Cultures of newborn and adult mammalian brain have been made from a number of areas using the roller-tube method (Costero and Pomerat, 1951; Lumsden, 1951; Hild, 1954, 1957a, b,c; Okamoto, 1958) and the coverslip method (Hogue, 1953). In order to obtain long-term survival of cultures and subcultures, and to be able to study the histological, cytochemical, and behavioral aspects of cells in such cultures, we (Geiger and Behar, 1953; Geiger, 1958a), used a combination of the Carrel1 flask technique and the lying-drop double-coverslip Maximow method, which will be briefly described. Cultures were made by planting tissue fragments of approximately 1-2 mm3 obtained under sterile conditions from the cerebral cortex, into a medium consisting of one-part chicken plasma, twoparts feeding solution [30%homologous serum, 30% Eagle’s solution

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R. S. GEIGER

(Eagle, 1955), 40% Tyrode’s solution (Tyrode, 19101, and one drop of chick-embryo extract-enough for clot forrnation-in 3.5cm or 5-cm Carrell flasks. After 24 hr, at 37”C, feeding solution was added as a supernatant phase, and was renewed every 72-98 hr, depending on the amount of tissue planted. To obtain relatively pure cultures of neurons, 0.01-0.05 mg/ml cortisone acetate was added to the feeding solution. Due to the sensitivity of the neurons, the continuous use of antibiotics is to be avoided (Geiger, 1958a). Generally, it is possible to make subcultures at any time after the cultures have been growing for 2-3 weeks. The subcultures were made in Carrell flasks and Maximow slides. By subculturing, it is possible to get a large number of uniform cultures for comparative studies on migration, regeneration, and drug effects. Such subcultures have been maintained in Carrell flasks for as long as 2 yr. These subcultures are useful for long-term survival studies, and also furnish material for subsequent subculturing. The Maximow lyingdrop cultures can be used for detailed observations of the outgrowth and for time-lapse microphotography. They can also be used for electron microscopy and for radioautograms, as well as for histochemical studies. After the second month in culture, the feeding solution was altered (W homologous serum, 30%Eagle’s solution, 3ox; Tyrode’s solution, lofx Tyrode’s extract of brain tissue). Better results were obtained when the media was supplemented with brain extracts. Feeding solutions should always be supplemented with brain extracts for subcultures beginning with the second passage. After a period of several days, reactive glial cells and some dendrites appeared at the edge of the initial explant. The perikarya of the neurons commenced to migrate out from the explants of adult mammalian brain after 8 days to 2 weeks or even longer (Geiger, lQSla, 1958a, c; Pomerat d al., 1957). Presumably, the period preceding migration is one of repair (following the removal of the tissue from the organism) and can be compared to the dormancy period in a healing wound. However, in all probability, the accumulation of catabolic products is also necessary, for, by allowing the pieces of cut tissue to incubate for 1 hr in a 0.001-0.005%trypsin solution before implantation, the perikarya of the neurons was induced to migrate out from the explant much sooner-after 4-8 days, After 2-3 weeks of migration and growth, a sufficient growth zone (consisting of glial elements, neurons, and at times mesenchymal elements) ap-

MAMMALIAN BRAIN CELLS IN CULTURE

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peared around the explant to enable subculturing. Both in the original cultures and in the subcultures, the neurons leave the explant very slowly as compared to other types of cells. Once they have migrated some distance from the explant, they assume a shape which they then retain for months. The axons may continue to grow in size and may become myelinized. Myelinization usually starts after a period of 3 4 weeks and is generally dependent on the presence of oligodendroglia. Associations can be established between the axon of one neuron and the perikaryon or dendrites of another (synaptic areas). Glial elements may become associated with the neuronal perikaryon, dendrites, or axon, or with one another, in a manner similar to that of brain sections (Scheibel and Scheibel, 1958). Ill. Description and Properties of Neurons

Neurons generally show a structural polarity. The axon hillock and axon can usually be distinguished from the rest of the perikaryon and dendrites (Fig. 1A)' (cf. Geiger, 1956a, 1960). Many neurons can be identified as pyramidal cells, giant pyramidal cells, giant polymorphous neurons, bipolar neurons, or purkinje cells. The cell bodies of the neurons vary in length from approximately 15 to 200 p. Binucleated neurons are encountered most often when cortisone or 2-aminotricyanopropene is present in the culture medium. Each nucleus usually contains one large nucleolus; however, 2 or even 3 nucleoli may be present. Nucleolar satellites (Barr and Bertram, 1951) or larger areas of heterochromatin (Hydkn, 1947), described for stained sections of brain can frequently be observed in the living cells. The nucleus is centrally or laterally located and appears round, oval, or streaming, depending on the position of the neuron in the clot, as well as on the physiological state of the cell. Viewed with phase-contrast optics, the nucleoplasm in the resting state appears clear, the nucleoli are dense and round, and the cytoplasm is granular. It is possible to distinguish mitochondria and other granules, such as Nissl granules. The Nissl particulates are absent from the axon hillock and from the axon but can be seen in the dendrites. In the perikaryon, the Nissl granules vary in location, size, shape, and density. The mitochondria are present throughout the perikaryon, dendrites, and axon, and are concentrated at axon end'All photographs are of living cells from subcultures of Maximow slide preparations except where stated.

a

m

FIG. 1 . A-F. Living I)inuclr;atcd pyramidal cdl from xail)culturc. sho\ving nuc1t.i ( X l ' ), nuclroli ( SLIC ), r;amif!ing tl(*ndritc. ( D ) , axons ( A ) , iixon hillock ( A H ). h'otc. ;Il)scmccb of tl(snsc. iwuroniil cytoplasmic granulrs ( KC<; ) from axon hillock, and also movernc-nt of granu1c.s h v n sides of axon hillock by comparing this iar(*;i in Fig. 1A and in Fig. 1F. singlc. friarnc-s 30 min upiwt from timc*-l;ipsvrnovic. Also, obwrw~slight cIiiingt-5 in sin, of i a x m I i i h k . 1'Ii:asc. contrast, 150 X . Fic. 2 . Siamt. t y w cell a h in Fig. 1, from frontal lolw of Iiumm. fifth p i ~ w g csulxwlturc~.stainvtl with .)y osmic acid vapor.

--l a x .

MAMMALIAN BRAIN CELLS IN CULTURE

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ings. They can further be identified in the living cells by vitally staining with J a m s green. The Nissl granules stain vitally with neutral red. Methylene blue first stains the Nissl granules and then the rnitocliondria in the living cells, thereby showing up the boutons terminaux (Geiger, 195Sa, 196%; HydAn, 1960). Neurofibrils can sometimes be identified in the living neurons (Figs. 1, 15, 16, and 18). When epinephrine is added to a culture, more neurofibrils become visible (Geiger, 195%; Geiger et al., 1960). These are seen to emanate from the nucleus, starting at the region oi the nucleolus and going into the cytoplasm (Fig. 1 6 ) . Weiss and Wang (1936) and Levi and Meyer (1936-1937) have described neurofibrils in living bipolar neuroii~in ganglion explants of chick embryo. Neurons may further be identified by their staining properties in fixed preparations of whole mounts of Maximow lying-drop cultwes or ofsections from Carrel1 flask cultures. Fixing and staining with osmic acid shows the presence of numerous osmophilic granules (Figs. 2 and 5) which vary in size and degree of osinopldia, and the presence of myelinized axons; the latter appear any time after 3-4 weeks in culture. Nissl substance can be demonstrated wit11 Fletcher thionine stain, gallocyanin-chrome alum, or cresyl violet ( Figs. 29 and 33). The Nissl substance appears as either particulates, strands, granules, or fine dust-like particles, and can be identified by staining in vacuoles during early chroinatolysis (Geiger, 1957a; 195Sa). Myelinized and unmyelinized axons can be found. Myelinized axons have been demonstrated in the living cell with plain light, phase contrast, or polarized light, and in stained preparations with Kliiver’s LUXOIblue or Sudan black B or demonstrated after osmicacid-fixation with the electron microscope ( Fig. 11) ( Geiger, 195621, l958a, c, 1960; Murray, 1959) . Actively secreting oligodendrocytes are usually present on the axon or in its vicinity (Fig. 1 2 ) . Hild ( 19S9) described myelinization ot axons in cultures of newborn kitten cerebellum in the absence of any oligodendrocytes. Bornstein ( 1958) noted the presence of oligodendrocytes during myelinization of fibers of newborn rat and kitten ccreliellum. De Robertis and Gerschenfeld ( 1961) reviewed extensively the relationship between myelinization of axons and the presence of oligodendrocytes in the brain cortex as observed by electron microscopy, based on the initial observations and hypothesis of Luse ( 1956). By Koelle’s method ( 1955). using acetylthiolcholiiie and butyryl-

FIG. 3. Oligodendrocytes encircled by branch of unmyelinatrd axon. Phase contrast, 5 0 0 ~(reduced ). FIG.4. A X . Purkinjr crll from subculture. Single frames from time-lapw movie, 40 min apart, showing astrocytc pulling away and undergoing division. Phase contrast, 200 x ( rcduced ).

FIG.5. Same type neuron as Fig. 4, from subculture of human cerebellum, stained with 2%osmic acid vapor. 2 0 0 ~(reduced).

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R. S. GEIGER

MAMMALIAN BRAIN CELLS IN CULTURE

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thiocholine as substrates in conjunction with specific inhibitors such as diisopropyl fluorophosphate and eserine, we were able to demonstrate the presence of cholinesterases in cultures and subcultures, some of which had been maintained over 6 months. True acetylcholinesterase was present as granules in cell bodies of the neurons and along the axons. High concentrations were found at axon endings, in the boutons terminaux at presynaptic endings, or in those ending around oligoclendrocytes (Figs. 6A-D, 7, and 8). Butyrylcholinesterases (Fig. 9) were concentrated mainly in oligodendrocytes and astrocytes (Geiger and Stone, 1962a, b ) . For localization in brain section, see Koelle ( 1955, 1957) and Hebb ( 1959), Occasional mitosis of neurons was seen and followed in the cultures with time-lapse, phase-contrast photography in living neurons and also after fixing and staining the preparations (Geiger and Behar, 1953; Geiger, 1956a, 1957a, 1958a). They were encountered most frequently in first-passage subcultures with cortisone in the media. Mitoses are not, however, frequent enough to sustain a culture. The neurons growing in cultures and in subcultures are primarily those which migrated from the transferred portion of the explant. During mitosis, neurons preserve the characteristic shape of their cell bodies. New neurites are produced by both cells following the telephase. During mitosis, neurofibrils remain present in the neurons, but may diminish in number and become somewhat coarser and more irregular. Continuous intracellular movements can be demonstrated in neurons by the use of time-lapse, phase-contrast microcinematography at a rate of 4 frames per min, and projecting the film at 24 frames per sec. The impression of streaming of the cytoplasm in the perikaryon and processes is created by slow, continuous, unidirectional movement of groups of mitochondria and other granules. Cytoplasmic granules also show other kinds of movements which are independent of the general direction of the cytoplasmic streaming, ~~

~

~

FIG. 7. Same type of preparation as Fig. 6A-D, showing distribution of acetylcholinesterase in cell bodies of neurons. 280 x . FIG. 8. Same type of preparation as Figs. 6 and 7, showing presence of aoetylcholinesterase in axon endings around oligodendrocytes. 280 x . FIG. 9. Nonspecific cholinesterase in glial satellites of subculture of rabbit cerebral cortex, incubated with butyrylthiocholine. Fixed 12 min after addition of Na barbital. 280 x .

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R. S. GEIGER

MAMMALIAN BRAIN C U L S IN CULTURE

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usually at a faster rate, to and from the nuclear membrane or to actively growing processes. Slow, pulsating movements of the cytoplasm, as evidenced by movement of the granules in the perikaryon and axon, can also be observed. These movements are enhanced by stimulation or injury. A continuous movement of granules occurs, together with accumulation, at the margin of the axon hillock (Fig. 1A-F), which is thereby sharply demarcated. This line of demarcation is further marked by the lack of Nissl granules in the axon hillock. Mitochondria and some other osmophilic granules do descend into the axons, travelling mostly near the inner surface of the cell boundaries. A streaming of the nucleoplasm and, on occasion, a pinching-off of the nucleoplasm into the cytoplasm, can also be seen; this latter phenomenon has also been described by Hogue (1953).Rolling movements and shifting of the nucleus, and changes in its size and optical density can be recorded. The nucleolus in the resting neuron is usually round and inert and shows a uniform optical density, but occasionally (due to a change in environmental conditions) it may spread out, move toward the nuclear membrane, and change its optical density and shape. The transfer of nuclmlar material to the cytoplasm has often been observed, but most regularly as a reaction to stimulation by electrical pulses or by drugs (Figs. lS, 16, and 19) (Geiger, 1954, 1956a,b, 1960). The neurons, after migrating out from the explant and establishing themselves in the growth zone, remain stationary. A slow, pumping movement of the neurons can be observed when the rate of the time-lapse photography is slowed to 2 frames per min over a period of several days and then projected at 24 frames per sec. A slow, shallow, gradual contraction seems to force cytoplasm down the axon. One cycle of contraction and expansion takes from 6-8 hr (Geiger, 1962b). FIG. 10. A-C. Changes in shape and size of boutons in presence of epinephrine. Single frames at 30-min intervals. Phase contrast, 350 x ( A and B ) , Fig. 10. C. Photograph of same area, 140 x , FIG. 11. Electron microphotograph from culture of rabbit cerebral cortex, showing myelin extending from small portion of oligodendrocyte to obliquely cut myelinating axon. Approx. 24,500 x . Electron microphotographs are from unpublished work of R. S. Geiger, E. Lang, T. Gibbs, and I. Karys. FIG. 12. Portion of myelinating axon. Insert shows same segment at different level of focus. Note presence of oligodendrocyte. Unmyelinated portion at end of axon shows branching. Plain light, 105 x ( reduced).

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R . S. GEIGER

IV. Interactions among Neurons

On neurons which have migrated and established themselves at some distance from the explant, axon endings coming from other neurons can be seen. These contacts ending on the perikarya and on dendrites (Fig. 14) resemble synapses as they would appear in histological preparations taken from brain. Some of these endings, when stained with Bodian’s protargol gold chloride, show a typical picture of boutons terminaux. Many mitochondria are concentrated in these endings. The boutons appear either as a solid mass or as a dark ring, in the living state. They stain very heavily with Janus green B or with methylene blue, These contacts, once established, have persisted for days or even months, in long-term cultures. When such “synapses”are observed with time-lapse, phase-contrast photography, the end-feet and their fibers move around within circumscribed areas on the neuron (Figs. 1, 4, 10, 18, 25, 26, and 28). In the synaptic region, the cytoplasmic granules of the neuron show rapid movements which persist; these synaptic areas are sensitive to environmental conditions, as will be described later. Axo-dendritic synapses also show continuous movement of the endings and fine fibers (Geiger, 1957a, 1958a, c, 196%). When chromatolysis occurs in the neurons, the presynaptic areas retain their character and the mitochondria remain in evidence. During chromatolysis, the mitochondria aligned around the boundaries of the vacuoles remain visible in the neuron itself long after dissolution of the Nissl granules. The neurofibrils also remain visible after the disappearance of the Nissl substance (Fig. 18A, B, C ) (Geiger, 1958b, Geiger ef al.. 1960). V. Properties of Glial Cells

As previously mentioned, the reactive glial cells, gemistocytes, and gitter cells are the first to migrate from the explant, followed by the oligodendrocytes. The more differentiated microglial cells and astrocytes migrate much more slowly, but they also retain their specific staining properties, and whole-mount cultures can be stained with Penfield’s stains for oligodendrocytes or microglial cells and Cajal’s stain for astrocytes. Oligodendrocytes also stain with Alician blue-p-aminosalicylate ( as do myelinized portions of axons ), indicating the presence of rnucopolysaccharide substance, a substance

M A M M A L I A N BRAIN CELLS IN C U L W E

15

FIG. 13. A-C. Subculture of human cerebral cortex. Single frames of timclapse movie at 2-hr intervals in presence of 2-aminotricyanopropenr. Noti. changes in astrocyte which is attaching to neuron i n region of nucleus. Phaw contrast, 140 x , FIG. 14. Axo-dendritic synapsc. Phastt contrast, 350 x .

probably containing hyaluronic acid since it is partially digested by hyaluronidase. Microglial cells stain positively for glycogen with p-aminosalicylic acid ( PAS ) , and the astrocytes may have mucoprotein inclusions (Lurnsden, 1958). Geiger and Behar ( 1953) also found a narrow band of material around nuclei of some pyramidal neurons as well as certain areas in the nucleoli which stain positively with PAS and are not digested by hyaluronidase or ribonuclease.

16

H. S. tiEICER

FIG. 15. A-E. Neuron from subculture of cerebellar cnrtex of rabbit. Single frames from time-lapse movie at 1-hr intervals. Note changes in nucleolus which extrudes material to cytoplasm. It goes through phase of swelling and contracting that rrstilts in grcutcar conclensiition of material around it. Observe visiblr nrurofibrils ( NF ) , Serotonin, 0.05 &ml present throughout sequence. Phase contrast, 400 x .

MAMMALIAN BRAIN CELLS IN CULTURE

Flc:. 16. A-D. 1~yr;iinitl;il nc'iirons p s e n c c of c.pinephrine

( 0.001

Iri)ni

s i ~ I ~ c u l t i ~i r) l~ -c.~~141r;il s c.orti.i

17

iti

pg/ml), sIio\ving clinngc-s in nuclius ;ind

nucleolus during neurofil)ril formation ;iritl incrcasct in Xis51 sul~st;ince.400 x . A and B. Single friimes from time-lapse movio, 60 initi and 85 min after addition of drug. Note incrrasc of fibrils i n nuclcus md cytoplasm, nlso increiiscin Kiss1 substancc~. C. Vitally staincd with ncutral red and Janus grrcn B, 60 min after i~cldition of drug. Xotc increased :imonnt antl tlistribution of Janus grem B staining m a t c h 1 prcwnt in nuclcw ( norm:rlly prcwnt iis thin ring around n u c k d u s ), as \vc-ll iis lorgc' amount of ncutr:il rcd-staining niatcrinl. D. Fixed antl stiiined with 2%osrnic :icitl vapor. ($0 min iifter addition of drug. Compare with Figs. 2, 5 , and 24.

18

R . S. GEIGER

FK. 17. Astrocytcs from subculture: of human subcortical whitc matter with processes ending around oligodendrocytes. 122 x . Fic. 18. A-C. Chromatolysis in presence of epinephrine. Vitally stained with neutral red arid Janus grcen B. Sequences at 2-hr intervals. Showing disappearance of neutral rcd-staining material brfort. mitcichontlria ( M ) or neurofibrils. Phase contrast, 400 X .

MAMMALIAN BRAIN CFLLS IN CULTURE

19

They possibly are sulfated mucopolysaccliarides,as Abood and AbulHaj (19%) and Young and Abood (1960) have demonstrated mucopolysaccharides in the perikarya and axons of neurons. The astrocytes encountered in cultures of the gray cortex are generally of the protoplasmic type, whereas those in cultures of pure subcortical white matter are more often of the fibrous forms. Glial elements also appear in intermediate forms. The cell bodies of oligodendrocytes are round or oval with a central nucleus, and a small amount of highly refractile cytoplasm. Microglial cells, in their highly diff erentiated form, have a cell body of only several microns in diameter and short, fine processes (Fig. 30). They also appear in rod forms, or gitter forms. The astrocytes may form a spcytium (Lumsden, 1958), or be present singly or as perineuronal satellites, often with elongated cell bodies and a clubbed end-foot, ending on a neuron ( Figs. 4, 13). The oligodendrocytes may appear free, or associated with axons (Fig. 3) or dendrites, or as perineuronal satellites, or associated with astrocytes (Fig. 17). The microglial cells in their differentiated form appear associated either with neurons or other glial elements. In their rod form or gitter form, they appear free in the immediate vicinity of the neurons or glial elements or in close contact with them ( Geiger, 1962b). When observed by time-lapse photography (Canti et al., 1937; Lumsden and Pomerat, 1951), the oligodendrocytes show pulsating movements of the order of 3-5 min for a cycle. During the contractile phase, the oligodendrocytes were seen to extrude material (Figs. 25 and :30).The microglial cells in the reactive state also were seen to give off granules and take up material. The astrocytes showed twitching movements and slight pushing movements against the neuron. Pomerat ell (11. (1957) observed the tips of astrocytes taking up fluid by pinocytosis. Reactive astrocytes ( gemistocytes) show fewer ameboid-type movements than do reactive microglial cells. Microglial cells, in their highly differentiated stage, are relatively immobile. In the rod form, their movement can be compared to that of worms: undulating waves of contractile movements. Oligodendrocytes can undergo mitotic or amitotic division in culture (Wolfram and Rose, 1957; Pomerat, 1958); astrocytes can undergo mitotic or amitotic division (Fig. 4 ) (Geiger, 1960; Geiger and Stone, 1961).

20

21

22

R. S. GEIGER

VI. Interaction of Neurons and Glial Cells

Experimental data collected in recent years strongly suggest that a constant exchange of material occurs between neurons and glial cells, and that this process is intensified during nerve activity, induced by stimulation or by various drugs. In a recent review, Hydhn (1960) discussed the available evidence for this, including the results of his own analysis of individual glial cells and neurons, obtained under various physiological conditions. In adult mammalian brain cultures and subcultures in which associations between neurons and glial cells lasted for days or weeks, we were able to observe and to photograph (time-lapse) direct transfer of material from glial cells to neurons, and to show the effects of the functional state of the neurons on their interaction with glial satellites (Geiger, 1960; Geiger et al., 1960; Geiger and Stone, 1961). In the presence of “resting” neurons, the differentiated oligodendroglial satellites show pulsating movements with some extruFIG. 19. A-C. Sequences showing transfer of nucleolar material in granular and fibrillar form ( with epinephrine), 400 x . FIG. 20. A-C. Single frames from sequence showing oligodendrocytes, which are aligned along a fiber, coalescing into one cell. 122 x . FIG. 21. Brain subculture fixed 12 min after addition of Na barbital and stained with phosphotungstic acid hemotoxylin ( as recummended by Mallory for demonstrating mitochondria), Note large size and dense staining of glial . elements, virtual absence of shin in cell bodies of neurons. 3 2 0 ~ Compare with Fig. 32. FIG.22. A. Neuron, 12 min after addition of Na barbital, fixed and stained with 2%osmic acid vapor. Note virtual absence of mitochondria in some areas of cytoplasm. Cell membrane shows increased osmophilia as do other cell structures. B. Same type neuron as Fig. 22A but without Na barbital, fixed and stained with 2%osmic acid vapor. 800 x . FIG.23. A-B. Two single frames from a time-lapse movie, 30 niin apart. Serotonin present. Note progressive increase in bulging of axon. Note also that structure of axon hillock can still be distinguished from the rest of cytoplasm of neuron’s perikaryon. Also, note contracted state of glial elements. 1 2 0 ~ . FIG. 24. Pyramidal cell with perineuronal glial satellites 10 min after addition of LSD-25, fixed and stained with 2%osmic acid vapor for 20 min. Note that satellites are in expanded state. 375 x . FIG.25. A-C. Single frames from time-lapse movie focused on surface of neuron, showing changes in size and position of boutons over a period of 3 hr in presence of eberine. 375 x .

MAMhlALIAN BRAIN CELLS IN CULTURE

23

R. S. GEIGER

1;~;. 26. A-C. Singlc friimcas from timc*-l;ipsr movie, focused on cncliiigs of synapses vitally st;iinc.tl with Jiiiius grcwi 13 in thc. p r cw n w of rsc*rinc~,civt’r ii ptariotl of 30 min. Fig. 26B. Endings nlrratly h;ivcA c.h;ingcd in size and tlrnsity, ;is wc4 a s I y p i to churigcb in color from grc-cn to rctl. 350 x . FIG 27. Single friimi. from timelapse movie showing rcutivc. glial &mcmts. 140 x . FIG. 29. Neuron fixed and stained with cresyl violet, 20 miri after addition of pentylenetctrazolc, showing Nissl substance, changes in iinclcus, absence of Nissl substance in axon hillock, presence of satellite glial cells. Compare with Fig. 33.

%Ox.

MAMMALIAN BRAIN CELLS 1N CULTURE

25

FIG. 28. A-I). Sing!(. frarnvs itt 2o-min intcrvals. E x c h i . 11rrsc.nt. Note incrcasc in mitochontlria in fibers and in endings of fibers on nwron, also changes in ncwronal cell body and glial crlls. 1 4 0 ~ .

26

R . S. GEIGER

FIG. 31A-C. See following page for figure legend.

FIC. 30. A-C. Single frames from time-lapse movie, showing transfer of material from oligodendrocyte to neuron in the presence of serum from schizophrenic patient. Observe oligodendrocytes in region of neuronal nucleus: B. Swollen; C.Contracted. 160x .

MAMMALIAN BRAIN CELLS IN CULTURE

27

FIG. 31. A-F. Single frames from time-lapse movies of subcultures of human cerebral cortex, showing glial capillary relationship. Note changes in density in areas of capillary cement, also in and around sucker feet, as well as in cell bodies of glial cells and along their fibers. These changes are due to continuous uptake and movement of phenol red. 320 x .

28

R . S. GEIGER

sion of material, movements around the neurons and along the axons, spider-like climbing movements, and movements by contraction and expansion. The astrocytes, when present as satellites, usually push against the neurons. When the glial cells are stimulated by drugs, after a short period of contraction, the movements around the neuron and around the neuronal processes are greatly enhanced and the transfer of material from glial cells to neurons becomes more frequent. Incipient chromatolysis attracts increasing numbers of differentiated and reactive glial cells to neurons and causes increased rate of movements and increased frequency of transfer of material from glial cells to neurons. Under these conditions the uptake of granules by reactive glial cells from their environment is more frequent. Narcotics reduced the activity of oligodendrocytes for an extended period, as did serotonin and LSD-25. The effects of these compounds will be described below in greater detail. In 1959, Chang and Hild reported that electrical stimulation caused glial cells to contract. Neurons and glial cells react differently to various chemicals, which presumably act on the structure of their membranes, and to changes in osmotic pressure in their environment. When isotonic sodium citrate is added to the medium, in the presence of a small amount of phenol red indicator, the effect can be seen on the microglial elements which turned pink within a minute after the addition of citrate. The oligodendrocytes and astrocytes also turn color within a very short time. The effect of the citrate on the neuron is seen a few minutes later than that on the glial cells. When distilled water is present at the bottom of a well of a moist chamber, at a short distance from the culture so that the water vapor can gradually dilute the culture medium, the neuronal satellites increased their activity, especially expansion and contraction, long before any changes in the behavior of the neurons could be seen. In the electron-microscopic studies of De Robertis and Gerschenfeld (1961), brain slices were exposed to hypotonic solutions. Swelling of astrocytes was observed before changes in neurons could be seen. . When neurons are not present in the culture (subcortical white matter), differentiated glial cells may show pulsating and even slight twitching movements and secretion of material into the medium, but they are stationary for long periods of time and do not move about as when neurons are present. They usually remain in a fixed position even when they are stimulated by epinephrine, norepinephrine, or aoetylcholine, which after a short time induce an acceleration of

MAMMALIAN BRAIN CELLS IN CULTURE

29

pulsating movements and secretion of material by the oligodendrocytes. The material extruded by the oligodendrocytes shows some uptake of Janus green B vitally; it is osmophilic, and stains with Nile blue sulfate and either Sudan black B or Lux01 blue. The role of oligodendrocvtes in myelinization has been discussed elsewhere. VII. Glial Cells and Capillaries

Capillaries were grown in a number of cultures. As time went on, they became surrounded with what appears to be a glial membrane. Astrocytes and oligodendrocytes, once associated with capillaries, remained in the same position for months. The capillary cement concentrated phenol red from the nutrient media, where it was present in very small concentrations as an indicator. Even when the media became slightly acid, through accumulation of metabolites, and turned yellow, the pink-staining material retained its color, indicating that some of the high molecular substances of which the capillary “cement” is composed are neutral or basic (most probably protein), and adsorb or bind phenol red. Recently, a glucoprotein that concentrates phenol red has been isolated from brain capillaries ( Watanabe, S., Otsuki, S., and Geiger, A., Personal communication, 1962). From the capillary cement, the phenol red was gradually concentrated further underneath the sucker feet of the astrocytes and then drawn up through them into the cell body. Subsequently, the whole astrocyte took on a reddish color (Fig. 31A-F ) . The transport of the phenol red from the capillary cement into the sucker feet was accompanied by slow wntractile movements (Geiger et ul., 1960). Waves of contraction could be observed in the longer glial cell fibers. As a rule, phenol red is used in the medium as an indicator but it is not taken up by any of the glial cells unless they are attached to capillaries (Geiger and Stone, 1961; Geiger, 196%; Schaltenbrand and Bailey, 1988; Tschirgi, 1952; Bakay, 1956; Bairati, 1958; De Robertis and Gerschenfeld, 1961) . VIII. Effects of 2-Aminotricyanopropene”

Grenell and HydCn (1961) have shown that injection of 25 mg/ kg of 2-aminotricyanopropene into rabbits increases the cxmcentration of brain nucleic acids by up to 301%within 1 hr. The addition ‘The drug (U9189) was kindly given us by The Upjohn Company, Kalamazoo, Michigan.

30

R. S. GEIGER

of this drug to adult cortical cells in culture, in concentrations of

2-4 pg/ml, caused rapid increase in the size of the nucleolus and increased the optical density of the nucleoplasm. The concentration of Nissl substance increased and neurofibrils became visible in the living neuron, The nuclei of the oligodendrocytes became larger and more dense and secretion of myelin or its precursor was observed. The astrocytes showed an increased number of granules in their bodies (Fig. 13A-C ). All types of cells increased in size over a period of 2 days (Geiger and Stone, 1961). The microglial cells, although they became larger, still remained differentiated; more binucleated neurons appeared in the cultures and myelinization was enhanced. The increased amounts of RNA in the nucleolus and cytoplasm was shown by histochemical methods. Staining with Bodian’s protargol gold chloride showed fibrillar aggregates in the nucleus, as well as thick neurofibrils throughout the neuron. IX. Effects of Cellular Narcotics (Sodium Barbital)

Sodium barbital, when added to the brain cultures, in concentrations comparable to those used for cellular narcosis in duo, affects the structural elements of the neurons (Geiger, 1956b, 1957a). These effects are reversible, when the narcatic drug is washed out. The most striking effect of sodium barbital consisted in the disappearance (within 2-10 min) of the neuronal mitochondria after the addition of the narcotic. These mitochondria gradually reappear 15-20 min later as very small granules, finely dispersed throughout the cytoplasm. After washing and feeding of the culture, the mitochondria reappear in their original size within about 15 min. The Nissl substance appears to be unaffected by barbiturates. Occasional processes of neurons, in the presence of barbiturates, show some retraction from synaptic regions. The effect of the narcotic drug on the mitochondria, observed with phase-contrast optics and time-lapse photography, was confirmed by fixing and staining with osmic acid or with phosphotungstic acid hematoxylin, according to the recommendations of Mallory for demonstrating mitochondria ( Figs. 21 and 22). The same picture was observed in the living cells with phase contrast or vital staining with Janus green B. Other osmophilic granula (not mitochondria) appear fewer, but larger, than in normal cells. The membrane of the neuron, in the presence of barbiturate, stains more deeply with osmic acid and phosphotungstic acid

MAMMALIAN BRAIN CELLS IN CULTURE

31

hematoxylin than under normal conditions. The perineuronal oligodendrocytes are also very deeply stained (Geiger, 1962a). A decrease of the acetylcholinesterase in the perikaryoti was observed. The oligodendrocytes stained heavily for butyrylcholinesterase (Fig. 9) (Geiger and Stone, 1962a, b). The narcotic stopped the pulsating tnovements of oligodendroglial elements and progressively increased their size for about an hour, as seen with time-lapse photography (Geiger and Stone, 1961). After this time, the pulsating movements began again at a sotnewhat accelerated rate, even when the narcotic retnained in the culture. These effects of the narcotic on the glial cells, namely the inhibited pulsations, increase in size, and heavier staining, may indicate an interruption of material-transfer from the glial cells to neurons. Pentylenetetrazole, in concentrations of 50-100 pg/ml, when added simultaneously with sodium barbital, inhibited the phenomena described above. Also, when added at any time to barbitaltreated cells, pentylenetetrazole reversed the barbital effect and mitochondria reappeared within a few tninutes ( Geiger, 1956a). A convulsant barbiturate, sodium 1,3-diniethylbutyletl1yl barbiturate, when added to brain cell cultures, did not affect the neurons or glial cells in the manner described for sodium barbital. On the contrary, its action was similar to that of pentylenetetrazole, described below. These data indicate that narcotic drugs act specifically on mitochondria of neurons by dispersing them, lending support to a hypothesis advanced previously that one of the effects of narcotic drugs may be the alteration of the relationship between the enzymes in the mitochondria and those in the rest of the cytoplasm, thereby modifying enzyme activity. It is well known that thorough dispersal and mixing of the enzymes of the particulates with those of the cytoplasm may lead in extreme cases to complete blocking of glycolysis in brain tissue (Geiger, 1939). Bain (1952) found no difference between barbital and 1,3-dimethylbutylethyl barbiturate in their inhibitory effect on oxidative phosphorylation of rat brain mitochondria. X. Effects of Stimulants ( Pentylenetetrazole 1

Pentylenetetrazole and electrical stimulation act primarily on those structures of the neuron which are rich in nucleoprotein,

VK;. 32. hlitochoiitlria iii iit*iiroii iintl glinl cc.115, 12 niin aftor addition of ptlntylrnctc~trazol~.Staincd with phosphotuiigstic ticid hrmotorylin according to Mallory ( for clrmonstrating mitcJcliondria). Cornparv with Fig. 21. 360 x .

32

MAMMALIAN BRAIN CELLS IN CULTURE

33

namely, the Nissl substance, nucleus, and nucleolus. After the addition of pentylenetetrazole (in concentrations of 50-100 pg/ml) the Nissl substance and mitochondria are rapidly collected and form a thick, dark band around the nuclear membrane. For the next 20 min, the Nissl substance appears to increase in concentration throughout the perikaryon and dendrites. The liponucleoprotein granules gradually aggregate into large, coarse particulates, most striking in the dendrites, which acquire a knobby appearance. In fixed and stained preparation, large osmophilic and Nissl aggregates are found ( Figs. 29 and 30). These changes are accompanied by an increase in the size of the nucleus and nucleolus. The nucleolus shows zones of increased density, and frequent transfers of nucleolar material into the cytoplasm may be observed. Two kinds of transfer of material were observed: One is a kind of snapping-off into the cytoplasm of particulates originating in the nucleolus; the other, a slow penetration of less well demarcated material and of granules and fibrous material into the cytoplasm. The transfer of material from nucleolus to cytoplasm and from cell to cell, which was recorded with time-lapse photography in living cells, confirmed the large number of observations made by earlier authors on histological preparations (see Hydhn, 1960; Einarson, 1960). The optical density of the nucleus increased, but streaming of the nucleoplasm was still observed. About an hour after the addition of pentylene-tetrazole, contractions of the neurons were seen. The rate of movement of the cytoplasmic granules of the neuron was also increased, while the ends of the neuronal processes, whether free or in areas of synapses, markedly swelled and their movements became more rapid, After a short time glial activity was accelerated (Geiger, 1956b, 1957a, 1958c; Geiger and Stone, 1961) . In sections of cultures, methacrylate-imbedded by the technique of Borysko ( 1956), electron-microscopic examination showed that neurons, synaptic areas, and myelinated axons, as well as glial cells, retained their basic submicroscopic structures in long-term cultures. Some studies have been done on the effects of pentylenetetrazole (in concentrations of 50-200 &ml) on cells which migrated out FIG.33. Neuron, 4 hr after addition of pentylenetetrazole. Stained with gallocyanin chrome alum according to Einarson, showing dense Nissl aggregates and nucleolus. 9 0 0 ~ .

34

R . S. GEIGER

FIG. 34. Electron microphotogruph of section of adult mammaiian brain subculture from migratory zone. Note Golgi apparatus below nuclear membrane slightly to left of center. Along the right side, especially at right center, can be scen sections of endings of processes. Double membranes, vesicles, electron opaque areas and empty spaces are visible. Magnification approximately 20,OOOX. Insert shows detail at 60,OOOx of sectioii of ending near nucleus.

MAMMALIAN BRAIN CELLS IN CULTURE

35

FIG.35. Same type of preparation as Fig. 34. Showing cytoplasmic structure. Note few ovoid bodies ( lysosomes ), also electron-opaque granules. Observe small unmyelinated fiber at lower right, showing longitndinal alignmriit of structure. Approx. 25,500 x .

R . S. GEIGER

FIG. 36. Sirmc. t y p prtpration as Figs. 34 antl 35, 1 hr after ;Iddition of pentylenetetrozolr. Note increase in size antl number of ovoid bodies, lysosomrs, also enlarged vesicles and change in niiclear membranc. Approx. 25,500 x .

MAMMALIAN BRAIN CELLS IN CULTURE

37

from subcultures of the precentral area of the cortex. Electron microscopy showed l hr after the addition of pentylenetetrazole that the nucleoplasm, nucleolus, mitochondria, and liponucleoprotein aggregates of the neuron had undergone considerable change (Figs. 34, 35 and 36). Although mitochondria in the living neuron (when stained with Janus green B and viewed with phase contrast) showed no appreciable changes other than an increase in size, they could be seen to be considerably changed when viewed with the higher resolution of the electron microscope, and appeared similar to the pancreatic cells in starved animals described by Palade (1959). Other ovoid bodies (possibly lysosomes) 0.5-1.1 p in length with a few cristae and many fine granules, were increased in number. Increased numbers of unusually large and dense osmophilic bodies, liposomes, were present. The Golgi apparatus increased in size and changes in the nuclear membrane were observed. Such changes are similar to those seen in overstimulated secretory cells. Considering the increased metabolic requirements of stimulated neurons, these changes can be attributed to the increased metabolism. An increase of the metabolic rate of brain and brain cells during convulsion has often been demonstrated. All these changes are reversible in living cells. XI. Effects of Serotonin

Serotonin, in concentrations of 0.5-2 pg/ml, affected most markedly the cytoplasmic movements in the neurons (Geiger, 195&, 1960; 196213; Geiger and Stone, 1961). The granules in the perikaryon of neurons at rest had slow rhythmic movements, indicating slight pulsatile movements of the cytoplasm. As previously stated, a time-lapse movie taken at half the usual speed showed some pumping movement in the resting neurons, in cycles of 6-8 hr. However, when serotonin was added, the rhythmic expansion and contraction of the whole perikaryon was greatly accelerated, imparting a massive rhythmic pumping movement to the neurons. The time required for one cycle of contraction and expansion now varied between 45 min and 1%hr, as against 6-13 hr in the resting condition. During this slow cmtractile phase, cytoplasm was forced down into the axon (Fig. 23A-B). When this occurred, the proximal portion of the axon bulged and the axon, as a whole, became stiffer. This may

38

R. S. GEIGER

be part of the mechanism for the flow of axoplasm from the perikaryon, demonstrated by Weiss and Hiscoe ( 1948). In the perikaryon and in the dendrites, the liponucleoprotein granules (osmophilic and Nissl) became more aggregated. Fine undulating membranes appeared at the edges of the neuronal perikaryon or its processes. The synaptic areas showed increased motility, and small undulating membranes also appeared on the fine fibers visible in this area, The rhythmic pulsating movements of oligodendroglia were gradually arrested in the contractile state; they remained immobile for a period of up to 1 hr. Murray (1958) has described a similar arrest in the contractile state of oligodendrocytes in cultures of fetal human brain, using 5 pg of serotonin per milliliter. Microglial activity was not immediately affected, nor were the movements of protoplasmic astrocytes whose membranes may show enhanced undulation and pinocytosis (Geiger, 1962b). XII. Effects of Epinephrine

The addition of epinephrine in minimal concentrations (0.0010.005 pg/ml) to brain cell cultures made neurofibril formation visible in the living neurons. The first effects to be observed when epinephrine was added to cultures were changes in the optical density of various nucleolar and nuclear areas (Fig. MA-D). Visible neurofibrils appeared first at the nucleoli, from where they extended into the rest of the nucleoplasm and then, later, into the cytoplasm. Increased amounts of Janus green B stainable material, a dense osmophilic substance, and acid phosphatase appeared in the nuclear-nucleolar apparatus. Histochemical studies with the use of specific enzyme digestion and organic solvents, showed that neurofibril formation is accompanied by marked changes in the distribution of DNA and RNA, as well as of the basic proteins and proteolipids. The rate of transfer of material from the nucleus to the cytoplasm appeared to be increased. Epinephrine also increased the secretion of material by oligodendrocytes and enhanced their rate of pulsation, and increased the rate of twitching of astrocytes after a short time. The boutons at synaptic endings increased in size and moved more rapidly within a circumscribed area (Fig. 1OA-C). Glial cells associated with neurons showed enhanced motility. The rate of spider-like movements of oligodendroglial elements, along the axons toward the perikaryon, increased. These elements also moved along the axons by contractile movements toward the peri-

MAMMALIAN BRAIN CELLS IN CULTURE

39

karyon. Norepinephrine in the same concentration has much the same effect as epinephrine. Adrenochrome has some of the effects of epinephrine and norepinephrine on the neurons and glial cells. However, it has a more toxic effect than these substances, and even in a tenth of the concentration of epinephrine, causes chromatolysis within several hours (Geiger, 1958b, 1960, 1962b; Geiger et al., 1960; Geiger and Stone 1961). XIII. Effects of Hallucinogens (LSD-25)

Lysergic acid diethylamide (LSD-25), in concentrations of 0.0001-0.001 pg/ml, when added to subcultures has a visible effect on a number of structural units of the living neuron. The Nissl granules become smaller and more uniformly dispersed throughout the cytoplasm. They move away from the nuclear membrane. The staining of such neurons with thionine shows a very diEuse Nissl pattern. After a time, depending on the LSD-25 Concentration (1-4 hr), vacuolization occurs in the cytoplasm accompanied by complete dissolution of Nissl substance. However, the nucleus and nucleolus undergo changes similar to those which occur during increased production and extrusion of nucleoprotein into the cytoplasm in response to stimulation with pentylenetetrazole, epinephrine, etc. TWO possible explanations come to mind: either the neurons on addition of LSD-25 are using an excess of Nissl substance and the cell is unable to elaborate it in sufficient quantities, or some step in the elaboration of Nissl substance is interrupted. The neuron, as a whole, shows a slight contraction. Pulsation of oligodendrocytes is arrested in the expanded state (Fig. 24), in contrast to serotonin, which arrests the pulsation of the oligodendrocytes in the contracted state (Fig. 12) (Geiger, 195713,1960; Geiger and Stone, 1961). Another psychotomimetic agent which can induce hallucinations, N-methyl-3-piperidylbenzilate(Abood et al., 1959), acts like a stimulant on brain cells in culture. This observation corroborates the findings of Abood and Biel ( 1961) that the drug is basically a central nervous system (CNS) stimulant. XIV. Effects of Acetylcholine and Eserine

Acetylcholine, in concentrations of 0.5 mg/ml, and eserine sulfate in concentrations of 0.0001-0.001 pg/ml, have much the same effects on brain cells in culture. Feldberg and Sherwood (1954) have shown that the main effect of eserine sulfate in these concen-

40

R. S. GEIGER

trations, when injected into the ventricles of the brain in V ~ V O is , to increase the concentration of acetylcholine. The chief difference noted by us between the effects of acetylcholine (in the high concentration mentioned) and eserine, was that acetylcholine increases the permeability of neurons to vital stains more than does eserine. Time-lapse photography showed increased movements of mitochondria down the axon, and increases in the size and in the movement of the boutons at synaptic areas (Fig. 25 and Fig. 28). Synaptic endings (boutons) stained vitally with Janus green, in the presence of eserine, changed the color of this dye from green to red within a short time (Fig. 26); the red color indicated the reduction of lanus green (Showacre and DuBuy, 1955). This quick change in color of Janus green in the presence of larger amounts of acetylcholine indicates an increased rate of oxidations in the synaptic areas. Movements of oligodendrocytes around the perikaryon or along the axon were accelerated by eserine, as were the pulsating movements of oligodendrocytes. Nucleolar and nuclear changes previously described as indicative of an active state of nucleoprotein synthesis, also occurred in the neuron. The nuclei of the oligodendrocytes also increased in size and increased amounts of material were extruded by the oligodendrocytes (Geiger et at., 1960; Geiger and Stone, 1961). XV. Effects of Brain Extracts

Pentylenetetrazole, serotonin, epinephrine, LSD, and norepinephrine, when used over an extended period of time (24-48 hr) without washing and feeding of the culture, often produced chromatolysis in neurons. Tyrode’s extract of brain, added to the cultures, protected the neuron against these damaging effects. Exposing brain cultures to a high temperature (44°C)for about 6 hr resulted in degeneration of all the neurons within 5 days; however, addition of brain extract to the feeding solution after the heating, prevented this degeneration. Tyrode’s extract made from rabbit brain was equally effective on cultures from humans and monkeys (Geiger 1958a, 1960). XVI. Enzyme Activity of Brain Cell Cultures

In studies on respiration of newborn chick spinal-cord cultures Abood et al. (1952) found high Qo, values for neuroglia. In comparing long-term subcultures from adult rabbit brain, gray matter and white matter, Abood found the cytochrome oxidase to be about

MAMMALIAN BRAIN CELLS IN CULTURE

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the same in all types of cultures. Malic dehydrogenase, succinic dehydrogenase, and adenosine triphosphatase ( ATPase ) were somewhat lower in the cultures of subcortical white matter. The whitematter cultures were predominantly oligodendrocytes; the gray-matter cultures were either mixed, (neurons and glial elements) or almost pure neuron cultures (Geiger, 1957a). Pope ( 1958) d’iscusses the data available from all sources on enzyme activity of neurons and glial elements. Hyddn (1960) found that concentrations of cytochrome oxidase, and succinoxidase which were higher for glial satellites than spinal ganglion cells in controls showed a reversed ratio on stimulation. David et aE. (1962) applied various histochemical methods to cultures of kitten and chicken nervous tissue. They localized alkaline phosphatase only in vascular tissue and neuroglia, and acid phosphatase in structures identified as “compound lipid globules” of classical cytology. Esterases were primarily localized in the cytoplasmic network of neurons, including the strands of the distal axonal segments. Cytochrome oxidase and succinic dehydrogenase appeared more active in mitochondria of neurons, especially within the synaptic end-feet. Coenzyme-linked dehydrogenases were found in the chromophore paranuclear regions of the neurons, but they had reason to doubt the validity of this localization. Some other dehydrogenases ( such as a-glycerophosphate) could be demonstrated in neuroglia but not in neurons. Geiger and Stone ( 1962a, b ) (Geiger, 1962b), using Koelle’s technique (1955), localized acetylcholinesterase ( AChE) in all neuronal elements and BuChE in glial elements in the explants and migratory zones of whole-mount adult rabbit and cultures of human brain cortex maintained up to 6 months. AChE was present in variable amounts in the perikarya and fibers of neurons, and was found to be highly concentrated in axonal endings, especially presynaptic endings. Cultures from human subcortical white matter after 2 months in culture showed an absence of AChE but the presence of BuChE in glial elements. AChE and BuChE could be localized in the corresponding gray-matter cultures (Figs. 37, 38A, and 38B). XVII. Some Uses in Neuropathology

Brain cell cultures may also furnish a tool for studying cellular mechanisms under pathological conditions. Normal brain cell cultures may also be used as test objects for studying the influence of

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FIG. 37. Living culture from subtemporal area of gray matter of human brain maintained for 2 months. Vitally stained with methylene blue 1:200.000 for 30 min. Unfixed. Photograph is of body of explant and shows staining of neuronal cell bodies, fibers, and boutons terminaux. c30 x

.

body fluids, such as sera. Sera taken from rabbits with experimental allergic encephalitis cause demyelinization of axons in the brain cultures of newborn kittens ( Bornstein and Appel, 1960). We have shown that when sera from untreated patients with acute schizophrenia are used instead of normal human sera in the feeding solutions for adult human cerebral-cortex cultures and subcultures, a gradual change occurs in the appearance of the neurons.

XIAhlhlALI.4h’ BHAIh’ CELLS IX CULlURE

43

FIG. 38. A. Shows presence and distribution of acetylclioliriestcrnsc in body of explant of culturc similar to that sliown in Fig. 97. 5 4 0 x .

The effect of these sera was followed over a period of 10 days. During the first 48 hours, an increased aggregation of Nissl granules, an increase in pulsatile and pumping movements of neurons, and the appearance of fine undulating membranes on the perikaryon, dendrites, and axon were observed. The spines on the dendrites became more noticeable and showed rapid retraction and extension. All types of glia, after a short time, showed increased motility, which persisted through the 10-day period. The initially increased cytoplasmic activity of the neurons gradually subsided and after 72 hr returned to normal; a t this stage, the dendrites showed a tendency to flatten out and sometimes retract. The liponucleoprotein aggregates

R. S. GEIGER

FIG. 38. B. Body explant of culture of subcortical white matter from an area adjacent to that shown in Fig. 38A and maintained for the same period in the same manner. Treated simultaneously with 38A to demonstrate acetylcholinesterase. Procedure described in Geiger and Stone (1962~).Note absence of neuronnl elcments. 540X.

gradually became smaller, more diffuse, and less numerous. The neurons at this point showed a complete absence of particulates stainable with Nissl stains. Addition of normal brain extract to the media, along with schizophrenic sera, can prevent the long-term changes induced in neurons by such sera. Geiger (1958c, 1960, 1962b), Heath et al. (1959), Froman et al. (1960), and Pennell et al. ( 1961) have described various properties of schizophrenic sera. In a time-lapse motion picture presented by the author a t a

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Symposium on Serological Factors in Schizophrenia ( New Orleans, June, 1961), the effects of a protein fraction obtained from sera of schizophrenics (Taraxein, kindly sent by R. G. Heath) on human brain cell cultures was clemonstrated. In some respects these effects were similar to those observed with schizophrenic sera. However, tlie onset was more rapid and often caused chromatolysis in neurons within 1-3 days. Kept at 4"C, Taraxein became inactive within 5 days. A portion of the Taraxein which was kept in normal human serum retained its full activity throughout the period tested (10 days). Not only did the normal human serum stabilize Taraxein hut it also rendered it less toxic to the neurons and its effects more closely resembled those obtaincad with sera from schizophrenic patients. Brain tissue obtained from pathological soiirces has also been studied in cultures. Pomerat (1951), who used human brain obtained from lobotomy of schizophrenic patients, saw no abnormality in the brain cells. The author's own experience, limited to cultures of tissue obtained from the temporal lobe of 2 scliizophrenic patients, indicated no abnormality in tlie ciiltures of such material, which was studied with phase-contrast microscopy ancl time-lapse photography. Cultures made from tissue obtained from temporal lobectomies of 4 patients with psychomotor rpilepsy also heliaved like normal brain cultures. In one case of Gaucher's dismse (acute infantile form) (Cumings, 1960), brain tissue obtained a short time after death was cultured. Some of tlie neurons in the explant had already degenerated; however, viable neurons and glial elements migrated out. Initially, the outgrowth was similar to that seen normally in tlie vicinity of the explant. A few neurons containing inclusions emerged from the explant ant1 degeneratecl witliin a few days. Other netirons which appearcd normal at the beginning, degenerated within a few weeks after migrating some distance away from the explant. Some showed osniophilic and PAS-positive inclusions. The migrating oligodendrocytes tlifferetl optically from normals ancl were more osmophilic than those in normal tissue. Usually, inore oligodendroglial satellites were present around the neurons th,m in normal cultures. In some cases, tlieir number increased a s tlie nenrons began to degenerate. Material transferred from glial elements to neiirons, as seen with time-lapse micropIiotograpliy, was morc freqiwnt than in normal

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cultures. Some oligodendrocytes penetrated the neurons as they degenerated. Addition of Tyrode’s extract of fresh rabbit brain increased the survival time of migrating neurons two- to threefold. The addition of nuclei, prepared from rabbit-brain homogenate by differential centrifugation, to the cultures was even more effective in increasing the survival time ot migrating neurons. Mitochondria and microsomes added together were more effective than mitochondria alone but less effective than nuclei. This indicates that diseased cells, emerging from the explant, suffer from some deficiencies which prevent their siirvival for periods comparable to those of normal cells. This deficiency can be at least partly corrected by the addition of components of normal brain cells, even if these are taken from other species. The neurons which remain in the explant survive much longer than the ones that migrate out. Lumsden (1959) summarized the results obtained in tissue culture of numerous types of tumors of the nervous system, starting from the first cultivation of gliomas by Fischer ( 1925). He discussed the value of tissue culture in the diagnosis of tumors of the nervous system and also presented evidence supporting the purely neuroblastomatous nature of medulloblastomas. Both neuroblastomas and medulloblastomas yield characteristic outgrowths of nerve fibers within 24 hr. Kersting (1961) reported on more than 300 cases of various brain tumors that were cultured by him. He also included a thorough review of the literature. However, on the basis of the pattern of outgrowth he observed in medulloblastoma cultures over a period of several weeks, Kersting questioned Lumsden’s conclusion that they are of neuroblastomatous origin. He suggested that the early outgrowth of neuronal elements observed by Lumsden and by himself was due to contamination of the explanted tumor tissue by normal brain and that the outgrowth after 10 clays to 3 weeks resembles more the pattern of outgrowth obtained in cultures from fibrillary astrocytomas. In this author’s experience, in the initial explants of tumor tissue cultured in uitro, the first cells to show evidence of regeneration are usually the neoplastic cells which may eventually be overgrown by other cells (Doljanski and Hoffman: 1940). Therefore, the material and conclusions presented by Lumsden (1959) appears convincing. ’R. S. Hoffman was the previou\ name of R. S. Geiger.

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XVIII. Conclusions and Summary

Through the years it has been established by a number of workers that various kinds of central nervous system tissue, such as embryonic chick spinal ganglia, fetal human cortex, cerebellum of newborn kittens and puppies, as well as adult brain tissue of rabbits, monkeys, and humans, retain certain basic cellular properties in cultures. Under suitable conditions, it has been possible to subculture adult mammalian brain tissue, and from migration of the cells of the subcultured explant, to obtain more uniform subcultures consisting mainly of the migrated brain cells which were originally present. Such subcultures are suitable for the study of drug effects, cell metabolism, regeneration, and interrelationships among neurons, between neurons and glial cells, and among glial cells. Neurons retained their structural polarity, making it possible to identify perikarya, dendrites, axon hillock, axon, Nissl substance, neurofibrils, and acetylcholinesterase. Neurons formed associations among themselves with structures which morphologically resembled very closely synapses observed in brain tissue, including synaptic knobs and rings, and specialized postsynaptic areas in the cell. Such synapses may be axodendritic or axe-somatic. Occasionally, axoaxonal associations were observed, as well as dendrito-dendritic associations, or an axon from the same neuron branched and associated with its own dendrites or perikaryon. Presynaptic axon endings stained intensely with the vital stains Janus green B or methylene blue, as well as with stains for acetylcholinesterase. With electron microscopy, synaptic membranes, and vesicles could be seen. Synaptic areas remained localized on the same postsynaptic areas for months a t a time. There was a continuous movement of the boutons on the postsynaptic region. The fine branching fibers lengthened or contracted and the boutons increased or decreased in size, and became more or less dense. Certain drugs showed marked effects on these synapses. Epinephrine and norepinephrine, metrazol, eserine, and acetylcholine increased the rate of movements and size of the boutons. Of these substances, pentylenetetrazole had the most marked effects on the rate of movements and size of endings in axo-dendritic synapses. With eserine and acetylcholine, the number of mitochondria a t the endings also increased, and when they were vitally stained with

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Janus green B, after a short time they changed color from green to red ( the most reduced form of Janus green B ), indicating a nlarkedly increased rate of oxidative metabolism in this area, The normally long period of the pumping movement of neurons, which is 6-8 hr per cycle, could be increased to 1X-2 hr per cycle by the addition of serotonin to the medium. Under these conditions, a bulging of the proximal part of the axon, as well as an increased rigidity of the axon, could often be observed during the contractile phase. This phenomenon may well be an acceleration of the normal mechanism by which the cell body of the neuron supplies essential material to its processes. This is consistent with our knowledge of fiber regeneration, as well as with the demonstration by Weiss and Hiscoe (1948) and Van Bremen ct at!. (1958) on axoplasmic flow. During the expancling phase, more material is likely to enter the neuron from the surrounding milieu, either cellular or extracellular. Giant astrocytes were seen in cultures and subcultures of normal adult mammalian brain. These had morphological and staining properties which made it possible to distinguish them from neurons. They did not show the polarity of neurons and their giant processes could readily be distinguished from neuronal processes such as axons or dendrites. The free, unattached endings of astrocyte processes sometimes showed undulating membranes and pinocytosis. However, when astrocytes were attached to other cell structures, neurons, glial cells, or capillary cells, they did not show (with a resolution of time-lapse phase-contrast movie up to 6 0 0 )~undulating membranes at the point of association. Such endings appeared similar to the sucker feet described by Ramon y Cajal (1913, 1934). The only movements which the sucker feet showed at points of association with the capillary were mild contractions and expansions. Astrocytes at times exhibited twitching movements (types of expansion and contraction ) which are markedly accelerated by epinephrine and norepinephrine. Astrocytes, in their highly differentiated state, were relatively immobile, especially in subcortical white matter, where neurons are not present. The movements of oligodendrocytes are also greatly affected by the presence of neurons. Increased rate of production of Nissl substance, as seen in the presence of eserine, epinephrine, and %amino-tricyanopropene, and in injury to the neurons, usually resulted in more oligodenclrocytes becoming perineuronal satellites. The pulsating movements of such oligodendro-

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CUL’rURE

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cytes were markedly accelerated. The contractile phase of the pumping movements of tlie glial cells was accompanied by the extrusion of material. Conversely, during cellular narcosis with sodium barbital, the oligodendrocytes stopped contracting for a limited period of time, during which they markedly increased in size, presumably due to a retention of material which they would normally extrude during their contractile phase. During this stationary period, they stained much more intensively for nonspecific cholinesterase. Transfer of materials from the glial cells to the neurons was directly observed by time-lapse photography. Oligodendrocytes, astrocytes, and microglial cells, when associated with neurons, were observed to transfer material to them. The acceleration of this process, at times when the neurons appeared to be very actively secreting material, permitted observation-directly on living cells-of the role of glial cells as suppliers of metabolites for the neuron. Astrocytes and microglial cells were also seen to take tip particulates directly from the neuron. Glial cells reacted more readily to sodium citrate and water vapor than did neurons. This would appear to indicate some difference in the basic membrane properties of these cells, in the sense described by Heilbrunn ( 1943). Occasionally, mitosis was observed to occur in migrating neurons. This mitosis was very infrequent and insiifficient to produce subcultures in tlie usual sense. At infrequent intervals binucleated neurons also were seen. Instances of the rare mitoses of neurons observed in the central nervous system in oim, have sometimes been encountered in the literature. I n the cultures where mitoses were observed occasionally, the selective blood-brain barrier was absent, and the architectural design of cellular relationships in the original tissue was missing. It has not yet been determined what causes a neuron to undergo mitosis. Culture material may serve for such studies. Enzymes can be localized in these preparations ( acetylcholinesterase, cholinesterase, etc. ) . This presents the possibility of studying the effects of various conditions and drugs on the activity of enzymes of various types of brain cells. For example, adrenaline applied in high concentration, 0.2 g / m l for 25 min, considerably reduced the staining intensity of the perikarya of neurons for AChE, indicating that adrenaline may affect AChE. At the same time, dense

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staining for AChE appeared in some of the fibers and boutons. The different effects of adrenaline on the AChE of the soma and of the axon suggest the possibility of a different subcellular binding of the AChE in these regions. REFEWENCES Abood, L. G., and Abul-Haj, S. K. ( 1956). J. Nerrrochem. 1, 119. Abood, L. G . , and Biel, J. A. (1962). Intern. Reu. Neurohiol. 4, 218. Abood, L. G., Gerard, R. W., Bank, J., and Tschirgi, R. D. (1952). Am. J . Physiol. 168, 728. Aboocl, L. G., Ostfeld, A., and Biel, J. H. (1959). Arch. Intern. p h u m codynumie 122, 188. Bain, J. A. ( 1952). Federution Proc. 11, 653. Bairati, A. (1958). In “Biology of Neuroglia” ( W . F. Windle, ecl.), pp. 85-98. Charles C Thomas, Springfield, Illinois. Bakay, L. ( 1958). “The Blood Brain Barrier.” Charles C Thomas, Springfield, Illinois. Barr, M. L., and Bertram, E. G. (1951). J. Anut. 85, 171. Bornstein, M. B. (1958). Anut. Record 130, 275. Bornstein, M. B., and Appel, S. H. (1960). Time-lapse motion picture, shown at 11th Annual Meeting, Tissue Culture Assoc. Borysko, E. (1956). J . Biophys. Biochem. Cytol. 2, 1. Canti, R. G., Bland, J. 0. W., and Russell, D. S. (1937). Assoc. Reseurclz Nervous Mentul Diseuse Research Puhl. 15, 1. Chang, J. J., and Hild, W. (1959). J . Cellular Comp. Physiol. 53, 139. Costcro, I., and Pomerat, C. M. ( 1951). Am. J. Anat. 89, 405. Crain, S. M. (1956). J . Comp. Neurol. 104, 285. Cumings, J. N. ( 1960). In “Modern Scientific Aspects of Neurology” ( J . R:. Cumings, ecl. ), p. 330. Arnold, London. Cunningham, A. W. B., and Rylandtrr, B. J. (1961). J. Neurophysiol. 24, 141. Cunningham, A. W. B., Dougherty, M., and Rylander, B. ( 1980). Nuture 180, 477. David, G . B., de Ameicla, D. F., de Castro, O., and Brown, A. W. (1962). Acta Neurol. Scnnd. Srippl. I , 38, 43. De Robrrtis, E., and Gerschcnfelcl, H. M. (1961). Intern. Rev. Neurobiol. 3, 1. Doljanski, L., and Hoffman, R. S. ( 1940). Nature 145, 857. Eagle, H. (1955). Science 122, 501. Ehrman, R. I., and Gey, G. (1956). J. Nntl. Cuncer lnst. 16, 1375. Einarson, L. (1980). In “Modern Scientific Aspects of Neurology” (J. N. Cumin& ed. ), pp. 1-67. Arnold, London. Feldberg, W., and Sherwood, S. L. (1954). J . Physiol. (London) 125, 488. Fischer, A. ( 1925). J . Cuncer Reseurch 9, 71. Froman, C. E., Latham, L. K., Czajkowski, N. P., Beckett, B. G . S., and Gottlieb, J. S. (1960). Federation Proc. 19, 83. Geiger, A. ( 1939). Biochem. J . 33, 877. Geiger, R. S. (1954). Am. J. Physiol. 179, 3. (Time-lapse motion picture.) Geiger, R. S. ( 195611).A.M.A. Arch. Ncurol. Psychicit. 75, 442.

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THE ELECTRICAL ACTIVITY OF A PRIMARY SENSORY CORTEX: ANALYSIS OF EEG WAVES' By Walter J. Freeman Department of Physiology, University of California, Berkeley, California

I. Introduction . . . . . . . . . . . . . 11. The Functional Subdivision of the Brain . . . . . 111. The Spatial Structure of Neuronal Electrical Fields IV. Distribution of Prepyriform Electrical Activity . . . V. Isolation of the Prepyriform Signal . . . . . . VI. Comparison of Evoked and Spontaneous Potentials . . VII. Correlation of Electrical Activity with Behavior . . VIII. Input-Output Relationships for the Prepyriform Cortex . IX. Conclusions and Summary . . . . . . . . . References . . . . . . . . . . . . . I

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53 54 59 66 76 79

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. 113

111

I. Introduction

There is unique value in the type of cerebral electrical activity termed normal brain waves, spontaneous EEG, background activity, etc., as distinct from spikes, seizure discharges, spindles, evoked potentials, and afterbursts. It provides an index of the activity of masses of cells, an industrious murmur made by the brain in the course of its normal operation, which, if deciphered correctly, could provide the basis for a major advance in understanding normal brain function. There are so many obstacles to interpretation that its potentialities are still largely unrealized, The purpose of this review is to describe the electrical activity of one cortical area of the cat brain and its relation to behavior, as the focus for discussion of these ob'This work was supported largely by the Foundations' Fund for Research in Psychiatry (59-204) of which the author was a Fellow (1958-1960), and in addition by a grant from the National Institute of Neurological Diseases and Blindness (B2537), U. S. Public Health Service. 53

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stacles and some techniques for surmounting them. The primary olfactory ( prepyriform) cortex is appropriate for an elementary review, owing to the relative simplicity of its anatomical, electrical, and functional properties. The choice of this structure for intensive study was by no means accidental, Its field was selected from among several large fields detected in hypothalamic recordings (Ward, 1953; Freeman, 1957) on the basis of the high amplitude of its wave form. While conclusions as to its pattern of operation are tentative and cannot be extended carte blanche to other types of cortex, certain basic concepts and methods of analysis now well established by the work of many neurophysiologists are relevant to all structures in the brain. It is with the exemplification of these concepts that this report is chiefly concerned. II. The Functional Subdivision of the Brain For purposes of observation the neurophysiologist implicitly or explicitly divides the brain into parts, each having some as yet largely indeterminate distribution and function. Each part is in eommunication with others and, therefore, has sets of input and output, including elements of feedback. The set of parts comprising the whole brain forms a chain, both in series and parallel, extending from sensory receptors to somatic and autonomic effectors. The problem for an understanding of brain function is to determine what the parts are, and what role each plays in the elaboration of behavior, i.e., to isolate conceptually each functional subdivision, measure or manipulate its input, infer its output or lack thereof, and describe the operation performed by that part on its input. This is not so much a statement of hypothesis as an operational view, which is shared in one form or another by most workers in physiology, whether using stimulation, ablation, administration of drugs, recording of potentials, etc. The specific problem for the electrophysiologist ( as opposed to the “electropathologist” interested in patternrecognition of disease) is to determine whether his signals can be used as the basis for functional subdivisions, and then further used to determine the function of each part. The freedom in choosing the size of any elementary functional part, in terms of single cells, groups of cell types (Hydkn and Pigou, 1960; Galambos, 1961a), nuclei, areas of cortex, or entire regions of the brain (Jackson, 1884), is limited mostly by the techniques

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chosen for observation and manipulation; but, in any case, the elementary part is rightly conceived in terms of its anatomical structure (Sholl, 1956). EEG waves are multicellular in origin, so that one EEG part must consist of a homogeneous population of cells having some common electrical characteristics, in order that their signals may add to produce detectable waves. For the same reason, they must occupy some localized volume in the brain, probably in contiguity with one another, and they must receive afferent fibers from the same sources in order that there be a basis for synchrony. Histological similarity in a group of cells is suggestive of homogeneity but does not demand it, so that an anatomically homogeneous population might consist of several functional parts, and vice versa. The prepyriform cortex in mammals might reasonably be expected to comprise an elementary part divided into two subsections -the frontal and temporal regions. Anteriorly, the frontal region is bounded by the root of the olfactory bulb; medially, by the lateral margin of the olfactory tubercle; laterally, by the rhinal fissure; and, posteriorly, by the crossing of the middle cerebral artery (Papez, 1929; OLeary, 1937). The temporal portion comprises the anterior third of the pyriform lobe. Both sections receive afferent fibers from the lateral olfactory tract crossing the surface. This terminates almost entirely within the cortex and serves to define it as a functional unit. Histologically, both parts are quite similar and differ strikingly from surrounding structures. There is a single layer of densely packed pyramidal cells which vary greatly in the shape of the soma (Ram6n y Cajal. 1909-1911) but each cell shares the common characteristic of a profusely branched apical dendrite-extending into the molecular layer (Ram6n y Cajal, 1955; O’Leary, 1937) where it forms synapses with collaterals from the lateral olfactory tract. Only the minority of cells, principally those in the deeper part of the layer of cell bodies, have basal dendrites as well. The axons, which most of these cells send into the underlying white matter, terminate throughout the lower pole of the caudate nucleus (Crosby and Humphrey, 1939; Papez, 1929), the claustrum (Berlucchi, 1927), the adjacent neocortex (Allison, 1953), the hippocampus ( Cragg, 1962), and through the anterior commissure to the contralateral basal forebrain, where they are lost to further tracing, (Ram6n y Cajal, 1955). Further synaptic transmission may be widespread (Allen, 1943; Kaada, 1951; Berry et al., 1952; Gastaut et al., 1952;

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Pribram and Kruger, 1954). There is no commissural system. Cells with short or ascending axons are sparse, and there are relatively few recurrent collaterals. Centrifugal afferent fibers have not been described in the lateral olfactory tract. Electrophysiological data imply the existence of agerents to the cortex in the anterior commissure (Freeman, 1959) and from the midline nuclei of the thalamus, bilaterally ( Drooglever-Fortuyn et al., 195!3), but these have not been adequately described histologically. In the main, then, this system is composed of a single sheet of pyramidal cells. Its receptive surface area is largely restricted to the superficial dendrites, and its efferent fibers project from the buried side of the sheet (Fox et al., 1944) with relatively few recurrent collaterals. The problem of its electrical activity in the first stage was to determine whether the whole of the cortex generated a single characteristic signal, or whether the cortex should be conceptually broken up into a group of stable or continuously varying subsections. For the second stage, some means was required for isolating signals of this cortex or its parts from those generated by surrounding structures, e.g., the orbital cortex, the basolateral amygdaloid nucleus, the pterygoid muscles, etc. The third stage was to determine whether this signal could be used to specify cortical input and output. The urgency to realize input-output relations is paramount and has largely determined the kinds of physiological interpretations usually placed on EEG waves. Some of these views and the nature of the parts assumed will be briefly discussed. In the most widespread view, the assumption is made that the electrical activity recorded from electrodes in a nucleus or area of cortex is generated by that structure. It is common, for example, to see sets of records of spontaneous activity taken simultaneously from various areas of the brain along with the presumption that each of the structures is thus being “monitored.” When efforts are made to go beyond presentation of time series, increases in amplitude or frequency are often equated with increased activity of the part and, therefore, with increased input, and probably increased output as well. Decreases in amplitude are often ascribed to desynchronization implying that hidden increases in amplitude are present, which would be unmasked by synchronizing stimuli. This assumption

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stems from the Sherringtonian concept (Sherrington, 1940) of the central excitatory state and apparently even before that from the doctrine of nerve energies (Muller, 1826; Darwin, 1872; Walshe, 1942; Fulton, 1949). One should not underestimate the association in the minds of electrophysiologists between amplitude and degree of activity, even though the possibility has been discussed that high amplitude may represent excitation, inhibition, or functional block as a kind of “busy signal” ( Hebb, 1954). The second assumption is materially reinforced by use of pulsed sensory inputs such as clicks or flashes of light, which evoke potentials in the brain having magnitudes roughly proportional to the intensity of input. Particularly persuasive is evidence of assimilation by some parts of the brain of the frequency at which the pulsed input is presented (John and Killam, 1960), the distributions of which support Pavlovian ( 1927) concepts concerning stimulus generalization and secondary inhibition, Electrophysiologists are gratified when the amplitudes of such impulse responses increase when paired with a variety of unconditional stimuli and are puzzled when they do not ( HernPndez-Pe6n et al., 1956; Jouvet and Michel, 1959; Horn, 1960; Al’tman, 1960; Hearst et al., 1960; Marsh et al., 1961; Galambos, 1961b). In reality both should be equally puzzling in view of the potentiation or “release” of evoked sensory responses by pentobarbital (Kerr and Hagbarth, 195!5), the reduction in amplitude of the direct cortical response by arousal (Purpura, 1956), and other related evidence (Livingston, 1959; Horn, 1960; and others). For rapid data production, this view of EEG waves in unparalled; all that is required is stereotaxic placement of electrodes and straightforward application of behavioral and statistical analytic techniques. Its difficulties stem largely from the untested assumptions required and the uncritical placement of electrodes ( see Section V ) , although it is useful as an early screening procedure. This same urgency has lead to the remarkable development of what has come to be called “evoked potential technique,” which involves the application of exteroceptive or electrical pulses to the nervous system, on the basis that the input is under more careful control. The electrical response is now a major analytic tool applied to the localization of functional parts, the determination of excitability characteristics, and the identification of sequential components in mixed populations as in the neocortex. Several recent reviews have

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been devoted to this subject, so the manifold data need not be summarized here. It will suffice to point out that although similarities between evoked and spontaneous brain potentials are widely recognized (Jasper and Ajmone-Marsan, 1952; Grundfest, 1957; Bishop, 1958; Purpura, 1959; Tower and Schad6, 1960; Delafresnaye, 1961; John, 1961; Morrell, 1961)-such as their tendency to occur predominantly in volumes heavily packed with dendritic branches, their similarity in form and time course, their common susceptibility to anoxia and ischemia and to the action of many drugs, the tendency for temporal and spatial summation to occur, and the absence of a refractory period-no serious effort has been made to determine the conditions under which spontaneous and evoked potentials might closely resemble one another in terms of spatial distribution, wave form, and relation to behavior. Emphasis has been placed instead on the resemblance of evoked potentials to the postsynaptic potentials of the spinal motoneuron, to the propagated soma1 and dendritic potentials in those or other large neurons permitting intracellular recording, to the negative or positive afterpotentials, or to other properties of vertebrate and invertebrate electrogenic tissues ( Fessard, 1948; Eccles, 1951; Chang, 1952; Albe-Fessard and Buser, 1955; Clare and Bishop, 1955, 1956; Grundfest, 1957; Purpura, 1959). These analogies are of genuine interest and importance, but they have failed to resolve the question of what the relation is that evoked potentials bear to normal brain function. To a large extent this seeming misdirection of comparison has arisen from the need to conceive the evoked potential in terms of cortical output as well as input. The arrival of an afferent volley in a dendritic layer is easily comprehended as producing dendritic depolarization. How might this phenomenon modulate cortical neuronal discharge to produce an efferent volley? Some plausible mechanisms have been proposed, and many attempts have been made to correlate unit activity with evoked potentials or spontaneously occurring slow waves, with encouraging but largely inconclusive results. There is not space to evaluate these data here, other than to state that much of the present uncertainty stems from the choice of the motoneuron or of neuronal networks based on cortical histology (Lorente de N6,1933a; Pitts and McCulloch, 1947; Sholl, 1956; Bartley, 1959) as the basis for analytic models, rather than the intrinsic properties of spontaneous waves.

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For example, it has been suggested that EEG waves are the sum of hyperpolarizing and depolarizing postsynaptic potentials ( PSP) generated by the dendrites of cortical and other neurons, which regulate cortical neuronal firing by the same electrotonic mechanism as that of the PSP's of the motoneuron (Purpura, 1959). This formulation was premature, in part because electrochemical analysis of membrane conductance and equilibrium potentials was not available, and in part because an independent measure of cortical output equivalent in stature to the muscle twitch was not employed. Furthermore, surface-negative cortical evoked potentials were found to be accompanied by deep-positive potentials, so that is was not possible to tell whether any given evoked potential having that polarity was a surface excitatory PSP or a basal inhibitoy PSP. In other words the amplitude and polarity of potential recorded extracelluIarly from one side of a mass of active cells is not a reliable index of their state of activity v i s - h i s membrane potentials, other parts of the brain, or behavior (Bishop, 1958; Eccles, 1960). Other kinds of analysis have emphasized the oscillatory character of the EEG and have suggested analogies with a variety of physical and electronic control devices involving scanning, phase comparisons, resonance or filtering, etc. Whether attention is directed towards cellular analogies or more general mathematical properties in physical analogs of biological systems, the primary concern in these approaches has remained the functional import of recorded signals, and the initial stages of EEG analysis have remained relatively underdeveloped. There is less need now for analogs of function than for analysis of the waves themselves, leading to criteria for electrode placement and judgment of validity of recordings. Validity here means conformity to some general concepts about the nature of EEG waves, leading to some clear ideas of what recordings ought to look like, other than what they have looked like in the past. I l l . The Spatial Structure of Neuronal Electrical Fields

The approach used in this study was an extension to spontaneous potentials of the system developed for nerve action potentials by Lorente de N6 ( 1947a) and Tasaki and Tasaki ( 1950), and for muscle by Craib ( 1928), Sugi ( 1940), Buchthal et al. ( 1956), and others. Emphasis in this method has been placed on wave form as the manifestation of currents generated in the volume conductor of the brain

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and determined by spatial or structural configuration of active neurons, more than on wave form as an expression of membrane phenomena. Volume conductor theory has been extensively applied to neuronal potentials-to the spinal cord by Lloyd and McIntyre ( 1949), Fatt ( 1957), Wall ( 1958) , and Brookhart and Fadiga ( 1960); to brain stem nuclei by de N6 Lorente (194713); to laminar structures by Adrian ( 1936), Brookhart et al. ( 1951), Chang ( 1952), Bishop and Clare (1952), Freygang (19Fi8), Tomita et al. (1960), Green and Petsche (1961); and to the distribution of potentials at the surface of the head or cortex by Walter and Walter (1949), Brazier (1949), Lilly and Cherry (1954), Shaw and Roth (1955), Cobb and Sears ( 1960), Cooper and Mundy-Castle (1960), and by numerous others. The basic aim has been to determine the distribution of the elemental components of activity generated by populations, in which the contributions of single cells are usually indistinguishable. All neuronal potentials recorded extracellularly in the nervous system have certain properties in common. In as much as the brain constitutes a conducting volume the existence of a potential difference between any two points implies a three-dimensional field of potential, in many instances extending in all directions to the scalp. This is as true for one neuron as for any larger number, A potential difference also implies in brain fluids a field of current comprising the movement of ions. Because the movement of these ions is not ordinarily affected by magnetic fields leading to rotational forces, they move in the direction of the maximum gradient of the field potential. To the extent that brain impedance can be regarded as homogeneous, one can construct from a map of the scalar distribution of potential ( V ) at any instant in the brain a vector representation of the concomitant spatial pattern and density of the current. This can be derived (Kellogg, 1953; Webster, 1955) from the product of the gradient, i.e., the vector s u m of the “first” partial spatial derivatives of potential with respect to rectangular coordinates, times the scalar value of specific resistance of the medium. The “second” spatial derivative of potential may be treated either as a vector or as a scalar product. The former defines an additional set of vectors representing vortex motion. In electrical fields with negligible transient magnetic phenomena, this quantity, known as the curl, is essentially zero. The latter defines the rate of “expansion”

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or “contraction” of the field with distance and is known as the divergence. The existence of a positive divergence in a homogeneous volume conductor constitutes a source, i.e., a region in which net positive charge is being added to the brain volume. Conversely, a negative divergence represents a sink or region of charge disappearance. In a volume containing neither sources nor sinks, the divergence at all points is zero. In accordance with core conductor theory (Katz, 1939; Hodgkin and Rushton, 1946; Rall, 1959),sources and sinks represent the net movement of positive ions out of or into neurons, so that the divergence given by the spatial distribution of potential across the cell membrane defines the relative density of transmembrane current (Lorente de N6, 1947a). Because neurons cannot store significant amounts of charge, it follows that the volume integral or sum of the divergence for any whole neuron or population thereof is essentially equal to zero. This relationship, which is described succinctly by Laplace’s equation, is as fundamental in neurophysiology as in the theory of potential, so it is surprising that it is not more clearly stated in every textbook on neurophysiology Every charged particle emerging across the closed surface of the core conductor (equivalent to the membrane of an active cell) must be replaced by another of the same sign entering, or one of the opposite sign leaving, at some other part of the surface (whether by resistive or capacitative transfer), with the exception of negligibly small amounts involved in changes of transmembrane potential. All extracellular fields take the form of closed current loops, in which charge moves in one direction through the volume conductor, i.e., the extracellular space; and in the op-

.

This was found to be 10%of the brain volume by Rall and Patlak (1962) using the rate of C“ h u h diffusion outward through the brain from the ventricles in viuo. The discrepancy between electron micrographs showing 3 4 % fluid space between cells and chemical determinations using thiocyanate, chloride, inulin, etc., suggesting a considerably larger space, has in the recent past been ascribed to special properties in glial cells including a high intracellular chloride content and low membrane resistance (for a review of this subject, see Tschirgi, 1960). On the present basis of present evidence it seems doubtful that the low specific resistance of the brain in comparison to other tissues (Kinnen and Kubicek, 1962) could be accounted for entirely on that basis. It is probable that brain cells undergo changes involving an increase in the number of osmotically active particles intracellularly within a few minutes after circulatory arrest. Estimates of the transmembrane osmotic gradient in a number of tissues ( M d y and Leaf, 1959) have shown that brain and testes are unique in having an in-

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posite direction through a closed surface or core conductor, i.e., cytoplasm. The source constitutes a locus of increased positive charge density, whereas the sink constitutes a locus of diminished positive charge density. These loci or poles represent, respectively, the locations of maximum values for positive and negative potentials. Between these two poles there lies a surface, which is the geometric locus of points at which charge density is equal to that at infinity and, therefore, defines the zero isopotential surface. For a cylindrical core conductor depolarized at one end, the sink at the active end and the source at the passive end are so arranged that the centers of gravity for net positive and negative charge are separated, and the zero isopotential intersects the cylinder at the site of reversal of transmembrane current. For a diffusely spreading core conductor, which establishes a source enclosing a sink as may a system of dendrites around the active soma of a neuron, the centers of graviq for positive and negative charge may coincide in space. To the extent that enclosure occurs, the zero isopotential is displaced away from the membrane locus at which reversal of transmembrane current takes place. If the two centers do coincide, there ward directed osmotic force across the membrane, but this only occurs near the end of a determination requiring several minutes. This was interpreted as being due to postmortem changes. The volume and chloride content of apical dendrites has been shown to increase a few minutes after circulatory arrest (Van Harreveld and Schadk, 1960), concomitantly with an increased specific resistance of whole brain (Van Harreveld and Ochs, 1956) and with a decrease in inulin space (Rall and Patlak, 1962). However, considering that membrane specific resistances characteristically exceed internal and externaI specific resistances by 10' (Miiller, 1958), and that ions appear to move preferentially through narrow extracellular clefts in Schwann cells rather than across the cytoplasm (Frankenhaueser and Hodgkin, 1956; Villegas and Villegas, 1960), the difference between 3% and 10%appears insignificant. Thus, although a significant fraction of the extraneuronal return current path within one-cell diameter of a neuron might be transgliaI (Adey et al., 1962) the path for large scale current spread leading to formation of EEG waves is most probably extracellular. Moreover, in cats with chronically implanted electrodes, measurements of the amplitude of spontaneous prepyriform activity and of transcortical prepyriform impedance by a modification of the technique of Freygang and Landau (1955) have shown that variations in the impedance of the extracellular return path do not contribute to the recorded potentials, i.e., changes in the recorded current x resistance drop are due solely to changes in current, not in resistance ( see Section VIII ) .

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is (unlike the dipole) no field of potential beyond the anatomical extension of the neuron (Lorente de N6, 1947b). The “dipole” and the “crater” are archetypes for neuronal fields with a range of variants possible between them. The equality of sources and sinks implies equal charge motion and not equal amplitude of potential, the latter being proportional to charge density. In brain fields unequal amplitudes are the rule rather than the exception. This may be due in some populations to coincidence of centers of gravity of source and sink accompanied by disparity in membrane current density with displacement of the zero isopotential surface to the vicinity of the larger area. This appears to account for the disparity in magnitudes of the positive and negative poles of the fields of spinal motoneurons (Fatt, 1957; Brookhart and Fadiga, 1960). The packing density of contributory branching fibers in a volume may be important. Related to this is the fact that, in a layer of cortical cells forming a curved surface or gyrus, such as the hippocampus (Eidelberg, 1961) or optic lobe ( O’Leary and Bishop, 1943), charge tends to be boxed into the concavity and dispersed over the convexity, resulting characteristically in higher amplitudes in the concavity. A similar restrictive effect results from the presence of the skull (or mineral oil) forming a resistive barrier over superficial cortex, increasing charge density over the surface, resulting in disparity of amplitude but in the reverse direction, i.e., amplitudes in the surface pole exceed those recorded in the deep pole (Freeman, 1959). No field of evoked or spontaneous potential is adequately described until both its source and sink have been identified. This may be tested for dipoles by multiplying the volume enclosed within a representative positive isopotential surface by the peak absolute amplitude recorded in the source within that surface. This product should equal the product of the volume enclosed by the negative isopotential surface having a magnitude equal to the first surface times the peak absolute value of potential recorded concomitantly in the sink within the second surface. This test may be used to rule out errors of measurement owing to faulty placement of the reference electrode during monopolar recording-especially if the zero isopotential surface is not stationary (see Section IV)--or to partial inactivation or distortion of the field by a penetrating electrode. The basis for this test is that potential represents charge content per unit

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volume, and that total charge content in each pole should be equal but opposite (Freeman, 1959). The classical theory of potential was developed for homogeneous media, whereas the brain and its coverings are neither homogeneous nor isotropic, This does not invalidate but rather accentuates the need for precise mapping. The difficulty may arise that at every impedance discontinuity across which current flows there are changes in gradient manifested as fictitious sources and sinks. A similar problem is encountered in electrostatics, when a field traverses the boundary of two media having unequal dielectric constants. This problem can be handled mathematically by assuming the existence of a fictitious dipole layer at the boundary (Webster, 1955) equivalent to a spurious source and sink in conducting media. Such boundary phenomena have sometimes been mistaken for sites of electromotive force; unless impedance measurements are made by application of exogenous currents across such discontinuities, they cannot rationally be distinguished from true sources and sinks (Tomita et al., 1960; Green et al,, 1960). The converse is also true. It follows from these properties that some electrical event in the brain overlap many others, so that a recording from a single electrode in the brain with respect to some suitable reference point might, in a sense, be said to monitor much of the brain. Clearly the distance between an active cell and the recording site plays a major role in determining the extent of contribution of that focus to the total recorded activity, but several other conditions may be equal or more important. The exploring may lie on or near the zero isopotential surface of the focus and, therefore, record little or no activity, even if that focus be adjacent to the electrode. The defect is not remedied by bipolar recording, because both eIectrodes may reside on the same isopotential of the field. Neurons arranged in symetrical palisades, characteristic of cortical and other laminar structures, offer a geometric basis for the addition of sources and sinks and must be expected to show greater amplitudes of electrical activity than nuclear or particularly reticular structures. Because cortical fields are dipolar their currents spread into the concavity of the brain at least as easily as outward to the scalp. Bipolar recordings from nuclear structures do not reject these distant signals but merely attenuate those signals, the isopotentials of which parallel the axis of the bipolar electrode (Walter, 1959; Freeman, 1957). Neurons

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having long processes extending outside the immediate vicinity of their dendrites and somas tend to broadcast their activity over larger regions [the “open” fields of Lorente de N6 (1947b)l in comparison to those structures in which all processes of the active cell terminate in the vicinity of the soma (the “closed fields). The possible role played by inhomogeneities in brain impedance [the “impedance vector” of Schmitt (1959)l in determining current spread is not sufficiently known to support comment, although the localizing value of impedance measurements has been recognized (Robinson, 1961; Brown, 1957). Perhaps most important, a high degree of synchrony in activity of a laminar population may establish extensive fields of potential leading to false impressions of widespread synchronous activity, especially if the gains on different channels recording from different sites are adjusted to give equal amplitudes of recorded signal. On the whole, anatomical distance is a very treacherous criterion for assessing the contribution of any given population of cells to an electrical recording, and merely placing electrodes stereotaxically in or near known or suspected multicellular generators is not likely to solve the problem. The further problem arises that although the positions of cells and of afferent fibers in the brain is fixed, reproducible fields require that afferent activity be repeated in the same way and not cumulatively alter excitability, i.e., the steady state must prevail. This cannot be assumed or demonstrated for spontaneous waves-obviously, not really spontaneous (Burns, 1958; Walter, 1959)-and continual changes in the pattern of input must lead to seemingly random changes in the distribution of potential. For this reason existing distributions at any instant are very difficult to plot, and success has been limited largely to surface distribution of prominent spikes Roth Bt al., 1938), slow waves during sleep (Brazier, 1949), and alpha waves (Cohn, 1948; Cooper and Mundy-Castle, 19f30). Furthermore, distinct populations may be interwoven as in the layers of the neocortex (Bishop and Clare, 1952). In any case, their electrical signs must overlap to some extent. The wave forms of such distinct populations might be very similar in their frequency components and amplitudes. The probable existence of unpredictably varying afferent activity renders the separation of signals from such populations highly uncertain solely on the basis of records of spontaneous

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changes, irrespective of how many recording electrodes are used, and the larger the number, the greater the degree of tissue damage and thus of distortion of electrical fields. The key to analysis lies in the use of controlled electrical stimulation of afferent fibers evoking activity reproducible in a population under study. This step requires introduction of a new elemental concept of analysis that remains invariant with respect to spatiotemporal patterns of activation. This is the geometric locus of possible distributions of electrical fields of a given type, within which changes in amplitude distribution may be described. The locus by virtue of its stability in relation to anatomical structures appears preferable to all other possibilities, including unique distributions of potential, gross or microscopic anatomical criteria, the shape of wave forms and their resemblance to those found in single cells by intracellular recording, and pattern-association with behavioral variables. However, its experimental determination depends on demonstrating the homology between evoked and spontaneous potentials for a given population in terms of spatial distribution, wave form, and behavioral correlates. These will now be described for the olfactory cortex in the next 3 sections. IV. Distribution of Prepyriform Electrical Activity

The concept of a locus for prepyriform activity arose from the finding that wave forms of spontaneous activity recorded simultaneously from 6-8 monopolar electrodes spaced through the basal forebrain were often so similar in form that they could be superimposed, provided the gains on the different channels were appropriately adjusted (Freeman, 1957). Maximum amplitudes for this common signal were found in the vicinity of the prepyriform cortex. Wave forms having similar shape and apparently identical distribution could be evoked by stimulation electrically of the olfactory bulb or lateral olfactory tract. Similar wave forms having somewhat different amplitude distributions and phase relationships occurred upon single shock stimulation of more lateral parts of the cortex not covered by the tract, of the periamygdaloid cortex, claustrum, midline thalamic nuclei, and occasionally of the anterior commissure ( Freeman, 1959). From recordings on the surgically exposed surface of the cortex, these evoked potentials took the form of waves travelling concentrically away from the stimulus site and, thus, varying in

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direction of movement depending on the activating site. They formed with spontaneous activity a class of wave forms loosely identified with this cortex. Systematic development of these observations required the development of a map. All such maps of neural activity involve the reduction of measurements of potentia1 in the 4 dimensions of space and time to 3, expressed either as families of curves in 2 coordinates, one being the value of potential at a given instant and the other a length, or as a set of maps consisting of isopotentials drawn in 2 spatial dimensions. The choice of the variable to be deleted depends on the nature of the field under study and has to be carefully selected for its simplifying value. Maps of potential around single nerve or muscle fibers have been based on the assumption of axial symmetry, such that volume distributions of potential can be shown in a single plane passing through the axis of the fiber (Tasaki and Tasaki, 1950). In the case of peripheral nerve, in which the amplitude of the spike and its conduction velocity were sufficiently constant, 3 dimensions were reduced to 2 by superimposing the time and longitudinal distance coordinates ( Lorente de N6, 1947a). In cortical mapping either the normal axis of potential distribution is omitted, leading to sets of maps showing the distribution of isopotentials at successive moments in time (Lilly, 1954; Lilly and Cherry, 1954),or axial symmetry is assumed and the changes in potential with time are displayed along only the normal axis (Green and Petsche, 1961; Spencer and Brookhart, 1961). Potentials have been mapped in the spinal cord by assuming segmental symmetry such that isopotentials can be plotted in a single coronal plane at each of a series of moments in time ( Fatt, 1957). For the prepyriform field, the decision was made to eliminate the time variable by mapping the distribution of peak amplitudes of either positive or negative potential irrespective of time of occurrence. This was based on the need to determine the volume distribution of potential, and was justified by the temporal characteristics of the evoked potential. Waves of potential, recorded from fixed electrodes in the brain, result from continuous changes in the positions and in the magnitudes of electromotive forces generating those fields. Though it is doubtful that any existing field is truly either dc or standing, a useful abstraction is the comparison of the moving dc field with the stand-

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ing ac field. Assume, for the sake of simplicity, the existence of 2 symmetrically distributed dipole fields with poles of equal and opposite magnitude (Fig. 1).In the first the values of potential remain fixed in time, and the whole field moves along the line joining the 2 peaks of potential (the dipole axis) with constant velocity. The second field does not move, but the value of potential rises, falls, and reverses in time along a sinusoidal curve such that the isopotentials represent the loci of maximum amplitude. As seen in Fig. 1, the form of the first spatial derivative or gradient of potential along the dipole axis for the 2 fields is identical. In the dc field, its position in space changes, whereas in the ac field its value and polarity change. The first temporal derivative of the dc field is zero, whereas that of the ac field is a cosine function, having a 90O-phase shift from the potential as a function of time. Monopolar recordings of potential as a function of time in these 2 fields would display diphasic wave forms somewhat resembling one another as in Fig. 2. In the nervous system wave forms correspond precisely to neither of these two as shown, so that single monopolar recordings do not suffice to distinguish between these two types of field. Suppose, however, that a series of monopolar recordings were made from electrodes placed along the path of movement of the dc field, The result would be a curve in time recorded at each point equivalent in form to the distribution of potential in space along the axis of movement, the only difference at successive points being latency determined by the velocity. Bipolar electrodes placed with their axis along the line of movement would show the difference in potential between 2 adjacent points in the field and, if the distance between the 2 electrodes were made much less than the dimensions of the field (“bipolar” recording must always be specified with respect to the field), the recorded potential would approach the first spatial derivative of the distribution of potential in space. By virtue of constant velocity and amplitude, the bipolar recording would approach the first temporal derivative of the monopolar recording (Lorente de Nb, 1947a). Monopolar recordings at different points in the ac field would not show differing latencies but instead would show differing amplitudes of signal. The amplitude of bipolar recordings would change with position in the field in accordance with the gradient or first spatial derivative of potential, provided the electrodes were suffi-

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CRITERION:

MOVING D C

FIELD STRUCTURE:

-

D I R E C T I O N OF M O T I O N :

A M P L I T U D E VS. T I M E : (WAVEFORM)

STATIONARY A C

0

0

+

A M P L I T U D E VS. DISTANCE:

SLOPE O F WAVEFORM:

0

+ SLOPE VS. D I S T A N C E : (GRAD I E N T )

o+

+ FIG. 1. Comparison of electrical fields. Two basic types of field are encountered in the nervous system, the moving dc field being characteristic of nerve and muscle potentials and the stationary ac field of cortical dendritic potentials, although the distinction is not rigorous. The spatial and temporal derivatives are compared as the basis for Figs. 2 and 3.

E L E C T R I C A L RECORDINGS: 1.

2.

3.

M O V I N G D C , MONO POLAR^

v = f (11

MOVING

D

C , BIPOLAR: d v'= (V)

dx

STATIONARY A C , MONOPOLAR-

v = f (1)

STATIONARY A C , B I P O L A R : v' = ddx (v)

-

FIG.2. Monopolar and bipolar recordings are shown as they would occur from the fields shown in Fig. 1. The numhers 1, 2, and 3 refer to displacement of the recording electrodes successively from the left to the right of the fields shown in Fig. 1. Positions 1 and 3 are taken to he in the poles of the ac field. In each case, the exploratory electrode is to the left of the reference electrode in bipolar recordings.

ACTIVITY OF PRIMARY SENSORY CORTEX

71

ciently close together, and the axis were parallel to the gradient. In neither type of field would the temporal derivative appear as a function of electrode positioning (with the exception of comparisons between intracellular and extracellular recordings noted in Section VI ). Monopolar and bipolar recordings of the prepyriform evoked potential were easily interpreted in terms of these schematics, provided the electrodes were placed in accordance with the requirements given. Stimulation of the olfactory bulb resulted in a diphasic surface wave moving with a velocity of about 2 meters per sec from anterior to posterior along the cortex, preceded within 0.75 msec by a triphasic action potential. The amplitude increased with distance from the bulb at the anterior margin of the cortex as shown in Fig. 3, remained fairly constant across most of the cortex, and diminished at its posterior end. The wave form was always initially negative except outside the superficial anatomical boundaries of the cortex. Bipolar recordings with the axis of the electrodes parallel to the direction of movement showed peaks of potential coinciding with maximum rates of change as would be expected in a moving field. Monopolar recordings from an electrode moved by small increments from the surface to the depth of the cortex showed no definable latency. The amplitude decreased to zero at the junction of the molecular and pyramidal cell layers, and a mirror-image diphasic potential appeared immediately below this level. The amplitudes of bipolar recordings were zero, both at the surface and depth of the cortex, and maximal at the zero isopotential, as would be expected for a standing ac field. These findings implied that the field manifested by the evoked potential was a diphasic, dipolar ac field, which was standing along the normal but moving along the surface of the cortex, The basis for movement was found to be successive activation by propagated potentials in the lateral olfactory tract and not cell-to-cell transmission within the cortex or propagation in intrinsic horizontal cortical cells, as revealed most clearly by the absence of an intrinsic initial positive wave in the surface, and by the invariable precedence by 0.75 msec of a propagated potential in the tract. The afferent fibers for both surface positive and surface negative phases were localized in the molecular layer by recording and by use of lesions, and the existence of an intracellular limb of the current loop was inferred from the existence of high amplitude oscillations in potential resulting from

12

WALTER J. FREXMAN

passage of a hook-shaped microelectrode across the zero isopotential surface (Freeman, 1959). As in other similar structures, such as the hippocampus (Eidelberg, 19Sl), the surface and basal waves concomitantly recorded were seldom precise mirror images. Attempts were made using superficially applied drugs, asphyxia, lesions, and

4

1

10

13

I6

I9 m r

Spread of evoked potenlial parallel to surface.

0

0.4

0.6

0.8

1.e

2.0 r m

Spread of same potential nOrI'IId to surface.

FIG.3. A comparison is made between the monopolar (upper tracing of each pair) and bipolar (lower tracing of each pair) recordings of the prepyriform evoked potential along the surface of the cortex (upper set of tracings) and along the normal (lower set of tracings). These show that the evoked potential manifests an ac field that is stationary along the normal and moving along the surface. (From Freeman, 1959).

stimulation at varying depths in the cortex to selectively suppress either the surface or basal component but without success. Considering the lack of afferent fibers required to account for simultaneous activation of the base of the cortex independently of the surface, these discrepancies were best explained in terms of the properties of voIume conduction previously described, combined with the delay in activation of the homogeneous population. Dissociation of the initial surface-negative phase (positive at the depth) from the following surface-positive phase (negative at the depth) did not occur during suppression of the evoked potential by asphyxia, curare, or locally applied procaine. The surface applica-

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tion of 7-aminobutyric acid had no effect on either phase (cf. Purpura, 1959). The amplitudes of both these phases increased with increasing stimulus intensity and decreased with pentobarbital, although the amplitude of the positive phase was more strongly depressed than that of the negative phase by pentobarbital and increased less with raised stimulus intensity. The correlation of positive and negative peak amplitudes in ink tracings during a train of singleshock evoked potentials was near zero, provided measurements were made from a fixed baseline, as noted for neocortical activity by Tunturi ( 1959). This however, resulted from the fact that 2 kinds of spontaneous activity were present. The kind having the same frequency components as the evoked potential tended to increase or decrease the amplitudes of the 2 phases together (because sine waves of the same frequency but differing in phase and amplitude add vectorially to produce a sine wave having the same frequency). That kind having lower frequency components changed the level of the entire trace and, thus, increased the positive peaks at the expense of the negative and vice versa. Thus, although it was not possible at this stage to determine whether the mass phenomenon was based on oscillatory potentials in each of a set of identical cells or on hyperpolarizing and depolarizing potentials in parallel sets of cells, this uncertainty did not invalidate the conclusion that the dipolar diphasic oscillatory wave form was the elementary cortical response. These several characteristics of the evoked potential led to adoption of a view similar to those proposed two decades ago by Adrian ( 1936), Libet and Gerard (1939), Bishop and O’Leary (1942), and others of this cortex as a sheet of parallel elements generating a standing ac field, which appeared to move only by virtue of the physical impossibility of activating all of the elements at the same time. Because the major source of activation would appear normally to lie in the olfactory bulb (Allison, 1953), this was the logical site to place a stimulating electrode for the purpose of mapping the locus of spontaneous activity. Planes for mapping were chosen approximately at right angles to the direction of maximal rate of movement, so that plots of isopotentials based on maximal amplitudes irrespective of time of occurrence would involve minimal distortion. Such distortion took the form of overlap of isopotentials near the lateral convexity of the cortex, where the latency of evoked potential

74 WALTER J. FREEMAN

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75

was greatest. This map, shown in Fig. 4, reflected many of the properties of volume conduction discussed in Section 111. Comparison of the amplitudes and phase relationships of evoked and spontaneous potentials, recorded simultaneously on a dual-beam oscilloscope from pairs of points widely scattered through the locus, revealed a prominent component spontaneous activity having the same spatial distribution as the evoked potential. Not all cortical evoked potentials are “standing,” particularly some of those evoked in the neocortex by thalamic stimulation (e.g., Spencer and Brookhart, 196l), for which several reasons might be considered. Neocortical pyramidal cells are staggered in array rather than packed in a single layer as in the olfactory cortex, hippocampus, dentate fascia, etc., which could result in activation delay along the normal. Potentials evoked by electrical stimulation usually greatly exceed in magnitude those of spontaneous activity, and the greater “dendritic” depolarization, thus implied, might convert a normally standing depolarization, resembling the local response of axon to a decrementally propagated wave of depolarization ( Lorente de Nb, 1947b), resembling the axonal action potential, which would have no spontaneous homolog. Some types of spontaneous waves may spread upwards to the surface, perhaps based on multiple sequential synaptic or ephatic interactions between cortical cells, that might be simulated using evoked potential techniques as suggested by Bishop ( 195S), Bartley ( 1959), Burns ( 1958), and others. However, the standing ac field is so characteristic of several types of simple cortex (Bishop and O’Leary, 1942; OLeary and Bishop, 1943; Brookhart et a,?., 1951; Green et al., 1960; Boudreau and Freeman, 1962) that it might be sought as one of the elementary components of more complex neocortical evoked potentials. It must be emphasized that the model for this analysis is the stable locus of fields of a cell mass, which may or may not be reducible to a qualitatively similar event in some “average” cell. Attempts to analyze the elementary basis for extracellularly recorded potentials by intracellular recording of unit wave forms have encountered ~~

~

FIG. 4. The distribution of isopotentials in millivolts for the prepyriform evoked potential is shown in 4 coronal planes specified in millimeters anterior to the interaural plane. The positions, amplitudes, and volumes of the surface and deep poles may be accounted for by the properties of volume conduction. (From Freeman, 1959).

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WALTER J. FREEMAN

many difliculties. Observations thus far have been restricted to relatively large cells capable of surviving micropuncture, and the numbers of recordings either from small cells or from cells displaying the same type of oscillations recorded from cell masses are few. Even for concomitant intracellular and extracellular recording the comparison of wave forms is not simple (Svaetichin, 1951; Tomita, 1956; Rosenfalck, 1957; McAlister, 1958; Freygang and Frank, 1959). Intracellular recordings tend to emphasize events occurring at or near the soma, and although it can be argued that the functionally important dendritic changes are detectable there, electrical events far out in the dendrites may be so attenuated, delayed, and distorted on reaching the soma as to provide little basis for comparison with extracellular potentials (Kandel and Spencer, 1961). Most importantly, however, EEG waves in some sense represent the combined activity of many cells, and it is precisely this combination that is of major interest in the analysis of molar brain function. Rather than ask whether single cells can generate sinusoidal oscillatory waves [which some can (Arvanitaki, 1939; Monnier and Coppee, 1939; Miiller, 1953; Sjodin and Mullins, 1958; Huxley, 1959), although usually under abnormal conditions] one should inquire into how multineuronal events should best be described. It is to be expected that the characteristics of a multicellular locus will be accounted for on the basis of single cell recordings, but that step is secondary to description, V. Isolation of the Prepyriform Signal

From this description there follows a basic rule for recording multicellular electrical changes in the nervous system that are stationary along a dipole axis. For optimal recording from any given population one electrode should be placed in the source and the other in the sink of the collective field, the signal being led off to a difference amplifier (Tonnies, 1959). If adjacent fields strongly overlap the desired field, the recording electrodes must, in addition, be placed as closely as possible to the same isopotentials of the unwanted fields. This is the basic requirement for obtaining good EEG data from the nervous system, and it follows directly from the characteristics of neuronal activity and of the difference amplifier. The achievement of t h i s aim for the olfactory cortex was strongly dependent on the use of the evoked potential, first for mapping the

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locus of the desired signal and then for guiding electrode placement. Placement required: first, that a monitoring electrode be placed stereotaxically near the cortex; then, that a stimulating electrode be placed in the bulb or olfactory tract; finally, that bipolar electrodes, with tips 1.5 mm vertically separated, be aimed toward the cortex and advanced stereotaxically, until the evoked potential recorded from the foremost electrode passed the zero isopotential surface and displayed a mirror-image evoked potential to that recorded on the hindmost tip. This technique permitted multiple insertions in each animal with an accuracy of placement with respect to anatomical landmarks equal to, if not better than, that provided by histological verification. There are many structures in the brain for which this technique of placement would be appropriate. The majority of recordings reported in the literature, however, are from electrodes implanted stereotaxically or in accordance with bony landmarks followed by histological verification. The choice of direction (vertical, lateral, or anteroposterior) and extent of separation of bipolar tips is largely determined by the technical problems of electrode manufacture and placement, and consideration is seldom given to the distribution of isopotentials across the recording sites. The cost of this casual approach is relatively minor in terms of ( a ) lowered signal amplitude by common mode rejection when both electrodes happen to be in the same pole, ( b ) uncertainty regarding anatomical location during prolonged behavioral studies, and ( c ) the lack of cross-check and of optimal phase measurements from two or more pairs having similar orientations with respect to the same population. The major loss lies in the possibility of the mixing signals from two or more discrete populations. No amount of computation can unmix two unknown signals. Since the elemental electrical signals of the brain are still largely unknown, it is imperative that every effort be made to obtain the simplest possible signals by careful electrode placement. The requirement of this is a map (however rudimentary) showing the distribution of sources and sinks in the desired field, and the rates, directions, and extents of representative movements. A field mapped by the use of evoked potentials merely determines a locus of possible distributions of spontaneous waves, within which multiple recording sites are necessary to define existing distributions of potential. The choice of number and position of elec-

I SEC

FIG. 5. The spontaneous electrical activity of the prepyriform cortex is shown recorded (same cat) from 4 transcortical leads spaced about 3 mm apart with tips 1.5 mm apart straddling the dipole, and a fifth monopolar recording near the root of the olfactory bulb. This set of tracings reveals the low-frequency respiratory waves synchronous with respiration, the bursts of sinusoidal waves associated with the slow waves, and the high degree of similarity between recordings from different locations in the cortex, i.e., the coarseness of the surface grain of spontaneous activity. The upper set was taken 30 sec after the lower set and revealed a shift in the distribution of a lower harmonic. Such shifts in the spatial distribution of a component have not yet been correlated with concomitant behavioral changes. The dominant, behaviorally correlated change is in amplitude of activity for the whole cortex. (From Freeman, 1960a). 78

ACTIVITY OF PRIMARY SENSORY CORTEX

79

trodes depends on whether measurements are to be made of the surface distribution of amplitude in a field irrespective of time of occurrence, or on the spread of activity through the field. The implications of this distinction between what might be referred to as the “profile” and the “wave” involve the more basic question of whether the cortex is organized as a set of parallel channels (Li et al., 1956; Mountcastle, 1957; Hubel and Wiesel, 1959; Mountcastle and Powell, 1959; Hubel, 1959,1960),in which short axon neurons and recurrent collaterals serve largely to maintain or refine the spatial and temporal distribution of cellular activity-patterns resulting from convergent afferent discharges (Jung, 1953, 1959), or is constructed around the principle of cell-to-cell dissemination with mixing of afferent patterns (Lorente de N6, 1933b; Pitts and McCulloch, 1947; Brady, 1954; Burns, 1958; Cooper and Mundy-Castle, 1960). In the former case, waves would be regarded as the accidental result of inequality of afferent fiber lengths causing systematic delays in the establishment of patterns over areas of cortex up to 100 mmy or more. In the latter case, the spread of waves would be regarded as a reflection of the local synaptic or ephaptic spread of unit-born information through the cortex, and time relationships would be more important than relative peak or average amplitudes. Electrodes for recording the former would consist of a small number (to minimize cortical damage) of transcortical bipolar pairs placed at intervals within the locus defined by the evoked potential. Electrodes for the latter would consist of a large number of monopolar surface leads ( L a y and Cherry, 1954; Walter, 1959), the dimensions being determined by the grain as well as the locus of the surface electrical patterns. A single reference electrode would be best located at the base of the cortex, distant enough to average the phase relations at the surface (particularly if located in the concavity of a gyrus) but close enough to minimize the contribution of extraneous fields. Prepyriform activity has been recorded entirely with the first type of electrode array, owing to the extremely coarse grain of pattern disclosed by multiple bipolar electrodes (Fig 5 ) . VI. Comparison of Evoked and Spontaneous Potentials

The existence of techniques for isolation of the prepyriform signal made possible refined comparisons of the wave forms of evoked and spontaneous activity in an animal with implanted electrodes,

80 WALTER J. FREEMAN

(d

.3

ACTIVITY OF PRIMARY SENSORY CORTEX

81

free from distortions introduced by anesthetics, surgical trauma, the absence of normal behavioral conditions, and admixture of signals from non-prepyriform generators. Two types of spontaneous wave forms were observed (Freeman, 1960a): one consisting of a diphasic, negative-positive, quasi-square wave following each inspiration with an overall duration of about 100-300 msec; the other, a sinusoidal wave form having frequencies ranging mainly from 20-60 cycles per sec, maximum amplitudes occurring in bursts coincident with the surface negative-to-positive shift in the respiratory wave

(Fig. 5). Autocorrelation functions (Dawson, 1953; Siebert et al., 1959; Brazier, 1960; Mercer, 1960) were calculated ( Boudreau, 1962) on an IBM-704 from 3-sec segments of records, sampled 160 or 200 times per second, which were taken under controlled behavioral conditions. The program used was based on digital analysis developed by Tukey (1957). Functions from some cats (Fig. 6, C 28) at some times displayed sinusoidal oscillation at a single frequency having a variable exponential pattern of decay, in addition to a rapidly decaying exponential transient. Most cats (Fig. 6, C 23) displayed more complex oscillatory patterns suggesting the presence of 2 or 3 sinusoidal components. Power spectra were obtained by means of the Fourier transform of the autocorrelation function (Wiener, 1930; Blackman and Tukey, 1958; Jennison, 1961) ,which characteristically revealed 1, 2, or 3 peaks of amplitude (Fig. 7 ) , thus confirming the impression from ink tracings of the presence Of a relatively small number of frequency bands. Both the modal frequencies and amplitudes of these peaks varied between cats and from one stage to the next in behavioral sequences for each cat, the variability of amplitude greatly exceeding that of frequency. However, comparison of spectra from the same behavioral stage of successive trials by any one cat revealed a high degree of stability. The bursts of sinusoidal waves strongly suggested the performance of a narrow band-pass filter subjected to random impulse bombardment (Rice, 1954), so it was expected that the response to single electrical impulses should be sinusoidal oscillation having an exponential decay. This was true only under certain conditions (Freeman, 1961a). The animals had to be awake and alert but behaviorally inactive; they had to be trained to respond to the electrical stimulus used as a conditioning signal, such that upon receiving

82

WALTEB J. FREEMAN

C23

queal“

0

20

40

60

80

100

Frequency in Cycles / Second

C 20

40

60

80

100

Frequency in Cycles / Second FIG. 7. Power spectra are shown for the autocorrelation functions in Fig. 6. Demonstration of the ‘<squeak”phenomenon in C28 would probably require “pre-whitening.” See Fig. 8 for a suggested explanation. (From Boudreau, lQ62).

the stimulus the animals would press a bar to receive milk; and the stimulus intensity had to be kept so near threshold that the average amplitude of evoked potential did not exceed that of spontaneous potentials concomitantly recorded. This implied the necessity for the computer to average 36-60 single-shock evoked potentials to obtain ,,A

-.,n-m,.vn,J

...

n..,

c,,

l 7 . , , -

:r.

An”, -n”+&n+,a

.4r,,..rnoCnr.*oo

-11

ACTIVITY OF PRIMARY SENSORY CORTEX

83

CosineRandomly Distributed SD=O.I

R

Differentio ted Exponential Q = 6.75

Heterodyne 24 : 34::3 : 2 Q = 3.73

SingleShock Evoked Potential C23 AB 3.7 V 0.01m. sec

FIG.8. Stages are shown in the reconstruction of an evoked potential averaged by a Mnemotron CAT computer. The upper trace represents the quadratic exponential decay of a mixture of undamped sinusoidal waves having a normal distribution around the mean with the standard deviation of f 10%of the mean. The second tracing represents an exponentially decaying trace that has been passed through a high-pass filter, resulting in phase shift of the entire transient without change in its value for Q. The third trace represents multiplication of the &st two traces, followed by addition of two such multiplied traces having amplitudes and frequencies in the proportions indicated. The fourth trace represents an averaged evoked potential obtained from the cat whose records are shown in Figs. 6 and 7. (From Freeman, 1962a).

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WALTER J. FREEMAN

the evoked potentials required complex graphic analysis for reconstruction in the same terms as spontaneous potential. Three steps were required (Fig. 8), corresponding at once to mathematical operations, stages in the development of analog devices, and assumptions about the cellular origin of the evoked potential. The basic pattern of oscillation revealed by this cortex was described (Freeman, 1961a) by the linear second-order differential equation representing the function of a harmonic system (Gille et UI?.,1959): m

dt2 + r d V + ICV

d2V

=

f(t)

The variables V and t represent respectively amplitude and time, and the constants m,r, and k, represent, respectively, equivalent inductance, resistance, and reciprocal capacitance. The solution to this equation for single impulse driving, f ( t ) = 6 ( t ) , as obtained with an analog computer, had the form of an exponentially decaying sinusoidal transient:

in which V , is a constant, 0 is the natural frequency in radians determined by ( k / m )lI2, c is the phase of onset determined by the initial conditions of the computer, and Q is the resonance factor equal to ( o m / r ) . The expression of decay in terms of Q instead of the damping factor, ( r / 2 m ) was based on the need to measure evoked potential either in terms of the ratio of amplitudes of successive peaks of negative or positive potential, or from the “peakedness” of frequency-response curves resulting from repetitive electrical stimulation of the tract and these measures were not strictly equivalent to the damping factor (Freeman, 1962a). The second stage of reconstruction required passing the analog transient through a parallel resistance-capacitance in series with a low resistance to ground, the output voltage being measured across the low resistance. This constituted a high-pass filter (McCleery, 1961) having the transfer function: w +B v = V(t)jjw +a

(3)

85

ACTIVITY OF PRIMARY SENSORY CORTEX

which replicated some of the passive electrical properties of nerve membrane (Freygang and Frank, 1959). The constant, p, represents the reciprocal of the time constant of the capacitance times its parallel resistance; the constant, a, represents the reciprocal of the time constant of the capacitance times the high and low resistances in parallel, and thus corresponds reciprocally to the time constant of neuronal membrane. When a non-decremental sinusoidal transient is passed through such a filter, V ( t ) = Yo sin ( a t ) , t> 0, there is superimposed on the oscillatory transient an initial exponential voltage change ( e - a t ) owing (McCleery, 1961) to the charging up to the capacitance:

v = vo

sin (wt)

+ cos (wt) -

e-"l

]

(4)

This complex form was not useful, inasmuch as neither the physical properties of the membrane nor the internal phase of onset, E , could be determined with extracellular recordings, so that in practice a simplified combination of Eqs. ( 2 ) and (4)was used: wt

v = Vo[sin (wt + +) - sin (+>e-af]e-s

(5)

This form provided a suitable approximation for the simplest averaged single-shock evoked potentials, in which the rate of exponential decay measured as Q gave values ranging roughly between 2.0 and 8.0. The value for the time constant, l/a, taken from the rising phase of initial negativity, was found to be strongly dependent both on recording site and on the choice of the point of origin in time for the evoked potential. Repeated measurements on 8 cats gave a mean value of 4.8 & 0.8 msec, which was higher (presumably owing to temporal dispersion of cortical activity) than values estimated for the membrane of the spinal motoneuron (Eccles, 1953; Rall, 1959; Eccles, 1961). The value for the phase of onset, +, was taken from the relative length of the initial negative half-cycle with respect to the average duration of all succeeding half-cycles (Freeman, 1 9 6 2 ~ ) In . cats attentive to the electrical stimulus and for stimulus intensities not exceeding 1.5 times, threshold values ranged roughly between from 45" and 80". This simplified treatment neglected the lack of data on the intra-

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WALTER J. FREEMAN

cellular phase of onset, possible distortions introduced by bipolar recording in the volume conductor, the broadening of the initial surface-negative peak introduced by conduction delay in the olfactory tract, and the fact that the initial peak actually consisted of two transients that were differentially sensitive to barbiturates and thalamic tetanization (Freeman, 1959). However, none of these analytic difficulties detracted from the usefulness of the finding that the form of the initial half-cycle could be treated as a boundary condition, and that it need not represent a neuronal event uniquely different from events manifested by succeeding peaks of the evoked potential. This was especially important for validation of maps based on amplitude measurements of the initial negative peak. The biological assumption represented by this step was that currents generated by each pyramidal cell had to cross the membrane twice, that both resistive (ionic) and reactive (capacitive) components were present, and that the relative magnitudes were reflected in the phase of onset. Thus, an increase in reactive current would be manifested by a shortening of the initial half-cycle with respect to subsequent cycles, and an increase in resistive current in a lengthening. The results of comparing intracellular and extracellular recordings from spinal motoneurons and other cells are still inconclusive, but it is clear that, as a first approximation, extracellular transients can be represented as attenuated and quasi-differentiated intracellular transients ( actually derivative plus proportional ) as would be predicted by passing a standing wave through the highpass filter, represented by Eq. ( 3 ) (Freygang and Frank, 1959). It is of interest to compare nerve axons and cell bodies in this regard. The peripheral nerve action potential is a moving dc field such that, at any point, the time derivative is zero and only the proportional component is present in the extracellular field. However, the field moves past a stationary extracellular recording-electrode at constant velocity, which, for a nerve having homogeneous properties along its length, means that the external action potential provides an estimate of charge density and, thus, of membrane current at each recording site as a function of time as well as distance (Lorente de N6, 1947a). Membrane current is equal to divergence, i.e., the scalar second spatial derivative of potential, which by virtue of constant conduction velocity along a linear core-conductor is also equal to the second temporal derivative of transmembrane potential.

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On the other hand, in a standing ac field recorded from a stationary electrode, the spatial distribution of current contributes to the amplitude of recording at any point on the surface of the cell, but not to the wave form. That is determined by the internal wave form and the distributed. electrical properties of the membrane. If those properties remain constant during the transient, i.e., if the membrane is “passive,” and if the distribution of active surface does not change, i.e., if it is truly standing, then an extracellularly recorded transient can be said to approximate the first rather than the second temporal derivative of intracellular potential ( Sat0 and Austin, 1962). This apparently holds for the prepyriform evoked potential, although confirmation is still lacking. It is not known whether this transient is produced by single cells in the cortex, parts of cells, or sets of two or more cells operating in fixed combination. Because oscillatory transients have been demonstrated in single axons (Sjodin and Mullins, 1958) and have been approximated by means of the Hodgkin and Huxley (1952) equations (Huxley, 1959),the simplest formulation was to ascribe to the dendrites of each prepyriform pyramidal cell the property of generating oscillatory potentials. This could be demonstrated with intracellular recording, if the property were inherent in the membrane, but not if it were dependent on reflected waves between the apical and basal dendrites, with depolarization in one tree inducing counter-depolarization in an opposing tree, and then reciprocally vice versa. Such a pattern would result in a nodal voltage point in or near the soma of each cell, such that intracellular recordings might not reflect the sinusoidal flow of currents through the soma. This could account for the low incidence of sinusoidal wave forms from intracellular recordings and for the lack of conductive or electrotonic delay along the dendritic axis. Solution of this problem will be required for measurement of the intracellular phase of onset, C. For simplicity in most of this work, was taken to be zero, corresponding to setting the initial conditions of the analog computer at zero current and zero voltage, and to the biological assumption that the resting membrane and equilibrium potentials for oscillation of the cortex remained constant and equal throughout the transient. Oscillation (equivalent to the existence of complex roots in the solution to a system of equations representing cortical function) can be expected for both the Hodgkin-Huxley system and the Eccles

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WALTER J. FREEMAN

model. In physiological terms, the time course of the cortical evoked potential clearly differs from that of the central excitatory state of the spinal motoneuron pool in having a prominent overshoot. This may be the result of electromotive forces (emf) in cortical dendrites resembling more closely those of the axonal action potential than those of the monotonic PSP; or this may be the manifestation of patterns of afferent activity and of intercellular connection in the cortex qualitatively different from those in the cord, the dendritic emf being basically similar. The PSP model would require activation, during the initial surface-negative phase of a single-shock evoked potential, of inhibitory recurrent collaterals leading in turn to the surface-positive overshoot. A continual input of background discharge in the olfactory tract would probably be required to account for the second negative peak and all subsequent cycles. It is important to realize that the existence of distributed delay in the feedback path, which would be expected in a population of cortical neurons having varying lengths of recurrent collateral axons, would act as a low-pass filter with a sharp cut-off, so that only the fundamental frequency of successive excitatory and inhibitory PSPs would emerge. On impulse driving, this system would display the intracellular wave form of an almost pure, damped sinusoid, having a frequency determined by the optimal feedback delay and a decay rate fixed by feedback gain. The currents for this system would pass through the cell membranes, with phase lead and attenuation of the extracellularly recorded signal in proportion inversely to frequency. Both systems require postulation of two or more parallel paths either in the forward or feedback loops or both (see below). However, experimental analysis of the PSP model requires opening of the loop or measurement of loop gain, neither of which has yet been convincingly done. In the absence of such data the least equivocal representation is based on an open loop transfer function, which explicitly does not show preference for a model based on the Hodgkin-Huxley formulation but defers the question for future study. The third stage of reconstruction required introduction of 2 types of distribution of sinusoidal frequencies for reconstruction of the majority of evoked potentials. The first type consisted of adding 1, 2, or 3 modal frequencies as suggested from the study of spontaneous activity. Cortical frequency-response curves were constructed

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89

by repetitive electrical stimulation of the tract using short bursts of stimuli at each of 18 stimulus rates. The mean absolute amplitudes of each of the trains of evoked potentials were determined by rectification and averaging, and these were plotted against stimulus rate in the form of a resonance curve (Freeman, 1961a). These curves showed multiple peaks of amplitude at stimulus rates in the range of those seen for spontaneous activity, although usually not corresponding precisely to modal spontaneous frequencies. The reconstruction of complex evoked potentials was undertaken by such repetitive electrical stimulation to obtain estimates of component modal frequencies in the single-shock evoked potentials (Fig. 9). Sinusoidal transients having these frequencies and relative amplitudes were generated by the analog computer, added, and adjusted until an optimal fit was observed between calculated and recorded wave forms. The second type of distribution, introduced by assuming a normal distribution, was deduced from the finding that the peakedness of frequency-response curves was lower when long trains of pulses were used, as compared with short trains, the reverse of the result to be expected from simple, nondistributed harmonic systems (Fig. 9). For a population of non-decremental oscillators activated by an impulse there would result sinusoidal oscillation at the mean frequency having an S-shaped decay (Fig. 8 ) in time of recordable amplitude, because the population would be in phase at the start of the transient and would go progressively out of phase with each cycle of oscillation (Freeman, 1962a). Thus, the envelope of decay for the single-shock evoked potential was taken as the product of the cumulative normal distribution curve (Campbell and Foster, 1942) and the curve for exponential decay, resulting in a quadratic exponent:

where u was the standard deviation expressed as a per cent of the mean. Correspondingly, the frequency-response curve ( Fig. 9) was shown to be the convolution of the Fourier transform of these wave forms, i.e., of the normal distribution and resonance curves (Freeman, 1962a). Combination with the discrete distribution of modal frequencies provided an equation suitable for describing all evoked potentials :

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WALTER J. FREEMAN

V=

V,,[sin(w,t

+ 4) - sin(~$)e-~]exp(-2a2t2 - wnt/2&)

(6)

n-=1,2,3

In practice, using graphic analysis, it was practicable to assume up to 3 sets of values for amplitude, V,, frequency, o,, and distribution, on, and single values for the remaining 3 variables of phase of onset, reciprocal time constant, a,and resonance factor, Q.

+,

Computer Analog

"4

of Evoked Potential HFistributed,

with

spontoneouf' activity. 0-0 Impulse input; transient state.

& /!I b \

steady state.

.-C a

10

20

30

40

Stimulus Frequency in Cycles / Second

50

FIG.9. Frequency-response curves were obtained from all cats using repetitive electrical stimulation over the frequency range indicated on the abscissa. Reconstruction of these curves was carried out by means of an analog computer simulating Eq. ( 1).A sinusoidal driving force applied to the computer set with parameters derived from the evoked potential in Fig. 8 gave a sharply peaked curve. Application of the same stimulation and recording conditions to the computer as were used for the cat, followed by addition of externally generated "spontaneous" activity from a sine wave generator, resulted in the dashed curve shown above. Convolution of this computed curve with the two normal distribution curves used for the reconstruction in Fig. 8 resulted in a computed curve closely resembling the frequency-response curve derived experimentally from this cat under the specified stimulus conditions. This final curve is, therefore, thought to represent the Fourier transform of the averaged evoked potential in Fig. 8. ( From Freeman, 1962a) .

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The assumption of a normal distribution of elements has also been made for peripheral nerve (Monnier and Coppee, 1939), but there was the difficulty here that the extent of distribution seemed to be variable and, therefore, could not be ascribed simply to variation in cell size as in nerve. The need for averaging also introduced the uncertainty of whether the variation about modal frequency was concomitant or sequential. Tracings of single-shock evoked potentials, superimposed on photographic film, indicated that sequential frequency shifts were present, and this was substantiated by the decay in amplitude with time of autocorrelation functions from spontaneous activity. Presuming that the prepyriform cortex is a population of sinusoidal oscillators in a random distribution about one or more frequency modes, there may be fluctuations in the modes and extents of distribution, or in the selection of elements active in the population at any moment to account for gross changes in wave form3 This problem could not be resolved by increasing the intensity of electrical stimulation, because the wave form of the evoked potential underwent progressive distortion involving reduction in modal frequency and especially in Q (Freeman, 1962d). By the time response amplitude had increased sufficiently to become stable in the presence of spontaneous activity in an ink tracing, the response bore only superficial resemblance to spontaneous wave forms, and displayed little of the individual variability with behavior of potentials evoked by near threshold electrical stimuli, There was no explanation for this change. Possible contributory factors were massive

’The instability of modal frequency has thus far prevented search for spectral “side lobes,” suggested by Wiener (1958) as indicative of coupling between elements in a distributed population. These might result in the cortex from electrical interaction among a set of cells having a distribution of frequencies, drawing those closest to the mean together to oscillate in unison at the mean frequency, allowing those farther away to oscillate at their natural frequencies. The resulting power spectrum would have the appearance of a high central peak and secondary peaks, one on each side. The existence of this pattern would probably be requisite for proof of ephaptic interaction or “mutual induction” among neurons, as postulated by Cragg and Temperley (1954), Cooper and Mundy-Castle (1960), Adey et al. ( 1960), and others, which as yet has only been clearly demonstrated in the invertebrate nervous system (Hagiwara and Morita, 1962). It should be noted, however, that this spectral form can ako result from inadequate mathematical or mechanical filtering ( Blackman and Tukey, 1958).

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antidromic activation of fibers in the tract causing some critical change in the bulb, direct electrical activation of dendrites or other cortical elements such as medial olfactory-tract fibers ( Cragg, 1962), or activation of small fibers possibly existing in the tract having diameters below the levels of resolution of light microscopy. Several features implied that the spontaneous waves were likewise standing oscillations in transcortical potential, activated by unit activity in the tract. These waves tended to occur in bursts following inspiration when olfactory unit-activity is maximal (Adrian, 1942). The signals recorded from several pairs of electrodes spaced at intervals of 3-4 mm along the anteroposterior extent of the cortex had virtually identical envelopes of amplitude of the basal or carrier frequency. The carrier showed a characteristic phase shift along the cortex with anterior signals leading posterior signals by phase angles compatable with a surface wave velocity of about 2 meters per sec, i.e., equivalent to that of the lateral olfactory tract. The amplitudes of respiratory waves, bursts of sinusoidal waves, and the evoked potential were higher in the anterior than in the posterior cortex, presumably due to the higher degree of synchrony near the anatomical origin of the tract. Finally, the frequency of oscillation in bursts was usualIy highest at the beginning of each burst and diminished to the carrier frequency after the first few cycles of the burst, a phenomenon known for the alpha rhythm and known as "squeak (van Storm Leeuwen et al., 1960). This shift, e.g., from 52S6 cps to 3842 cps, appeared in ink tracings to be continuous. However, computed power spectra (Boudreau, 1962) in most cats usually revealed a bimodal rather than continuous frequency distribution in this range, with on the average a low amplitude peak at the higher end and a high amplitude peak at the lower end (Fig. 7 ) . This suggested that the higher frequency component might be the spontaneous equivalent of the foreshortened initial negative half-cycle of the evoked potential, in which case (from the relative periods of the 2 components) the phase of onset of spontaneous activity could be estimated as about 50". No evidence was found for intracortical foci or pacemakers as the origin for spreading waves (cf. Garoutte and Aird, 1958; Walter, 1959; Cooper and Mundy-Castle, 1960, for comparison with the aIpha rhythm). It was conchded that spontaneous waves were regulated in their frequency and rate of decay by the

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intrinsic characteristics of cortical cells, and in phase and amplitude by the number, velocity, and degree of synchrony of unit activity in the tract. The formal identity of evoked and spontaneous potentials was now clear. Both could be viewed as the sum of a small number of sinusoidal components often occurring in approximate integral ratios. Each could be expressed as a frequency spectrum or as an oscillatory wave form having an exponential envelope. Alternatively, both could be described as the algebraic sum of exponential terms having both real and imaginary exponents (Cole, 1941). They did differ in appearance, however, and this could be ascribed to the differences in their sources of activation (random pulse trains vs. single impulses) and to differences in their frequency spectra (the constants in the equation). In any one behavioral state, each cat displayed characteristic spontaneous and evoked wave forms dependent on the component frequency-bands present. The bands for evoked wave forms varied with stimulus intensity and with the number of weeks of daily electrical stimulation, whereas the bands for spontaneous wave forms did not. The result was that initially the bands could be made to coincide by adjustment of stimulus intensity, but after several weeks of stimulation they could not. It was this plasticity of wave form that enjoined the use of frequency analysis as the basis for comparison of data from the same or different cats. Objection might be raised to the use of a linear equation to describe neuronal function, which in general is highly nonlinear. In fact, no physical system is truly linear, but many display limited ranges of function in which linear approximations are both valid and useful. Properties of excitation and electrotonus in peripheral nerve, for example, have been described for many years in terms of a linear equivalent circuit. Postsynaptic potentials can be approximated by 2 first-order rate processes involving cable theory, a linearized model. The basis for assuming linearity for such data, as well as for the prepyriform evoked potential, was the tendency for two evoked responses overlapping in time to add algebraically, implying that the first response did not detectably change the excitability of the system to the second stimulus (Freeman, 1960b). Thus, for a limited stimulus range near threshold, 2 successive responses to electrical stimuli of equal magnitude produced the same wave form as a single response added graphically to itself displaced in time, the interval of

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displacement being equal to the interval between the 2 successive stimuli. Increasing stimulus intensity to the cortex drove the evoked potential into the range of distortion, and this property then no longer held. Additional justification was derived from cortical frequency-response curves, which displayed basic homologies with single-shock evoked potentials implying that each was the Fourier transform of the other, as would be expected in a linear system (Jennison, 1961) . However, the most compelling circumstance for adoption of a linear, harmonic model as opposed to one based on relaxation oscillation, was the sinusoidal form of spontaneous activity. The close relationship between spontaneous and evoked potentials further indicated that the physiological function of this cortex also lay in a Iinear range, insofar as observations were based on EEG recordings. The limitations of a linear approximation are more taxing in dealing with responses from cats naive or habituated to the evoking stimulus (Freeman, 1960b; 1962a), Superposition fails, although it is the amplitude and to a lesser extent the phase of the second of 2, superposed responses that appear altered rather than the frequency or decay rate. The sum of 2 heavily damped sinusoids still suffices to represent the majority of averaged responses, and the dominant of the 2 lies in the range of frequency of spontaneous activity. The nondominant of the 2, which is %-% the frequency of the other, either has an initial surface-positive wave concomitant with the dominant negativity (nonminimum phase) ; or it is delayed 10-20 msec in onset after the beginning of the negative wave and does not have a rapid rise time. It is not clear whether the development during habituation of the nondominant component reflects elaboration of a distinctive cellular process in the cortex essential for the rejection of an irrelevant input, or is a mathematical artifact resulting from application of linear computation to an increasingly nonlinear response. In such cases, linear techniques suffice to describe and to focus questions but not yet to provide satisfactory answers. Another objection might be to the implied existence of inductance in the cortex. This implication is not valid. The inductive constant, m,in Eq. (1)implies only that in some element of this system changes in current tend to lag behind changes in voltage. It is well known that the apparent inductance of axonal membrane (Cole,

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1941) can be attributed to sodium and potassium conductance changes in response to changes in membrane voltage (Hodgkin and Huxley, 1952; Sjodin and Mullins, 1958), and that the assumption of energy release rather than energy storage is a more satisfactory way to account for the voltage-current characteristics of the membrane. Nevertheless, the property can still be treated as an inductance (Bromser, 1925), and an equivalent is required for reproduction of “action potentials” by analog devices (Miiller, 1958; Teorell, 1958; Crane, 1W).The Hodgkin-Huxley equations, furthermore, can be reduced to a simple linear equivalent provided external stimuli are well below threshold for the propagated action potential of axons, i.e., there is no clear boundary between harmonic and relaxation oscillation ( Huxley, 1959), although the full nonlinear equations are required for the greater range of function. Correspondingly, while linear at first, with increasing stimulus intensity the prepyriform response becomes progressively nonlinear, and at high stimulus intensities there is onset in all cats of repetitive relaxation oscillation constituting a petit mal-like electrical seizure. Considering the flexibility of the Hodgkin-Huxley equations or the Miiller (1958) equations all of these cortical wave forms probably could have been described in either system by selection of appropriate constants. The decision to use the simple electrical equivalent was based in part on the limited range of cortical electrical function being explored, and in part on the lack of data bearing on ionic movements and conductances in the cells of this cortex. The apparent inductance was the price paid for this simplification. Considering the complexity of the underlying system and the small number of parameters introduced, it can be argued that there is inadequate guarantee that any of these parameters has significance at the cellular level. This is true. However, this criticism reflects misapprehension of the aims of this work. The difEculty to be overcome here was that autocorrelations or averaged evoked potentials from the same site in the same cat were uncommonly exactly alike, and that superimposed on this seemingly endless variability was variation depending on individuality4 and behavioral state. An

’It is not clear whether such differences might have borne any relationship to individual behavior. The principle barrier to exploration was the lack of objective criteria for quantifying relevant personality differences among cats ( Pavlov, 1927; Gates, 1928). Additionally, the reduction in modal frequencies

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effective system of description had to provide a general statement applicable to all examples, sufficient accuracy to reproduce each one, and the bases for separating those variables peculiar to each cat irrespective of behavior from those common to all cats and related to behavior. The use of Eq. (6) basically implied no more than a transformation of parameters to achieve these aims, such that averaged tracings were plotted as distributions of amplitude against frequency rather than elapsed time. Exponents were sought rather than heights, latencies, and numbers of peaks. The effectiveness of Eq. (6) depended primarily on the degree to which the desired signal could be isolated and the appropriateness of the signal, not on the mechanism of the signal generator. In fields other than neurophysiology there appears to be little question about the superiority of presentation of this type of data in spectral rather than wave form ( Blackman and Tukey, 1958). VII. Correlation of Electrical Activity with Behavior

Isolation of the prepyriform signal and identification of its wave forms provided the bases for quantitative correlation of this signal with behavior, Some of these correIates were previousIy observed in rabbit ( Maclean et al., 1952),man (Lesse et al., 1955), dog (Domino and Ueki, 1960), and cat (Lesse, 1957, 1960), and have since been confirmed in the rat (Sutton, 1961). In order that no essential element be overlooked flow sheets were used for each of the 2 correlates. On the one hand, electrical changes were characterized: first, in terms of wave form; then, in terms of the surface distribution of each frequency component; and, finalIy, the amplitude of each distribution as a function of time. Behavioral variables (Spence, 1956) were subdivided into sensory input (including modality, quality, and intensity) , attention (orienting, habituation, conditioning, and extinction), motivation (hunger, thirst, rage, fear, sex, parturition, that occurred upon prolonged exposure of cats to electrical stimuli (Freeman, 1962b) suggested that (if frequency was in any way related to membrane capacitance) neurons might resemble muscle, kidney, and other cells in undergoing hypertrophy in response to metabolic demands. Thus, power spectra might reflect olfactory experience rather than specific odors or character traits. It seemed more likely that the latter information was present either in unit form in the cortex or in total cerebral patterns of activity, neither of which was accessible solely from the prepyriform EEG.

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play, evacuation, etc., including manipulation of intensity where possible in terms of deprivation, satiation, fatigue, habituation to fight-provoking stimuli, etc. ) ,and response onset or decision-making (conditional versus unconditional, locomotor versus oral, etc., with provision for quantitative measurement of probability, latency, magnitude or rate, and duration of single responses). Measured responses included maze-running, hurdle-crossing, bar-pressing, work in an ergometer for food, and walking or running on a treadmills5 These flow sheets qualitatively revealed a remarkably simple pattern. The wave form dominantly related to behavior was the sinusoidal component, and although minor changes in the frequency and surface distributions of this wave form occurred continuously (Fig. 5), the major variable was the mean absolute amplitude (closely approximated by rms amplitude). This was measured over epochs 3-10 sec in duration corresponding to epochs required for behavioral measurement. Because of the superimposability of recordings from different parts of the cortex, following adjustment of gain and phase, any single pair of electrodes su5ced to measure the activity of the whole cortex. Therefore, behavioral correlation was based on the rms amplitudes of signals recorded from that bipolar pair giving the highest amplitude in the range of frequencies roughly up to 60 cps and excluding the low frequency respiratory waves (Freeman, 1960a). Variations in modality and the quality of sensory stimuli were poorly correlated with changes in amplitude compared with variations in the degree of relevance of the applied stimulus to behavior, i.e., the extent to which the stimulus elicited attentive or purposive behavior. Likewise, the inferred type of motivation and, thus, the ‘The exact form of these flow sheets was less important than the degree to which they covered all aspects of overt behavior. Initially, the forms were vaguely conceived; as preliminary observations progressed, certain key correlates emerged that set these forms along the lines indicated. Obviously, estimates of all variables depended on observation of overt behavior, but the classification of variables depended primarily on their electrical correlates in the cortex. While it has been traditional for many behavioral psychologists (e.g., Skinner, 1938) to avoid any reference to so-called “intervening variables” ( Hull, 1943), this restriction appeared nonsensical in the face of data obtained directly from the central nervous system. Classification of behavior is the essence of inductive analysis of EEG. The best recent example is the re-instatement in psychology by neurophysiologists of the concept of attention ( Magoun, 1958).

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pattern of purposive behavior was negligibly correlated with amplitude changes whereas its magnitude was highly correlated. The type of response was less significant than its occurrence or nonoccurrence and its duration. In the most general terms, the following sequence of events took place irrespective of whether the response under study was eating, attack, flight, coitus, grooming, etc. The presentation conjointly of motivating and goal-object stimuli increased amplitude; performance was preceded or accompanied by decreased amplitude; achievement brought an increase and then reduction back to the resting level. Slight changes in frequency-always in the reverse directionaccompanied the changes in amplitude. There was no specificity of correlation in ink tracings between various odors and either frequency or spatial distribution; although autocorrelation will be required to explore this further, there was no question about the predominance of correlates in areas of behavior other than that of olfactory discrimination. Four major conditions were correlated with amplitude changes, which are summarized in Fig. 10. In this test situation (Freeman, 1962c), which also served to define operationally the mzjor behavioral variables, each animal was placed in the starting box of an ergometer for 1 min. Measurements of rms amplitude were made during 5 out of each 8 sec with 3 sec for “read out.” During the last 5 sec an auditory warning signal or an odor of food in a constant air stream was delivered to the starting box, following which the gate opened and the cat was free to emerge and proceed down the 2meter runway to consume food placed at the far end. If the cat did not emerge the door was shut after half a minute and a new run was begun. The base line for amplitude was best taken a few minutes to an hour after satiation with the food used for rewarding ergometer work, at a time when the animal was awake but torpid, and its electrical activity showed neither slow waves nor bursts of fast waves. Behavioral activity such as grooming, defecation, or exploration was accompanied by amplitudes above this base line. Reduction below this base line to a minimum was achieved by closure of the nostrils either ipsilaterally or bilaterally, irrespective of whether mouthbreathing, attempted inspiration, or efforts to free the nostrils occurred, with prompt increase in amplitude upon release.

99

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but not in a satiated cat. These same stimuli delivered to an animal with already increased amplitude produced either no change or a decrease. Finally, during work there was diminution in the variance of amplitude with deprivation, and during food consumption there was a major decrease in amplitude even though respiratory activity was demonstrated to continue during sniffing, lapping, licking, or chewing.

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These observations indicated that the 4 major behavioral variables related to prepyriform activity in this test mode were: olfactory input; degree of attentiveness to olfactory stimuli or other modalities of stimulation signifying food, as measured by probability of response to any given stimulus; degree of motivation, as measured by duration of deprivation; and the performance of work to obtain or consume food, as measured by the average force of pull in the ergometer. These changes were thought to result either from variation in magnitude and degree of synchrony of input via the lateral olfactory tract, or from changes in cortical excitability to the existing level of input. A controlled input was required to test this. Use of olfactory stimuli was ruled out by the lack of adequate control of the time of onset, intensity, and duration of odors deIivered to the nose, and by uncontrolled variables introduced by neural factors operating in the olfactory bulb and mucosa (Kerr and Hagbarth, 1955). Electrical impulses were, therefore, used to stimulate the lateral olfactory tract, with adjustment of stimulus parameters so that the wave form of evoked potential (revealed either by computer averaging or by repetitive stimulation and construction of frequency-response curves) closely resembIed the wave forms of spontaneous activity. By this means it was found that the changes in rms amplitude of evoked potential closely paralleled the changes in spontaneous activity in 3 of these 4 conditions, the exception being that closure of the nostrils had no significant effect on the evoked potential. Training the animals to respond to the electrical stimulus with pressing a bar to receive milk whenever the stimulus was delivered resulted in an increase in vaIue for Q of the evoked potential, manifested either as an increase in peakedness in the frequency-response curve or as an increase in the number of cycles of oscillation detectable after a single shock. Extinction followed by habituation (Freeman, 1962b) decreased the value of Q (Fig. 11).The rise in Q increased the rms amplitude of the singly or repetitively evoked potential and reproduced the circumstances that olfactory, visual, or auditory stimuli signifying food were capable of increasing the amplitude of spontaneous activity, whereas signals to which the animal had been habituated were not. With increasing deprivation, the magnitude of the evoked potential increased in parallel with the amplitude of spontaneous activity, but with no consistent change in wave form, specs-

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ACTIVITY OF PRIMARY SENSORY CORTEX

cally in the value for Q, thus reproducing part of the increase in amplitude of spontaneous activity, although to less than half the full extent. With the onset of work to obtain food or consume food there was a decrease in persistence of the evoked potential (reduction in Q ) irrespective of whether the animal was attentive, habituated, or naive to the evoking electrical stimulus. The average decrease in amplitude of the evoked potential was sufficient to account for more

A

Time after Extinction!

15 min

RV

60 min

W-75 rnin

30 min

W

140 m sec I

FIG. 11. The effect of prolonged stimulation without reward on the averaged evoked potential of a normally attentive cat (C28) is shown as a function of time of habituation after performance of the final response. While not representative of the majority of evoked potentials in form, this example best illustrates the principle upon which analysis has been based. (From Freeman, 1962b).

than half, but less than 80%, of the concomitant average decrease in amplitude of spontaneous activity, implying that changes in the magnitude and phasing of olfactory input (particularly during lapping with its alteration of respiratory rate and depth) must also play a role. Therefore, of the 4 major types of change in amplitude with behavior, three were attributable predominantly but not wholly to changes in cortical excitability rather than entirely to changes in the level of olfactory input. Alterations in evoked potential induced by exteroceptive stimuli have indicated that the effects of auditory and

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visual stimulation likewise result primarily from changes in excitability to olfactory stimuli, and not from addition of excitation to this cortex from those sensory systems (Freeman, 1962b, c ) . This is consistent with the effects of closure of the nostrils on spontaneous activity, and with the anatomical facts that the dominant set of afferent fibers to this cortex is olfactory in origin, and that according to Winkler ( 1918), Allison (1953), and Sholl (1956) following transection of the lateral olfactory tract there is extensive transneuronal degeneration of the pyramidal cells in this cortex. VIII. Input-Output Relationships for the Prepyriform Cortex

Apart from pattern-recognition in the diagnosis of disease, systems of EEG analysis can be seriously considered only if they provide bases for specification of neuronal input and output. All organ systems must be treated in these terms if knowledge of their physiology rather than structure is desired. This step was taken immediately from the data described in Sections VI and VII. The equation used to describe evoked and spontaneous potentials was used as a transfer function, i.e., a formal statement of operations performed by the cortex on its input to produce its output. The logic of this step may be seen in the fact that the equation had its own formal propertiesfithat could be used to analyze the effects on one variable of These could, as desired, be expressed in some physical model, of which the analog computer used in this study was one type, although such models have their own peculiarities that may obscure instead of clarify basic similarities (Dainty, 1960). The use of a descriptive equation as a transfer function (cf. Segre, 1962) for the purpose of factorial analysis was justified by the degree of order it introduced into correlative data and by the fact that theory was bound stringently to observation. The basis for this move was the extension of the time span of measurement of behavioral and electrical variables beyond unique or momentary events to epochs of seconds or minutes corresponding t o the time scale on which both cat and man must live. Most of the behavioral and electrical measurements correlated in this study were averages of rates (e.g., rate of stimulation, rate of work, rate of transcortical current manifested as potential), reflecting the behavior of the whole animal and of the whole cell assembly coextensively in time especially in response to impulse driving, i.e., 6( t ) (Dirac, 1958; Jennison, 1961; McCleery, 1961). When treated in this way, the function of the cortex became predictable or determinate, and the partial differential equations of classical physics could apply. Were the period of averaging shortened, as would be implied by the sole use of ink tracings of EEG, or were the spatial dimension restricted to the study of single cells, a biological

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alteration of another (Gille et al., 1959).Changes in parameters of the equation that reproduced changes in evoked potential served to categorize behavioral factors in terms of single parameters or combinations of these. For purposes of clarity, only the simplest formulation is presented, which suffices to synthesize the most important variables of rins amplitude, stimulus input, attention, motivation, decision, and response. For this approximation, Eq. (1) was expressed as follows:

d2V mdta

+ (y - z) dV - + kV dt

=

z6(t)

(7)

In this form m and k were constants representing fixed reactive properties inherent in the electrochemical generator of the cortex in a restricted dynamic range (Section VI). The symbol S ( t ) again represented an impulse driving force owing to single-shock electrical stimuli (Fig. 12). The variable z, modifying the input multiplicatively ( Hull, 1943), was found to vary directly with motivation, specifically food deprivation. This factor accounted for the tendency of the evoked potential to increase in amplitude, without change in form, upon increasing the duration of deprivation. The variable z indicated a negative resistance, the sign implying an energy source in the cortex by means of which transcortical potential oscillations following a single impulse were decrementally sustained. It was related to attention, specifically an increase in z accounting for the increase in amplitude of spontaneous activity and in the Q of potentials evoked by stimuli, when these stimuli were employed as conditional signals. The variable y was a positive or damping resistance implying energy expenditure by the cortex. An increase in y was related to the onset of food consumption or work for food by an equivalent of statistical mechanics would be required (Bohr, 1960). It is instructive to note in this context that the major developments in thermodynamics were only secondarily justified by statistical mechanics ( Schrodinger, 1960, p. 65).The choice between a set of first, second, or higher order diiferential equations (Teorell, 1958), Bessels functions, matrices (Sholl, 1956, Chapter VII), or nonlinear equations (Graham and McRuer, 1961) was less significant than that the equation chosen provided a simple reliable guide through the labyrinthine complexities of behavioral and EEG analysis. A linear, orthogonal, secondorder function of wide applicability in physical systems (Jonnard, 1959) was well-suited to this role.

104

WALTEX J. FREEMAN (motivotlonl

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v/p TO brain

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FIG.12. A schematic diagram of the function of this cortex is shown, which is based on Eq. (7). The cortex is presumed to have a primary input via the lateral olfactory tract and subsidiary inputs from other as yet uncertain parts of the brain. Two outputs are shown, V ( t ) being relevant to the observer, and V / Q being relevant to the animal.

animal and was manifested by reduction in the amplitude of spontaneous activity and in the value for Q of the evoked potential during lapping. Variation in the relative dominance of x or 'I/ accounted for the variable change in amplitude at the onset of ergometer work (Freeman, 1960a). In essence it is proposed that the prepyrifonn pyramidal cell be described mathematically as a band-pass filter (the dendritic membrane) cascaded into a high-pass filter (the axonal membrane) and then into a low extraneuronal resistance across which the output

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voltage is read (Fig. 12). The pass bands for cells in the cortex are thought to cluster around one or more means, with dispersal tending to occur as the result of prolonged exposure to electrical stimuli (Freeman, 1962b). Variability in means for natural frequencies can readily be seen to account for the distinctive wave forms characteristic of each cat and each recording site. This set of filters is subjected ordinarily to bombardment by quasi-random pulse trains, f ( t ) , but in addition can be driven by single volleys, 6 ( t ) . It has 3 variable parameters controlled presumably by other parts of the central nervous system. z serves to augment the Q of the band-pass filter to selected patterns of input, y to “load” the system presumably by altering the high-pass filter, and z to increase the input as well as both x and ‘y. Formulation of a statement regarding cortical output involved two problems. First, an alternate measure of cortical output was required. The only meaningful measure was the output of the whole cat, because unit or wave activity recorded from this cortex, the spinal cord, or any intervening stage could only be defined with reference to this same standard. In behavioral terms it was clear that olfactory stimuli could lead to work to obtain food, that the response was modulated by deprivation and also by visual, auditory, and gastrointestinal stimuli operating to determine the excitability of the whole animal to olfactory stimuli, and that each stage of the olfactory system could be regarded as contributing to the end result. It was, thus, predicted that quantitative measurements of prepyriform output should be correlated with quantitative measurements of response. The response measure chosen was “rate of work done to get food in an ergometer,” on the basis of preliminary results showing that the amplitude of spontaneous electrical activity was correlated with rate of work, but not with the latency and probability of response (Freeman, 19SOa). It was also postulated that the olfactory cortex would contribute to the response to a varying extent, owing both to the presence of serial intervening stages in the motor system of the brain stem and to the presence of other sensory systems operating in parallel on the same final common path. Thus, it was anticipated that the correlation between cortical output and rate of work would be low. The second problem was the selection of an appropriate electrical measure to represent cortical output. Several observations on

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spontaneous activity indicated that the value for Q would form a critical part of this measure. The amplitude of spontaneous activity tended to diminish with the onset of work to obtain food or with food consumption. So also did the Q of evoked potentials (Freeman, It increased with increasing deprivation during anticlSeOb, 1961~). ipation of both work and consumption but not significantly during them. This implied the operation during those activities of a factor tending to diminish spontaneous amplitude, this factor increasing with deprivation. The amount of reduction in Q of evoked potentials with the onset of food consumption was found to increase with increasing deprivation. The amplitude of spontaneous activity was found to increase in correlation with increasing rate of work at low levels of deprivation, the reverse relationship probably holding at high levels of deprivation, again suggesting the operation of a damping factor related to rate of work and also to deprivation (Freeman, 1960~).This posed the question: Would Q of the evoked potential show a negative correlation with rate of work? Measurements of Q were made during each of a series of runs in an ergometer by 3 cats, and it was found repeatedly that for each cat an inverse correlation existed between the value for Q measured during each run and the rate of work done during that run. The variability of measurement was high, so that irregular curves resulted from adding 3 sets of 30 runs each by each cat, A smooth curve (Fig. 13) was obtained only by pooling the data from the 3 cats. Although this step was provisional until faster means of data-handling could be developed to surmount the inherent variability, there was no specific contraindication. Use of this correlation to formulate a measure of cortical output required construction of a working hypothesis concerning the cellular mechanism of this cortex. It was postulated that the dendritic system of each pyramidal cell sustained an oscillatory emf that underwent damped sinusoidal oscillation when triggered by impulses in the lateral olfactory tract. The currents generated by this emf were postulated to form a closed loop passing in one direction across the dendritic membrane and in the opposite direction across the soma and initial segment membrane of the pyramidal cell axon. This current was thought to consist of resistive and capacitive components, the ratio of reactive to resistive being proportional to the phase of onset of the evoked potential. The magnitude of cortical

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output was presumed to be dependent on the number of propagated action potentials leaving the cortex per unit time, as opposed to the quality of output dependent on the spatiotemporal pattern of discharge. It was assumed that the probability of discharge was determined by the magnitude of resistive current flow across the initial segment of each cell. Finally, it was assumed that with the onset of work there was an increase in conductivity along some part of the

-20

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0

10

20

% Mean Rate of Work of Cat

FIG. 13, On the abscissa rate of work is plotted as % deviation of blocks of trials from the overall mean. On the abscissa (also shown as % deviation from the mean) are changes in Q of evoked potentials from attentive cats as a function of rate of work done concomitantly, average amplitude of spontaneous activity during the same runs, and the ratio of these two. For reasons given in the text, V/Q is taken to represent the output of this cortex (Freeman, 1962e).

current path, either in the dendritic membrane or in the membranes of surrounding glial cells-possibly involving the shift of a nodal voltage point from the soma into the dendritic shaft-resulting in an increased resistive, ionic current across the initial segment, and thus in the range of voltage fluctuation across that part of the cell, with alteration of its probability of discharge. This mechanism served to account for the amplitude changes in evoked and spontaneous activity with onset of work, and also for the low correlation between spontaneous amplitude and rate of work, because a decrease in Q was associated both with a decrease in electrical amplitude and an increase in rate of work. It could also be

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extended to account for the increase in frequency of the evoked potential, on the basis of shunting somewhere in the system of the membrane capacitance that in part determined the resonant frequency of the population. Several predictions have been tested to evaluate the utility of this scheme, a brief description of which may indicate the kinds of detailed observation that can be organized by Eqs. (6) and (7) into a coherent pattern. a. The resistive component of dipole current was thought to depend in part on a conductance increase measured as a reduction in Q and described as an increase in y, and in part on an increase in total current measured as an increase in over-all amplitude, V , and described as increase in both 2 and z. The measure chosen for cortical output was the ratio of the rms amplitude of spontaneous activity, V , to the mean value for &, both measured concomitantly during each of a series of ergometer runs (Fig. 13). Both the ratio, V / Q , and the average rate of work done during each run were expressed in terms of the per cent deviation of each estimate from the means of the total runs by each cat on each day. A positive correlation was found for each cat, and the combined data showed that the regression between the 2 variables had a slope of about 45", implying on the average a 1:l relationship between them. The relationship shown in Fig. 13 represents testing in circumstances in which the variability of input to the animal was minimized, so that the change in V with increasing rate of work was also minimal. Covariation in V and rate of work was easily induced either by varying the duration of deprivation, using different kinds of food for reward, or extending the number of runs in the ergometer to the point of exhaustion. In each of these cases there was on the average for each 3%increase in rate of work a 1%increase in V . b. Because of the postulated increase in ionic current during a behavioral response, it was predicted that the phase of onset of the evoked potential during a response should decrease. This was sought in 3 cats which were attentive to the stimulus, and which had each a single modal frequency in its evoked potential. The phase of onset was found to decrease during consumption of food obtained following a conditional response to the electrical stimulus (Freeman, 1962c), as compared with that measured during anticipation. It was also shown to decrease in these cats with the onset of effort unrelated

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to the electrical stimulus (Freeman, 1962b). Because both the tangent of the phase angle and the value for Q might roughly be said to be proportional to the ratio of the lumped equivalent reactance to resistance in this system, it was thought that the two might change proportionately. However, this was true only within a rough order of magnitude, i.e., on the average tan 9 decreased 40% from 2.72 to 2.03 and Q decreased 34% from 5.20 to 3.43,and the correlation between Q and tan $ from consecutive runs was low. Therefore, the use of lumped circuit parameters as an extension of the mathematical model was regarded as an unjustified oversimplification at present. The existence of a resistive load also led to the prediction that, whereas the natural frequency of the single-shock evoked potential increased by 3.5%, the peak frequency of frequency-response curves from these same cats should not increase as much, inasmuch as the resonant frequency of a repetitively driven damped oscillator is less than the frequency of free oscillations on single-impulse driving, and the difference increases with increased damping (Gille et al., 1959). In fact, the frequency-response curves showed no increase in peak frequency upon lapping, leading to the further prediction-verified by Boudreau (1962)-that the frequency of spontaneous activity during lapping as compared to waiting should increase by 3 4 % .This was by use of autocorrelation functions transformed to obtain power spectra from a different group of cats. c. Because the factor relating to decision was nonspecific whereas that relating to attention was selective, it was predicted that electrical stimuli would “pass through the cortex in proportion to their value for Q. This was tested in a group of 6 cats naive to the electrical stimulus and without orientation by stimulating both the left and right cortexes repetitively during alternate pairs of runs in an ergometer (Freeman, 1961~). The values for Q in these cats averaged 1.06 & 0.12. The rate of work during stimulated runs exceeded that recorded during nonstimulated runs by an average of 4.5%.Following orientation and habituation, values for Q fell to an average of 0.82 k 0.11. The stimulated runs then averaged 2.3%less than the nonstimulated runs. Three of these cats were subsequently trained to wait in the ergometer starting box for an electrical stimulus delivered only to the left cortex before beginning work. Their average value for Q rose to 1.28 rt 0.13. During alternate pairs of runs, the

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unilateral electrical stimulus used as the go-signal was continued throughout the run. Such runs exceeded in rate of work by 2.5% those runs in which the stimulus was terminated after initiation of the response. This potentiating effect of electrical stimulation, which served approximately to double the average recordable amplitude of prepyriform electrical activity, also supported the concept of a quantitative, causal relationship between sensory cortical function and molar behavior ( Herrick, 1933; Stanley-Jones, 1957 ) d. A cortical impedance decrease was predicted in association with food consumption. This was sought either by passing an alternating current of 100-1000 cps at amp between one bipolar pair of electrodes in the cortex and measuring the amplitude of this highfrequency signaI recorded from an adjacent bipolar pair, the spontaneous activity being removed by a band-pass filter; or by passing a current of amp across one bipolar pair of electrodes and measuring the combined tissue-electrode impedance with a Schering bridge. Measurements were made of the magnitude of the impedance vector to +-1%and of the phase angle to &0.1”. No behaviorally correlated change was found, although the technical difficulties of this measurement suggested that a negative result should not be taken seriously, Impedance changes similar to that predicted in their time of onset, direction, magnitude, and duration have been found by Adey et al. ( 1962) in the hypothalamus (cf. Brown, 1957). This negative result did serve to show, however, that changes in amplitude of spontaneous and evoked potentials resulted from fluctuations in the magnitude of current flow across the extracellular space of the cortex and not from concomitant fluctuations in transcortical impedance. This cellular model incorporated a number of functional features well established in other neuronal systems (Frank, 1959) and required only the new assumption of an unspecified conductance increase to account for electrical changes related to the decisionmaking process. A precedent should not be expected at the spinal level, where variable delays of seconds to minutes do not occur. It must be realized, however, that re-description of “loading” in terms of single cell activity essentially means translation of an electrical into an ionic equivalent, i.e., of the resonance factor into an ionic conductance in the same fashion that Hodgkin and Huxley (1952) translated Cole’s (1941) inductance term for squid axon tnembrane

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into the sodium inactivation and potassium activation factors. The basic limitation on interpretation of EEG data is that this step cannot be taken without supporting data from intracellular microelectrodes. On the other hand, the interpretation of single cell recordings is severely limited in correlations with molar behavior by problems of population sampling. The realm of effective use of EEG data lies not in analysis of the cellular mechanisms of the cortex, but rather in synthesis of the framework within which analyses can be carried on, in the fashion that Sherringtonian reflex physiology provided the essential molar concapts on which the analysis of the electrical properties of the spinal niotoneuron has been based (Eccles, 1953). The study of unit activity in the prepyriform cortex, for example, cannot be done in the context merely of evoked and spontaneous waves, but in addition to afferent stimulation must include control or measurement of factors related to motivation, attention, decision, and output. These are all essential factors in cortical function; they can no more be neglected than could proprioception in the analysis of spinal mechanisms. That described is one of several plausible schemes and serves only to show that, once formulated quantitatively, EEG data can be translated from electrical into cellular terms at any time with ease for the accomplishment of specific aims. However, so little being known about cortical mechanisms, it is suggested that permanent translation is not yet justified, and that if done prematurely by use of analogies with better known systems, some flexibility and scope of prediction are thereby sacrificed. IX. Conclusions and Summary

The basic unit for analysis of EEG should be the locus of multicellular electrical fields generated by a homogeneous population of cells having common electrical response characteristics, location, and afferent connections. Specification of a field requires knowledge of the position of its poles and zero isopotential surface, of the rates, directions, and extents of representative movements, and of the basic wave form on impulse driving within the physiological range. Analysis may be directed toward the spread of waves in that locus or towards the spatial distribution of amplitudes in that locus irrespective of time of occurrence. The choice will depend on the type of behavioral correlate being sought as much as on the theory of cortical

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function being considered. In either case, it is most important that electrodes be placed with respect to electrical fields rather than anatomical or stereotaxic landmarks, thus providing isolation of desired signals at their sources. Not all neuronal populations can be expected to generate spontaneous potentials detectable with gross electrodes, but those that do can be expected to pass currents through otherwise silent structures. The use of evoked potentials is essential for mapping loci and for guiding the placement of electrodes. It is also useful for measuring cortical excitability, provided only that the wave form is basically similar to that of spontaneous activity and that one or more neuronal relays do not intervene between stimulus and recording sites. The former condition implies the necessity for averaging of repetitively evoked potentials, because evoked activity will then differ from spontaneous activity neither in wave form, frequency, nor amplitude, but only in its known time of onset. The use of empirical equations is essential for an adequate degree of precision in description, for the achievement of general statements in the face of widely variant wave forms from a single population, and as the basis for formulating transfer functions, designating cortical input and output, which constitute the only meaningful aim of EEG analysis other than pattern-recognition. Some conclusions relevant to the prepyriform cortex (although parallels exist with other signals such as the alpha rhythm) are that the amplitude of spontaneous electrical activity reflects primarily the magnitude neither of the cortical primary input or output, but rather the degree of synchrony in that input and the level of cortical excitability determined by subsidiary inputs. Changes in amplitude reflect cortical operations rather than informational content, these being variable intensification (related to motivation) ,selective filtering (related to attention) , and variable delay (related to decisionmaking based on exteroceptive cues), The output depends on the conjunction of 4 types of input, can be estimated from the evoked potential but not from spontaneous activity, and is stochastically related to the output of the whole animal. The surface grain of activity is so coarse, that 2 pairs of electrodes (one for stimulation and the other for recording) s&ce to provide almost all the information on the function of the cortex that the EEG is able to convey relevant to behavior. Integration of the 4 types of input is not additive, as for

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the spinal motoneuron, but multiplicative, and as a result the terms excitation and inhibition do not have clear meaning for this cortex. A linear second-order equation can be used to describe cortical function in terms of input, output, and wave form of spontaneous and evoked potentials. The potential usefulness of this equation lies in the possibility that it can be tested as a special case in the HodgkinHuxley or Eccles systems, and that it can also be used in the context of cybernetics. Thus, it might eventually serve as a bridge for extending membrane theory to the study of molar behavior. REFERENCES

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Shaw, J. C., and Roth, M. (1955).Electroencephulog. and Clin. Neurophysiol. 7, 273. Sherrington, C. S. (1940). “Man on His Nature.” Cambridge UNv. Press, London and New York. Sholl, D. A. ( 1956). “The Organization of the Cerebral Cortex.” Methuen, London. Siebert, W. M., and Communications Biophysics Group. ( 1959). “Processing Neuroelectric Data,” Tech. Rept. 351. Research Lab. Electronics, Massachusetts Inst. Technol., Cambridge, Massechusetts. Sjodin, R. A,, and Mullins, L. J. (1958).J. Cen. Physiol. 42, 39. Skinner, B. F. ( 1938).“The Behavior of Organisms.” Appleton-Century-Crofts, New York. Spence, K. W. ( 1956).“Behavior Theory and Conditioning.” Yale Univ. Press, New Haven, Connecticut. Spencer, W. A,, and Brookhart, J. M. ( 1961).3. Neurophysiol, 24, 26, 50. Stanley-Jones, D. ( 1957).J. Neroous Mental Disease 125, 591. Sugi, Y. (1940).Japan. I. Med. Scl. 6, 293. Sutton, D. (1961).Ph. D. Thesis, Department of Psychology, University of California, Berkeley. Svaetichin, G. (1951).Acta. Physiol. Scand. 24, Suppl. 86, 23. Tasaki, J., and Tasaki, N. (1950).Biochim. et Biophys. Actu 5, 335. Teorell, T. (1958).Exptl. Cell Research Suppl. 5, 83. Tomita, T. (1956).Japan. J. Physiol. 6, 327. Tomita, T., Murakami, M., and Hashimoto, Y. (1960).J. Gen. Physbl. 43 (21,81. Tonnies, J. F. (1959).Electroencephalog. and Clin. Neurophysiol. 11, 608. Tower, D. B., and Schad6, J. P. (1960).“Structure and Function of the Cerebral Cortex.” Elsevier, Amsterdam. Tschirgi, R. D. (1960).In “Handbook of Physiology,” Sect. 1: Neurophysiology (J. Field, ed.), Vol. 3, Chapter 78. American Physiological Society, Washington, D. C. Tukey, J. W. (1957).J. Cycle Research 6,31. Tunturi, A. R. (1959).Am. J . Physiol. 196, 1175. van Harreveld, A.,and Ochs, S. (1956).Am. J . Physiol. 187, 180. van Harreveld, A,, and Schadb, J. P. (1960).In “Structure and Function of the Cerebral Cortex” (D. B. Tower and J. P. Schade, eds.), p. 239. Elsevier, Amsterdam. van Storm Leeuwen, W., Kamp, A., and Kniper, J. (1960).Electroencephulog. and Clin. Neurophysiol. 12, 244. Villegas, R., and Villegas, G. M. (1960).J. Gen. Physlol. 43, 73. Wall, P. D.(1958).J. Physiol. (London) 142, 1. Walshe, F. M. R. (1942).Brain 65,48. Walter, W. G. (1959).In “Handbook of Physiology” (J. Field, ed.), Vol. I, Sect. 1, Chapter XI. American Physiological Society, Washington, D. C. Walter, W. G., and Walter, V. J. (1949).Ann. Reu. Physiol. 11, 199. Ward, J. W.(1953).Am. J . Physiol. 172, 462.

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Webster, A. G. ( 1955). “Partial Differential Equations of Mathematical Physics” (S. J. Plimpton, ed. ), 2nd ed. Dover, New York. Wiener, N. (1930). Actu Math. 55, 117. Wiener, N. (1958). “Nonlinear Problems in Random Theory.” WiIey, New York. Winkler, C. (1918). In “Opera Omnia,” Vol. 5, p. 397. E. F. Bohn, Haarlem, Netherlands,

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MECHANISMS FOR THE TRANSFER OF INFORMATION ALONG THE VISUAL PATHWAYS By Koiti Motokawa Department of Physiology. Tohoku University School of Medicine. Sendai. Japon

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I Introduction I1. Retina .

A . Receptor

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. . . . . . . . . . . . . . . . . IV. Centrifugal Fibers within the Optic Nerve . . . . V . Lateral Geniculate Body . . . . . . . . . A. Anatomy of the Lateral Geniculate Body . . . . B. Electrical Activity of the Lateral Geniculate Body . C. Excitability Cycle of the Lateral Geniculate Neurons D . Binocular Interaction at the Geniculate Level . . B. Electrical Activity in the Secondary Neuronal Layer

C. Lateral Conduction 111 Impulse Conduction in the Optic Nerve

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VIII Corticipetal and Corticifugal Nonspecific Effects A Corticipetal Influence . . . . . . . B. Corticifugal Influence . . . . . . . IX Color Vision . . . . . . . . . . . X . Pattern Vision . . . . . . . . . . XI Summary . . . . . . . . . . . References . . . . . . . . . . .

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I Introduction

The fundamental problems of vision may be divided into 3 categories-reception. conduction and central processes. The present review is concerned mainly with the conduction along the visual pathway . Since the process of conduction can never be completely isolated from the other visual functions. receptive and central processes will also be considered. but only as they augment the under121

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standing of conduction. For convenience of presentation, separate sections will be devoted to the retina, the lateral geniculate body, and the visual cortex. Other topics, such as color vision and pattern vision, are not amenable to such anatomic classification so that separate sections will be devoted to such topics. In these sections, an attempt will be made to show the form in which the relevant visual information is transmitted between, and recordable from, various loci along the visual pathways. Subjects which are not directly related to the conduction, even though they are important for physiology of vision, are omitted. This review is also limited to properties of the vertebrate visual system. For topics omitted in the present review the reader is referred to recent reviews of other visual processes (Granger, 1959; Muller-Limmroth, 1959; Straub, 1961). An extensive review has been published by Crescitelli (1960). Brindley (1960) also has published a monograph which covers most fields in the physiology of the visual system. II. Retina

A. RECEPTOR POTENTIAL Undoubtedly, the first infiuence of light upon the visual system is a photochemical reaction in the receptor cells. Many of the details of photochemical reaction are now firmly established. Much less is known of the subsequent events which occur in the retina and lead to the impulses propagated from the ganglion cells. This ignorance applies especially to processes by which photochemical events are transformed into electrical events. By illuminating a solution of rhodopsin, a solution containing a suspension of rod outer segments or a retinal homogenate, Hara (1958) has shown that a change in electrical conductance occurs in parallel to the bleaching of the rhodopsin. The change is an increase in resistance, which is of the opposite direction to what would be expected from a temperature rise due to illumination by intense light ( cf. Tasaki, 1960). Although Hara considers that this phenomenon is linked directly to the initiation of nerve impulses, this conclusion may be somewhat remote. Nevertheless, the observation does seem to indicate at least the existence of some kind of electrical event within the receptor cells which accompanies the photoactiva-

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tion of rhodopsin. Concerning the possibility of a photically induced electrical response of the receptor cells, Brindley (1956) reported that in addition to spikes from the ganglion cell, spike potentials could be recorded from the outer end of the receptor layer in the frog retina. He suggested that the receptor cells might be the structures from which such spikes originated. In contrast to the spike potentials reported by Brindley, Oikawa et al. (1959) reported slow potentials recorded from the receptor layer of isolated fish retinas. It appeared that these slow potentials were from receptor cells, since ( a ) they could be recorded ody with extremely fine diameter microelectrodes (tip diameter less than 0.2 p ) and ( b ) they showed very little area-effect. A similar sustained negative potential has been recorded near the receptor layer of the cat retina (Motokawa et al., 195%; Griisser, 1957). Brown and Wiesel (1959), however, assigned the locus of the origin of this slow potential not to the receptor layer but to a region somewhat proximal to the receptor layer; this was further confirmed by electrode marking methods (Brown and Tasaki, 1961). Brown and Wiesel (1961) assigned the origin of the a-wave of the ERG to the receptor cell layer. Thus the pure, isolated a-wave, when not contaminated by other components of the ERG, must be the summated potential of the receptor cells themselves, that is, the “receptor potential.” Brown and Watanabe ( 1962) have devised two ingeneous means of recording the summated potential of the receptor cells isolated from other components of the ERG. Both methods involve recording from small areas within the retina of the intact mammalian eye. One method involves recording from the fovea of cynomolgus monkey; this area contains only receptor cells and, when illuminated locally, should give responses only from the receptor cells. The other method (Brown and Watanabe, personal communication, 1962) involves selectively blocking the retinal circulation in the cat’s eye, functionally isolating the receptor cells which are supplied by the choroidal circulation. The responses recorded under such conditions are undoubtedly receptor potentials in that they reflect the summated potentials of many receptor cells. However, one cannot yet deduce the action of the individual unit from knowledge of the mass response. Thus, there is an obvious gap in our understanding of how visual information is transferred from

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the first to the second neuronal units along the visual pathway. It appears that the needed information must sfill be obtained by recording from single receptor cells.

B. ELECTRICAL A m m IN THE SECONDARY NEURONAL LAYER It has been reported that spike potentials can be recorded from a microelectrode inserted into the inner nuclear layer (Ottoson and Svaetichin, 1953; Brindley, 1956). Brown and Wiesel (1958, 1959) have also recorded spikes from the inner nuclear layer of the cat retina. That such spikes are actually from the inner nuclear layer appears almost indisputable. In the cat retina, the ganglion cells constitute a single layer. Thus, the second spikes which are recorded during the course of a penetration from the internal limiting membrane must be from the inner nuclear layer. The measured depth of the layer where the second spikes were recorded corresponds well to the histologically measured depth of inner nuclear layer. Furthermore, when the second spikes were recorded, the polarity of the b-wave was negative, indicating that the tip of the recording electrode was in the inner nuclear layer. The origin of these spikes from the inner nuclear layer was also confirmed by electrode marking methods (Brown and Tasaki, 1961). Byzov (1959), however, recorded spikes from the inner nuclear layer in frog, but all of the spikes responded to the antidromic stimulation of the optic nerve, It was concluded that such spikes were all from the Dogiel's cell, primarily a ganglion cell which has been displaced into this layer. Antidromic stimulation of the optic nerve was not attempted in the experiments of Brindley and Brown and his associates, so the possibility that such spikes recorded by these authors were from the Dogiel's cell cannot be excluded completely. Tomita et at. (1981) have succeeded in recording the intracellular spikes from the inner nuclear layer of the frog retina but, in agreement with Byzov spikes responded again to antidromic stimulation of the optic nerve fibers, The possibility that an antidromic electric shock could stimulate directly the neuron in which an recording micropipette was impaled, was excluded in this experiment. It seems safe to conclude that the single unit-spike discharges are recorded in the inner nuclear layer of the vertebrate retina. But, it is unclear which type of cells in this layer generates such spikes-the bipolar cells, horizontal cells, amacrine cells or Dogiel's cells are all

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possible origins of the spikes. It does not appear to be technically feasible in the near future to distinguish from which of these cells records are taken. Sustained negative potentials accompanied by a fairly large negative dc potential shift can be also recorded from the secondary neuronal layer. Since the first report of the so-called cone action potential ( Svaetichin, 1953), there has accumulated a large number of investigations concerning this potential. These have been well reviewed by Brindley (1960) and Crescitelli (1960). Such slow potentials have been recorded from the retinas of fish (Svaetichin, 1953, 1956; Mitarai and Yagasaki, 1955; Motokawa et al., 1957b; Tomita, 1957; MacNichol d d.,1957), turtles ( Furukawa; Tomita, personal communication), frogs ( Tomita, personal communication ) and the cat (Motokawa et al., 1957b; Griisser, 1957; Brown et al., 1959, 1961). Although it was originally considered to reflect the activity of the photopic system, this sustained negative response seems to be common in all species regardless of the cone or rod retina. Svaetichin ( 1953) originally claimed that this negative sustained response was recorded from inside of the cone myoid or pedicle, but its origin in the cones at all has subsequently been questioned. Motokawa et d.(1957b) were inclined to regard the receptor cell as a possible origin, but they stated that a multiphotoreceptive mechanism within a single cone would have to be assumed to explain the multiple humps in a spectral response curve. The receptor origin of this potential was doubted by Tomita (1957). It was demonstrated that the depth at which the maximal response was obtained was in a region proximal to the receptor layer. Now, the problem of the origin of the so-called cone potential has been completely solved by many investigators using refined techniques of electrode marking ( MacNichol et aZ., 1957; Mitarai, 1958, 1960; Oikawa d al., 1959; Tomita et al., 1959; Gouras, 1980; Brown and Tasaki, 1961). Since it became clear that the origin of the Svaetichin potential is not the receptor cell, the term “cone potential” is not appropriate; thus, this term has been substituted by the term “S-potential,” following a suggestion of the present reviewer ( 6.Tomita, 1959). Whether or not the S-potential is really intracellular has been an important problem. Major criticism against the intracellular nature of the S-potential is drawn from the following observations: ( a ) The S-potential can be recorded with a large electrode; ( b )The response

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tolerates a large displacement of the electrode; ( c ) Anodal and cathodal polarization applied through a microelectrode does not alter the recorded potential; and ( d ) only a poor correlation exists between the associated dc shift and the amplitude of the S-potential (Tomita, 1957; Gouras, 1960; Tasaki, 1960; Watanabe et al., 1960). However, Svaetichin and his associates have consistently asserted the intracellular nature of the S-potential. According to their most recent reports (Svaetichin et al., 1961; Laufer d aE., 1961; Mitarai et al., 1961), they have succeeded in marking the electrode tip within an individual cell and concluded that the L-type of response (luminosity type) originates from a horizontal cell and the C-type of response (chromatic type) is recorded from a Muller fiber. Both of these cells are considered to be glial cells surrounding a nerve cell. Consequently, they have pointed out the importance of glial function to the nerve cell activity and discussed the glia-neuron interaction. If the origin of the S-potential is indeed glial cells, such as Miiller fibers or horizontal cells, the unusual behavior of the S-potential as an intracellular response may be explained by anatomical characteristics of these cells. CONDUCTION C. LATERAL Gouras ( 1958) has described a spreading depression which occurs in the excised frog retina; the depression appears similar to that which has been observed on the cerebral cortex (Leso, 1944). The retinal spreading depression is characterized by the spontaneous appearance of a misty, grayish wave which travels concentrically from its origin at a slow rate of 1-2 mm/min. Concurrent with the appearence of the color changes of the retina are changes in the retina’s excitability which is reflected in ganglion cell discharges and ERG. As the grayish wave reaches the recording electrode, an increase in spontaneous ganglion cell discharges occurs and a pronounced depression follows. Couras considered the spreading depression to have no physiological significance and considered the process attributable to ephaptic transmission. Evidence for lateral interaction in the excised carp retina was demonstrated by Motokawa et al. (1959b). Approaching from the receptor side of the retina with a micropipette electrode, they recorded a positive slow potential within the small illuminated area and a negative slow response from the surrounding region which

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was not illuminated directly by stimulus light (Fig. 1). This negative potential could be recorded as far as 3 mm from a light-dark boundary, but this effect was not possible when the illuminated and dark areas were separated by an incision in the retina made by a razor blade. A maximal amplitude of the positive potential was re-

FIG.1. Lateral distribution of slow potentials in response to microillumination of 60 p in diameter centered on the point labeled 0, in experiments on carp’s isolated retina. Positive going potentials are plotted upwards. Intensity of illumination: 7,000 lux. Representative records taken at 3 different points are shown in inset. (From Motokawa et al., 195913. )

corded at the receptor surface, but the negative potential was obtained maximally at the depth of 100 p from the receptor surface. Motokawa et al. (1959a) measured the conduction velocity of the negative potential and found it to travel across the retina at 67-160 mm/sec. Motokawa et al. (1959c, d ) also discovered that a slowly propagating negative potential, in many respects similar to that induced by photic stimulation, could be elicited by electrical stimulation of the retina. The electrically induced negative potential was found to have a maximal amplitude at a depth of 100 p from the receptor surface and to propagate laterally at a rate of 120 mm/sec. This observation suggests that both a photically induced negative potential ( outside an illuminated region) and the electrically elicited response

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are generated in the same layer of the retina. In the later experiments (Motokawa et al., 1961a), it was shown that the positive potential recorded at the illuminated area and the negative potential obtained from outside the illuminated area are antagonistic to each other with respect to ganglion cell discharge. As shown in Fig. 2,

FIG. 2. Relation between slow potentials of fish retina and spike discharge of a retinal ganglion cell. Numerals indicate lateral distances between center of illuminated area and electrode tip. Size and intensity of stimulus were 0.4 x 0.4 mm and 1,250 lux, respectively. Onset and termination of light stimulus are marked on slow potential records. (From Motokawa et al., 196la.)

the positive potential is related roughly to an on-inhibitory and off facilitatory, and the negative potential corresponds to an on-facilitatory, off-inhibitory discharge pattern. The physiological significance of these propagated potentials will be discussed in the section on pattern vision. As our present knowledge concerning the electrical activities of the receptor celIs and bipolar cells is quite limited, the vertical conduction from the receptor cells to the ganglion cells cannot be discussed in detail. Much is known about the electrical activity of the

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ganglion cells, but this is essentially the same as the activity of the optic nerve or tract. It will be described in the latter sections. 111. Impulse Conduction in the Optic Nerve

In classifying the conduction velocity of fiber groups in the cat optic tract, Bishop et al. (1953)recorded the monophasic antidromic action potentials at a point 6 mm from the crushed end of the optic nerve following stimulation of either the contralateral or ipsilateral optic tract. Figure 3 shows a series of records obtained at successive Crossed 0

-

Uncrossed A

e - A -

Combined id&

b-

FIG.3. Antidromic compound spike potentials from cat's optic nerve of opposite and same side using common stimulation site in tract for each pair of records a and e, b and f, etc. In the third column these have been superimposed. Conduction distance (crossed): 19.5 mm. Time interval: 0.2 msec. (From Bishop et al., 1953.)

depths through the optic tract. From these records it was concluded that: ( a ) There are two groups of fibers which have different conduction velocities; (b) The more rapidly conducting fibers are encountered much more frequently in the lower than the upper portions of the optic tract (left column in Fig. 3), (c) The more slowly conducting fibers are encountered more frequently in the ipsilateral than in the contralateral optic tract (middle column in Fig. 3). When these two sets of records are superimposed and compared, it is seen that the peak of the action potential of the ipsilateral slowfiber spike falls in the trough between the two contralateral action potentials. Thus, so far as conduction velocity is concerned, it might be said that there exist 3 different groups of fibers in the cat optic

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nerve, Conduction velocities, measured by Bishop d al. (1953) in this experiment, were 30-40 metedsec (mean 34) for the fastest, 17-23 metedsec (mean 21) for the intermediate, and 15-20 meters/sec (mean 18) for the slowest fibers. Chang (1952) reported somewhat different values for the conduction velocities of fiber groups in the cat optic nerve: 70,30, and 17 meters/sec for the fastest, intermediate and slowest groups, respectively. In the same study it was also reported that there are 3 peaks of distribution lying in the region of 1, 4,and 9 p of the fiber caliber spectrum. It was shown later (Chang, 1956) that the 3 peaks of the antidromic action potential could be recorded when both ipsiand contralateral optic tracts were stimulated simultaneously; this is to be expected since all of the fibers are contained within the same nerve bundle. Bishop and Clare (1955) could not confirm Chang’s observation, however, Constructing a fiber diameter histogram, they could find only one peak in the region of about 1p ; the number of larger fibers decreased as the diameter increased. In further analysis of this fiber group, they multiplied the number of fibers ( N ) in each size group by the 1.5 power of the average fiber diameter ( 0 )of that size and thereby constructed the distribution curve of the product ( N x D1.5) against the fiber diameter. This procedure also failed to demonstrate any peaks which corresponded to the 4 deflections of action potential which had been recorded by these investigators. A close correlation between the distribution curve of N x D1e5against D and recorded compound action potential has been shown to hold for peripheral nerves. Nevertheless, they concluded from the electrophysiological data they had collected that the fibers of the optic nerve show four groups which differ in conduction velocity. They suggested that: The most rapidly conducting fibers carry information to the visual cortex with synapse in layer A of the dorsal nucleus of the lateral geniculate; The next fastest fibers travel to layer B of the same nucleus and then to the lateral nucleus of the thalamus; The third fastest group innervates the pretectal area; the slowest fibers go to the superior colliculus. Thus, two different theories concerning the grouping of the fiber in the optic nerve have been reviewed; one correlates the fiber groups with their destinations in higher centers, while the other correlates them with color vision (Chang, 1952; Lennox, 1958). It

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should be noted, however, that neither of these theories offers a convincing account of the functional significance of the fiber groups which are anatomically observable in the optic nerve. To clarify the functional difference of these groups of fibers, a different experimental approach has been attempted by Motokawa et d. (1957a). The antidromic action potentials of the cat optic nerve were recorded in combination with an orthodromic volley of spikes elicited by a light stimulus. Figure 4A shows an example of

FIG.4. Suppression of antidromic potential of cat’s optic nerve by a preceding flash of light: A, Control without flash; B, Asynchronous discharge caused by flash of light alone; C, Suppressed antidromic potential. Time mark: 1 msec. (From Motokawa et al., 1957a.)

the antidromic response of the optic nerve, when recorded from the retinal surface. The stimulus was a single shock applied to the contralateral optic tract. The response consists of fast and slow components as shown. Since the recordings were made from a rather gross electrode (tip diameter 2 0 5 0 p ) , only irregular oscillations could be recorded following photic stimulation of the retina (Fig. 4B). When the effects of the interaction of the anti- and orthodromic volleys were investigated (by presenting first the light flash and then the shock to optic tract), the amplitude of the antidromic response was decreased (Fig. 4C).Under no conditions was it possible for the flash to suppress the mass response completely, but complete suppression could be seen when records were made from single units. Since a complete suppression is due to a collision of antidromic and ortho-

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dromic impulses somewhere in the optic nerve fiber, the probability of such collision should be proportional to the frequency of the ganglion cell discharges to the flash when a distance between recording and stimulating electrodes is constant; the amount of suppression of the antidromic mass response would be expected to be proportional to the sum of the collision probabilities of individual elements. Thus, the decrement of the mass response may be used as an index to investigate the effect of the photic stimulation upon the retina. In order to study the time course of discharge frequency change, an interval between the conditioning stimulus (flash) and test (antidromic) shock was varied systematically and the degree of amplitude suppression was measured. The results of such measurements were shown in Fig. 5 with respect to 4 different colored stimuli.

6. 4.

b

B

2

100

200

--.

I L

400

500

mseo

FIG.5. Temporal patterns of suppression of antidromic potential by preceding flash of colored light in cat's optic nerve. Ordinate: Magnitude of suppression. Abscissa: Time from onset of light to arrival of antidromic volley at retinal ganglion cell layer where pickup electrode was placed. 0: orange. Y-G: yellow-green. G: green. B: blue. (From Motokawa et aE., 1957a.)

Recording single ganglion cell discharges in the cat, Donner (1950) had shown that the frequency maximum in the discharge of ganglion cells occurs 100 msec after stimulation with red light, after 200 msec for green, and slightly longer for blue. Motokawa et d.

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( 1957a) adjusted the intensities of orange (0) ,yellow-green ( Y-G) , green (G) and blue (B) lights to be equal in bleaching visual purple, thus eliminating the influence of the rod system. Several elevations in the time course of percentage suppression were found in these interaction experiments (a, b, c in Fig. 5). In agreement with Donner, a marked difference between processes 0 and B was noticed, while the difference between G and B was very slight. All of these findings seem to support the hypothesis that the retinal information for color vision is transmitted as the change of temporal pattern of impulse frequency. The antidromic action potential recorded by Motokawa et al. (1957a) consists of 2 components which represent the fast conducting fiber group and the slow conducting group. The hypothesis, suggested by Chang (1952) and Lennox (1958), proposes that the fast group conducts information of the “red” and the slow group conducts the “blue.” It would follow from this hypothesis that, in the interaction type of experiment, there should be a selective suppression of the slow or fast component of the antidromic response which would be dependent upon the wave length of the photic stimulus, the fast component being suppressed by a flash of red light and the slow component being suppressed by blue light. But, this was not found to be the case (Motokawa et al., 1957a). In Liberman’s experiment ( 1957), it was demonstrated that some units of the frog retina responded with spike discharges of high frequency and short duration to red light but with low frequency and long duration to blue light. This characteristic difference to red and blue lights was maintained over a wide range of stimulus intensity, but Liberman reported that the elements which showed such different reactions to red and blue lights were encountered extremely rarely. Orlov (1961) recorded so-called neurograms from the frog optic nerve; these were integrated mass responses recorded by an ac amplifier with a simple intergrating network at the output, In the darkadapted state, no difference was found between neurograms for red and blue lights. However, a marked difference was observed between the green and blue neurograms under orange background illumination, or between the red and blue neurograms under green background illumination; the duration of the neurograms was long

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for the blue and short for either the green or red. The difference in time course of the green and blue, or red and blue neurograms was not altered either by changing the intensity of the background illumination or by even a hundredfold increase of the stimulus light intensity. A common factor which may be noted in all of these experiments is that whatever coding the retina may impose upon color information it seems to appear along the dimension of temporal patterning. IV. Centrifugal Fibers within the Optic Nerve

There has been considerable anatomical evidence which indicates the existence of efferent fibers in the optic nerves of vertebrates. Ram6n y Cajal (1955) described the fibers which terminate on the amacrine cells, and suggested that these fibers originated from somewhere outside the retina. Similar conclusions were drawn by von Monakow (1889), Dogie1 (1895), Polyak (1957), Johnston (1906), and Maturana (1958). That there has been no report of medullated fibers remaining in the human optic nerve following removal of the eye does not necessarily exclude the possibility of the existence of efferent fibers, for autopsy is usually carried out at considerable periods following removal of the eye, and such fibers may well be expected to disappear by retrograde degeneration. The lateral geniculate body, superior colliculus, ganglion isthmi, and hypothalamus have been suggested as possible central loci from which these centrifugal fibers originate, but the evidence implicating any of these centers does not seem conclusive. Recently, Fillenz and Glees (1961) investigated the occurrence of degeneration of the cat optic nerve fibers during the period between 3-325 days after enucleation of the eye or removal of the retina by suction. Two different groups of fibers could be distinguished which degenerated along 2 different time courses. The majority of the fibers which belong to the first group were completely fragmented by 11 days and their debris was removed by 162 days. The second group consisted of fine fibers of 0.5-1 p in diameter and showed a much slower time course of degeneration. It is quite conceivable that these fine fibers are centrifugal fibers to the retina. Fillenz and Glees may be close to the demonstration of a positive experimental solution of this previously controversial question. From the general principle of cybernetics, it would be natural to postulate

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some descending influence through centrifugal fibers for control of retinal functions. However, the physiological significance and functional role of such influence has not been demonstrated, in spite of the abundance of anatomical and electrophysiological investigation. Granit ( 1955a), recording from retinal ganglion cells, investigated the effects of stimulation of the tegmental region of the reticular formation, which corresponds to Magoun’s reticular activating system, upon both spontaneous and photically elicited spike discharges. He also recorded antidromic spikes produced by stimulating the pretectal optic nerve fibers. Responsiveness to photic stimulation was found to increase following repetitive antidromic stimulation of the pretectal fibers at the rate of 200300/sec, the magnitude of effect increasing with the duration of stimulation ( posttetanic potentiation). Occasionally, posttetanic inhibition was observed, Stimulation of the tegmental part of the reticular formation was also found to induce posttetanic potentiation for photically induced spikes. Such potentiation and inhibition were similar to those following antidromic stimulation, but there were no spikes induced by reticular formation stimulation. Since it is hardly conceivable that such long lasting posttetanic effects, either potentiation or inhibition, can be attributable merely to antidromic stimulation, Granit concluded that the potentiation or inhibition, initiated by stimulation in the tegmental part of the reticular formation or even by stimulation of the optic tract, is due to stimulation of the centrifugal fibers. Motokawa and Ebe (1954),by stimulating the optic nerve antidromically, found a similar depression of photosensitivity. The depression lasted for several hundreds msec after delivering a single shock. Such long-lasting alteration of photosensitivity cannot be interpreted as a consequence of the excitability cycle of the ganglion cell, since the latter should be much shorter than the former. Since recurrent collaterals have not been described for the retinal ganglion cells, the antidromic impulses cannot be used to explain the long duration of decreased photosensitivity. The action of centrifugal fibers cannot be excluded as a possible explanation. Other investigators ( Muller-Limmroth, 1954; Dodt, 1951; Marg, 1951),using the ERG as an indicator, have shown that illumination of one eye can influence the response of the other. Monnier (1949) criticized such experiments as demonstrations of centrifugal influence

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because of the possibility of mediation through the ciliary muscle and associated innervation, but this criticism was circumvented by showing that the interaction occurred even when the anterior portion of the eye had been removed. However, the possibility still remains that scattered light may penetrate the tissue between two eyes and stimulate the retina in which a recording electrode is located. Motokawa et al. (1956a) demonstrated in human subjects that monocular and binocular illumination produced different configurations of the ERG. The negative swing which followed the b-wave was found much deeper for binocular stimulation than for monocular stimulation. Since in this experiment both eyes had been atropinized and eye movements were carefully controlled, binocular neural interaction must be considered as a possibility. Jacobson and Gestring (1958a, b ) have shown the effect of the optic nerve section upon the ERG in the cat and monkey. A central stimulant (Metrazol) depressed the ERG amplitude while a central depressant (barbiturate, for example) increased the ERG. These effects were abolished by cutting the optic nerve. Confirming another observation of Jacobson and Gestring, Abe (1962) demonstrated that that section of the optic nerve caused a remarkable increase in the rabbit ERG which was maximal (up to 3OE) 3 4 hr after sectioning. It may be difficult to explain such effects, except as an inhibitory influence through centrifugal fibers. Stimulating the optic nerve and recording from the inner surface of the rabbit retina, Dodt (1956) found delayed spikes, which he believed reflected the arrival of centrifugal impulses at the retina. In addition to having much longer latencies, the “delayed spikes” differed from the ordinary antidromic spikes by: ( a ) disappearing during light adaptation; (b) failing to follow repetitive stimulation at rates above 6/sec, ( c ) being inhibited by Myanesin; and (d) being facilitated by strychnine, Similar delayed spikes were recorded by Motokawa et a2. (1956b) in the cat eye, but the latter experiment could not prove that this slowly conducting spike came from centrifugal fibers. Later, Granit and Marg (1958) also distinguished similar delayed spikes from the fast-conducting antidromic spikes, However, they failed to confirm the observations of Dodt, and concluded that these delayed spikes were ordinary antidromic spikes recorded from h e fibers which belonged to small

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ganglion cells. Since antidromic stimulation of the optic nerve was found to have no influence on the ERG pattern, Granit and Helme (1939) concluded that the ganglion cells do not contribute to the generation of the ERG. Recently, more refined techniques have made it possible to place fine microelectrodes within the retina of the intact eye and thereby record the intraretinal ERG from restricted loci within the retina ( Brown and Wiesel, 1959). By the use of such techniques it has become evident that at least some components of the ERG can be influenced by antidromic stimulation of the optic nerve. With a microelectrode placed immediately adjacent to the retinal side of the Bruch's membrane, a maximal amplitude of the c-wave was recorded by Tasaki et at. (1962), who found that repetitive stimulation of the optic nerve (2Wsec) caused a reduction of the amplitude of the c-wave (Fig. 6). Since the c-wave is

FIG. 6. Intraretinal ERG of cat recorded just on inner side of Bruch's membrane. Left, Control. Right, During optic nerve stimulation at rate of 2OO/sec. (Lower trace of right record was caused by stimulus artifact.) Positivity upwards: Horizontal bar indicates period of illumination (0.5 sec). (From Tasaki et d.,1962.)

considered to originate from the pigment epithelium (Noell, 1954; Tomita, 1959; Yamashita, 1959; Brown and Wiesel, 1961; Brown and Tasaki, 1961), it is likely that the pigment cells are influenced by activity of the centrifugal fibers. But before we can draw this conclusion, alteration of blood flow within the retina following the optic nerve stimulation must be excluded. Although there now exists a relatively large accumulation of electrophysiological evidence which indicates centrifugal influence upon retinal activities, none of the available studies seems to show directly or conclusively on just what system nor in what manner this influence operates. It is still too early to accept a theory of centrifugal control.

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V. Lateral Geniculate Body

A. ANATOMY OF THE LATERAL GENICULATE BODY The lateral geniculate body (LGB) is the relay station for the visual pathway between the retina and visual cortex, and it can be compared with the thaIamus and its role in the somatosensory pathway. The LGB consists of a number of cell layers, in which the crossed and uncrossed optic nerve fibers terminate separately. By the careful observations of Le Gros Clark (1941) on the synaptic connections within the LGB of the monkey, it became clear that there are 6 layers distinguishable in this region. Numbering these layers from inside toward outside as 1, 2, . . . and 6, the ipsilateral optic nerve fibers terminate in the layers of 2, 3, and 5, and contra&era1 fibers end in Iayers 1, 4, and -6 (Fig. 7 ) . An ending of each

FIG.7. Diagram of a transverse section through right lateral geniculate body of monkey. Layers are numbered from ventral to dorsal. IpsiIateraI retinal ganglion cells are shown on upper left, and contralateral ones on lower right. (Modified from Glees, 1961.)

fiber is divided into 5 or 6 branches within a single layer, and each ending makes a synaptic connection with one cell. Thus, the ratio of terminal boutons to lateral geniculate cells appears to be 1:l.This one-to-one correspondence is considered to be a characteristic feature in the monkey, but it has not been found in the cat. When one optic nerve was cut, the degenerating optic nerve fibers could be

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seen in the layers of 2, 3, and 5 of the ipsilateral geniculate body, and in the contralateral 1,4,and 6 layers. There seemed no anatomical interconnection between individual layers. By an examination of degenerating fibers induced by small lesions made on the retina, it was also found that the central part of the LGB corresponds to the retinal fovea, and the nasal and temporal side of the lateral geniculate receive fibers from upper and lower peripheries of the retina, respectively. These anatomical findings have been confirmed recently by Glees (1961). It has also been described that the lateral geniculate cells within layers 1 and 2 are larger than those of the other layers. But no functional differences between cells of different sizes have been discovered.

ACTIVITYOF THE LATERAL GENICULATE BODY B. ELECTRICAL Following illumination of the retina or electric stimulation of either the optic nerve or tract, single-unit responses as well as summated mass responses can be recorded by microelectrodes inserted into the LGB. Photic and electric stimuli give different patterns of the mass response, but these vary considerably with the site of the recording electrode. If one optic nerve of an anesthetized cat is stimulated with sufficiently strong single shocks, typical responses of the LGB are recorded with an electrode placed within the dorsal layer, as is shown in the bottom of Fig. 8 (Bishop and McLeod, 1954). The optic tract response is a triphasic action potential, t,, consisting of positive, negative, and positive phases as shown in Fig. 8, a,b. If the stimulus is maximal, a tract response tl is followed by a second wave tz,these 2 waves indicating the action potentials of 2 groups of fibers conducting at different rates. In the LGB response, tl and t z are followed by 2 additional waves, rl and rz (in Fig. 8, g,h). The notations t and T are adopted from Bishop and McLeod (1954). The latter negative waves, r1 and rz were interpreted as the action potential of the LGB cell (Marshall and Talbot, 1940; Bishop and OLeary, 1940; Bishop and McLeod, 1954). Tasaki et al. (1954) introduced a hyperfine capillary microelectrode into the cat LGB, and classified 4 different types of extracellular potentials, A, B, C, and D (Fig. 9). A was considered to be the action potential of a presynaptic axon with high conduction velocity, since its latency was fairly short (0.44.8 msec) and it followed stimulating shocks up to 100 shocks per sec with unaltered response

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FIG.8. Evoked potentials at optic tract and lateral geniculate body in response to electrical stimulation of cat's optic nerve. Time mark:0.2 msec. (From Bishop and McLeod, 1954.)

patterns. The response B appeared after a latency of 1.2-2.2 msec. Although this response resembled that of presynaptic fiber, it had a tendency to respond with double or triple spikes. Thus, Tasaki et al. (1954) suggested that these spikes were from postsynaptic medullated axons. The third type of responses, C, is probably from the cell

FIG. 9. Four different types of unitary responses taken with extracellular microelectrode from lateral geniculate body of cat. Vertical bar: 2 mv. Time mark: 1 msec. (From Tasaki et al., 1954.)

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body. The initial positive phase is followed by a relatively long, negative spike potential. The initial positivity may be attributed to an outward-directed current through the surface of the cell body, caused by synaptic activity. Due to subsequent depolarization of soma membrane, a cell body behaves as a sink, while inactive dendrites behave as a source: thus making the external medium purely negative with respect to a remote point. In this type of response, as in the case of postsynaptic axons, single shocks to the optic nerve induced multiple spike discharge. The fourth type of the response, D, is a slow diphasic spike, initially positive and followed by a prolonged negative phase. Such a pattern was interpreted as representing externally recorded potentials from the dendrites of a geniculate neuron. Although this interpretation lacks direct evidence, Hild and Tasaki (1962) confirmed this view by demonstrating all-or-none propagating impulses along the fine strands of dendrite in cat brain tissue culture. Vastola ( 1957) investigated orthodromic and antidromic responses of the LGB in the decerebrated cat. Antidromic responses, elicited by stimulation of the optic radiations, are triphasic action potentials. The initial deflection appears after a latency of 0.3msec and is recorded as positive above the dorsal margin of the LGB and as negative within the LGB. Vastola identified the &st deflection with the axon spike on the following grounds: ( l a ) a short latency; ( b ) a short absolute refractory period (as revealed by double shocks); and ( c ) ability to follow high frequency repetitive stimulation (up to 432/sec). The second deflection of the antidromic response has a latency of 0.6 msec and a duration of 1.2 msec; its polarity is opposite to the first deflection, the reversal of polarity occurring at a point slightly deeper than that for the first deflection. It was concluded that this second deflection arises from depolarization of cell bodies, from the following observations: ( a ) the second deflection behaves similarly to the first one, and follows repetitive stimulation up to 432/sec; ( b ) it has a longer latency ( for antidromic stimulation) than the first deflection; ( c ) its absolute refractory period is usually about 0.8-1.0 msec longer than that of the first deflection; and ( d ) following circulatory block (section of abdominal aorta), it decreases in amplitude within 3 sec, while the first deflection remains unchanged.

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The third deflection in antidromic response has the same polarity as the first, but it has a considerably longer duration. Although this deflection could be attributed to dendritic activity, Vastola finds no reason to support this view. The orthodromic response elicited by optic nerve stimulation has a considerably longer latency than that of the antidromic response. This response consists of multiple deflections of negative and positive waves. The slow negative component among these deflections was suggested to be due to depolarization of cell bodies. It was also considered that, during the course of this negative deflection, an electric current, which resulted from active depolarization of cell bodies, might flow into somas so as to decrease the membrane potential of the axons. With an adequate interval between anti- and orthodromic stimulations it was possible for the k s t deflection of antidromic response (axon spike) to fall on exactly the same phase as the above-mentioned orthodromic negative deflection. Under such conditions, a slight decrement in the amplitude of the first deflection of antidromic response (axon spike) was observed. Such an observation is consistent with the previous assumption of depolarization of the ascending axons. A reverse relation holds true for the positive phase of orthodromic response, The first deflection of the antidromic response increased, if it fell on the slow positive phase of orthodromic response. Thus, the orthodromic positive phase may be explained as a hyperpolarization of the cell body. C. EXCITABILITY CYCLEOF THE LATERAL GENICULATE NEURONS In the preceding section, identification and the criteria of LGB responses have been described. In addition, the following points will now be considered. Hubel (1960) demonstrated that single, extracellular spike potentials could be recorded both from axons and cell bodies and that these two types of spikes were distinguishable from each other. Recorded with tungsten microelectrodes, axon spikes were usually found to be purely positive, while soma spikes were biphasic (positive-negative or negative-positive depending upon relative position of recording microelectrode to the cell). In some cases, a prepotential was seen on the rising limb of spikes. Griisser-Cornehls and Griisser (1960) also recorded spike potentials preceded by prepo-

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tentials; under certain recording conditions only the prepotential was recorded, without being followed by a spike. Thus, this prepotential must be a synaptic potential elicited by impulses entering the LGB neuron. Bishop and Davis (1960b) showed an after-negativity of 10 msec duration following a neuron spike in the cat LGB; this was followed by a long-lasting positivity. An after-positivity lasting for 100-200 msec, which resembled the after-potential of the axon, was also demonstrated by Vastola ( 1959). This after-positivity disappeared during a period of repetitive stimulation of the optic radiation at 10-150/sec. Such slow potentials seem somewhat analogous to the well known after-potentials of peripheral nerve, in which there exists a close relationship between the recovery cycle and such after-potentials. Since the time course of the after-potentials, especially the positive after-potential, is much longer for these central neurons than is the case for peripheral nerve, a direct homologous relationship is perhaps questionable. The possibility of inhibitory postsynaptic potentials from cells with short axons should not be excluded. Using the technique of twin-pulse stimulation of the optic nerve to investigate the excitability cycle of neurons of the LGB, it was shown that the previously mentioned negative and positive after-potentials correspond, respectively, to periods of super- and subnormal excitability. In these experiments, special precautions must be taken to avoid possible summation due to overlap of subliminal fringes; the recovery cycle at the stimulating site (optic nerve) must also be considered (Bishop and Davis, 19SOa). The effect of subliminal fringes can be eliminated by the use of supramaximal stimuli. When such precautions are taken, the supernormality observed at the LGB neurons is characteristically of high magnitude and relatively long duration. Strong presynaptic bombardment upon LGB neurons and absence or scarcity of recurrent inhibition of Renshaw type is a possible reason for such high supernormality. The subnormal phase appeared after the supernormality, reaching a maximum at about 19 msec. Recovery to 87% of normal excitability required 220 msec and 2 sec for full recovery. Bishop and Davis (1960a) also reported a second supernormal period, less marked than the &st, which appeared several seconds after stimulation.

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D. BINOCULAR INTERACTION AT THE LATERAL GENICULATE LEVEL Histological studies (Glees, 1941; Minkowski, 1920; Silva, 1956.) have firmly established that optic nerve fibers from two eyes terminate at different layers in the LGB. More recently, however, Hayhow (1958) has shown that considerable overlapping of contraand ipsilateral optic nerve fibers occurs in the interlaminar zones and Interlaminaris medialis of the cat LGB. Recent electrophysiological studies, aided greatly by microelectrode techniques, have also expanded the evidence of binocular interaction at the geniculate level. Bishop et al. (1959) reported that 8.5%of the LGB units from which they recorded produced spike discharges following electric stimulation of either optic nerve. Some of these dually responding units have a relatively short latency (about 1.2 msec) and are apparently innervated by fibers from both of the optic nerves. Others have a much longer latency (about 300 msec); these probably are innervated via multisynaptic routes. Erulkar and Fillenz (1960) also found a few LGB units which in their study would respond to photic stimulation of either eye. For even more LGB units, photic stimuIation of one eye was found to modify the response to stimulation of the other eye. The latter type of interaction was usually inhibitory. A possible explanation for this interaction may be the inhibitory action of short axon cells which was reported to exist within the LGB ( OLeary, 1940). Griisser and Saur (1960) did not encounter LGB neurons which responded with spike discharges by diffuse illumination of either eye, but in 8 units they observed that the discharge frequency for binocular stimulation was lower than that for monocular stimulation. Hubel and Wiesel ( 1961) made a similar experiment with restricted illumination and found that the receptive field of the LGB neuron resembled that of the retinal ganglion cell and that the receptive field around the area centralis was small. But binocular interaction was not reported. Vastola’s experiments (1960, 19Sl), although not directly concerned with single unit activity, suggested a possible relationship between after-positivity and dc responses of the LGB. These studies examined the effect of ipsilateral optic nerve stimulation upon the contralaterally evoked spike responses at the LGB and optic radiation. A decrease in amplitude of postgeniculate spike appeared fol-

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lowing the ipsilateral conditioning stimulus and required 300 msec for recovery. The depression was generally more marked in the radiation spike than in the geniculate spike, A close correlation was shown to exist between the depression and the after-positivity which was evoked at the dorsal nucleus of the LGB by a conditioning stimulus. Further discussion of the nature of the after-positivity suggested that even though the conditioning stimulus was not sufficient to fire the geniculate neurons, a potential change appeared in the same way as the after-positivity caused by strong stimulation. Thus, it was concluded that the after-positivity is not an after-potential of the axons, but a hyperpolarization of the geniculate neurons resulting from cells with short axons. A subsequent study (Vastola, 1961) disclosed that by increasing the repetition rate of the conditioning stimulus above lO/sec there occurred abatement of after-positivity, and spike potentials to the test stimulus were no longer depressed. With a further increase in repetition rate, the direction of dc response changed, and the amplitude of spike response was augmented. In conclusion, we have now anatomical and physiological evidence that visual information from the two eyes generally arrive independently at the LGB, but there exists some binocular interaction at this level. The implications of such interaction with respect to visual perception are, however, still unanswered. VI. Nonspecific Afferents and Visual Transmission

One of the most significant developments in contemporary neurophysiology has been the demonstration of nonspecific af€erent pathways function in parallel to the well-known specfic pathways. The possibility has been suggested (Adrian, 1954) that some controlling mechanism may act upon the transmission of sensory information at a subcortical level. Selective attention is common to our everyday experience. Such sensations as touch or pressure are often lost if one is attending to auditory stimulation, That such selective attention is reflected at the subcortical level has been demonstrated by H e r n h dez-Pe6n et al. ( 1956). These investigators stimulated an unanesthetized cat with a series of auditory clicks and recorded the potentials evoked from the dorsal cochlear nucleus while the cat’s attention was diverted from the auditory stimulation to visual stimulation (in this case, a live mouse) ;the amplitude of the response was greatly reduced at the cochlear nucleus. Similar experiments have

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been performed in the visual system of the cat (Hernhndez-Pe6n et al., 1957). Responses to photic stimulation were recorded from chronically implanted electrodes in the optic tract, lateral geniculate, and visual cortex. It was found that the responses from these sites in the direct visual pathway were markedly suppressed when the animal's attention was diverted to nonvisual stimuli. Naquet et al. (1960), however, have pointed out that such experiments are easily misinterpreted, if the size of the pupil is not carefully controlled. They demonstrated that when the EEG shows an activation pattern, the pupil also dilates; the evoked potentials recorded along the visual system are thus enhanced, due to the subsequent increase of light intensity falling upon the retina. Naquet et aZ. (1960) concluded that only at the visual cortex does the amplitude of the evoked potential depend upon the state of activation; here the amplitude decreases during the activated state. At the optic chiasma and LGB, the amplitude of photically induced responses is independent of the activation state and is influenced only by the size of the pupil, the latter being a secondary effect. As autonomic functions (e.g., pupil size) can contaminate the demonstration of the subcortical nature of a nonspecific influence upon the specific visual pathways, a similar contamination has been shown to be induced by more gross behavioral functions (Horn, 1960). Horn recorded from chronically implanted electrodes in the visual cortex of unanesthetized cats. He found that the potentials evoked by light flashes were indeed reduced when the animal was distracted by nonvisual stimuli. Such reduction of the cortical evoked potentials, however, occurred only when the gross behavior of the cat showed apparent searching responses. Such experiments as discussed above, although in themselves interesting in showing the overall integrative functions of the nervous system, fail to answer the question of whether activity of the nonspecific activating system can influence the subcortical centers of the specih visual pathways. It should be noted that behavioral methods of altering an animal's attention, such as presenting a live mouse, are difficult to interpret when one wishes to assay the effects of such a stimulus at some specific subcortical relay center. It may also be pointed out for the previously mentioned experiments that, although the evoked cortical potentials would change in amplitude

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as the arousal state was changed, the latency and duration of the components of the evoked potential were not affected. It, thus, seems unlikely that the depression of the evoked potentials was merely an apparent decrease in amplitude due to desynchronization of the incoming volley. Thus, a number of investigators have attempted other types of experiments, using more direct neurophysiological methods, to examine the effects of the nonspecific arousal system on transmission of impulses in the specific visual pathways, This was accomplished by electric stimulation of the ascending reticular formation rather than by using extraneous sensory stimuli to arouse attention. Suzuki and Taira (1961) applied a single electric shock to the brainstem reticular formation (RF) as a conditioning stimulus and recorded both the mass responses and single unit discharges of the LGB which had been elicited by electric stimulation of the optic tract. A marked enhancement of the mass response was found to occur when the interval between the conditioning and test stimuli was about 100 msec (Fig. 10); the enhancement was even greater when

FIG. 10. Effect of mesencephalic reticular stimulation (single shock) upon maximal and submaximal LGB responses. A, Control response to submaximal stimulus; B, Response under reticular stimulation (to be compared with A ) ; C, Control response to maximal stimulus; D, Response under reticular effect (to be compared with C ) . Each record was obtained by superposition of 5 traces. Vertical bar: 500 pv, Time mark: 1 msec. (From Okuda, 1962.)

the intensity of the test stimulus (at the optic tract) was submaximal. This fhding suggests that the LGB neurons within the subliminal fringes were activated by stimulation of the RF.

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When microelectrodes were used to record from single LGB units, for a given submaximal stimulus intensity it was possible to assign a probability value to the event that a unit would be fired by a test shock delivered to the optic tract. It was found that, at a constant stimulus intensity, the probability that a unit would fire was increased by stimulation of the RF ( Fig. 11) .

FIG. 11. Responses of single optic radiation fiber of cat to 10 successive threshold tract stimuli. A and C give control responses taken before and after B; B gives responses conditioned by reticular stimulation (single shock) delivered 100 msec prior to tract stimulation. Response probability is obviously higher in B than in A and C. Voltage calibration: 5 mv. Time mark: 1 msec. (From Suzuki and Taira, 1961.)

Since the component tl in the mass response (action potential of the tract fibers) was not influenced by the reticular stimulation, it seems reasonable to attribute the effects of RF stimulation to an increase in the efficacy of synaptic transmission at the LCB. Such facilitation was marked when the synaptic transmission was relatively inefficient following administration of a small dose of anesthetics such as barbiturate. Furthermore, since the probability of the firing of a LGB neuron is nearly equal to unity when the optic tract is stimulated at supramaximal intensity, the facilitatory effect of RF stimulation appears to be limited. In addition to the mesencephalic reticular formation, other subcortical nuclei were also found to facilitate synaptic transmission at

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the LGB. These included the bulbar reticular formation, Nucleus (N.) centrum medianum, N. centralis lateralis, N. commissurae posterioris, N. subthalamicus, and Zona incerta. The facilitatory effect following stimulation of these nuclei was found to be about 70%as great as the facilitation following stimulation of the mesencephalic RF. Inhibitory action at the LGB synapses was found only following stimulation of the N. ventralis anterior ( Okuda, 1962). In addition to the electric stimulion of the optic tract, a natural stimulus (photic stimulation) was adopted in the experiments of Taira and Okuda (1962), who also investigated the effects of RF stimulation on transmission along the visual pathway. Taira and Okuda recorded on- and off-responses from the cat LGB and visual cortex, while concurrently monitoring the pattern of the EEG. An activated EEG pattern could be obtained by either repetitive stimulation of the RF or nonvisual sensory stimuli. In agreement with other investigators, it was found that during periods of EEG activation there was a slight attenuation of the evoked cortical potentials. However, by superimposing the cortical evoked responses, it was found that the cortical response showed far less variability in both amplitude and duration following stimulation of the RF than was the case when the EEG showed a relaxed pattern. Since a similar finding was obtained at the LGB, it does not seem likely that in the relaxed state the evoked cortical responses were contaminated by algebraic summation with the highly fluctuating background EEG. It seems more reasonable that stimulation of the RF imparts a greater uniformity and stability, or less variability, upon the transmission of agerent impulses through the LGB. Such action of the R F upon synaptic transmission at the LGB was also studied by recording responses from postgeniculate single fibers. For this purpose, a “response impulse number” was obtained in the following manner: During a 0.5-sec period following the onset or termination of photic stimulation, the number of spontaneous spikes was subtracted from the total number of spikes recorded, the difference served as a response impulse number. As is shown in Table I, the number of impulses attributable to the effects of the photic stimulus increases during the alert state. But a more distinct feature of the RF stimulation is the remarkable decrease of variability during the activated state. Thus, it was concluded that visual

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information is transmitted from the optic tract to the higher center with high probability and is not modified by accidental factors. Space does not permit a detailed discussion of each of the many experiments which have investigated the effects of RF stimulation. Many of the differences in results which have been reported appear to reflect differences in experimental conditions. TABLE I

EFFECTSOF NONSPECIFXC STIMULI UPON DISCHARGE OF LCB NEIURONS IN RESPONSE TO PHOTIC STIMULUS Number of response impulses over 0.5 sec Nonalert ( b e d on EEG)

Alert (based on EEG)

Type of discharge

Mean

S.D. (%)

Meen

S.D. (%)

on on on on on

10.1 18.6 16.5 24.2 8.0

33.6 16.7 24.8 14.0 47.5

11.7 15.1 14.1 29.2 11.0

22.2

Off

10.3 8.6 10.1 8.0 9.6

25.2 25.6 33.6 36.2 36.4

14.3 15.0 14.6 12.5 15.8

12.6 9.3 13.0 20.0 10.8

Off

Off Off Off

7.3 17.7 10.0 23.6

Type of stimuli

R. F. R. F. R. F. R.F. Auditory R. F.

R.P. R.F. R. F. Olfactory

There is good agreement on the enhancement of the evoked responses elicited by electric stimulation of the visual pathway (Dumont and Dell, 1958, 1960; Bremer and Stoupel, 1959; Long, 1959; Suzuki and Taira, 1961; Okuda, 1962). The photically evoked responses, on the other hand, are usually reported to be reduced following R F stimulation ( Hernhdez-P&n, 1955; Hernhdez-Pebn et al., 1957; Bremer and Stoupel, 1959; Steriade and Demetrescu, 1960; Taira et d.,19s2). The difference between electric and photic stimulation has been considered to be due to the fact that an electric stimulus evokes a synchronized volley, while a photic stimulus gives rise to a temporally dispersed train of impulses. To explain why the response to electric stimulation is increased while the response to photic stimulation is decreased, Bremer (1961)

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suggested that R F stimulation results in a synaptic occlusion which is more easily overcome by the synchronous impulses caused by electric stimulation than by the dispersed pattern of impulses arising from photic stimulation. The diverse observations as to the effect of the R F stimulation upon the photically evoked potential may have originated from the different experimental conditions used. To compare the results from different studies, one must pay careful attention to variables in the state of the experimental animal (such as whether it is in the resting or waking state; is chemically immobilized, physically restrained, or unrestrained; or is anesthetized, and if so by what type of anesthetics) as well as to the stimulus variables. In the latter respect, Steriade and Demetrescu (1960)found that the opposite results were obtained depending upon the rate of flickering light stimulation; and Long (1959)also reported that the direction of the effect was determined by the frequency at which the R F was stimulated. VII. Visual Cortex

The cortical area in which the optic radiation fibers terminate is Brodmann’s area 17. The representation of the retina upon the visual cortex has been studied extensively by anatomical, physiological, behavioral and clinical means. It is now firmly established that the fovea centralis is represented on the posterior extremities of the striate cortex; the upper quadrant of the retina corresponds to the upper lip of the calcarine fissure, and the lower quadrant of the retina to the lower lip of the calcarine fissure. The area of the visual cortex devoted to the macular region is very large compared with the area for the periphery. By electrophysiological means (recording from the area of maximal evoked cortical potential for illumination of restricted retinal points) Talbot and Marsha11 ( 1941) and Thomson et al. (1950)confirmed the previous anatomical conclusions on the projection of the retina upon the cortex. However, at least in the cat, Doty (1958) could not confirm the precision of topographical retinocortical relations; he found that a cortical strip along the marginal gyrus adjacent to, but probably not within, the striate area yielded by far the highest amplitude potentials elicitable by photic stimuli. But, unilateral extirpation of this area caused only insigdcant retrograde degeneration in the LGB. Thus, the high amplitude responses pro-

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duced in this region seem to be inherent in its organization and relatively independent of the manner of afferent activation. When the more distal visual pathways are stimulated electrically, four surface positive deflections of about 100 kv in amplitude and several milliseconds in duration, followed by a slower surface negative wave can be recorded from the cortex in the cat (Fig. 12).

FIG. 12. Visual cortex response to stimulation of cat’s optic tract, S: Stimulus artifact. Positivity downwards. (From Malis and Kruger, 1956.)

This pattern of the evoked response has been consistently reported in the experiments carried out by several investigators (Bishop and Clare, 1951, 1952, 1953; Chang, 1952; Chang and Kaada, 1950; Clare and Bishop, 1952). Opinion has been divided on the question concerning the origins and functional significance of the 4 surface-positive deflections, As to the surface-negative wave, most writers favor the view that the negative wave is the result of cortical activity, but there is still doubt whether this activity is a slow conducting depolarization of the dendrites, or an electrical

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variation resulting from multisynaptic transmission, or perhaps the mixture of the two. Chang (1952) concluded that the first three surface-positive deflections were spike potentials of the 3 different groups of the radiation fibers which had digerent conduction velocities, and that the fourth positive deflection and the slow negative wave were the intracortical events. He reported that each of the 3 surface-positive deflections could be potentiated separately by illuminating the eyes with lights of different wavelengths. From these observations, Chang postulated a hypothesis that color information is transmitted from the retina to the visual cortex through different channels; the fastest conducting fibers carry the “red,” the next group carry “green,” and the slowest fibers the “blue” information. Chang’s hypothesis has been supported by Lennox ( 1958). But Malis and Kruger (1956) have provided evidence challenging this hypothesis. They found ( a ) that the surface-positive components 1 and 2 were not affected by topical application of procaine, while the components 3 and 4 were reduced in amplitude, and ( b ) that, using paired stimuli, excitability cycle of components 1and 2 was Merent from that of components 3 and 4. Thus, Malis and Kruger concluded that only the first and second positive deflections represented the arrival of d e r e n t volleys of impulses from the fast and slow conducting fibers, respectively. In still another attempt to clarify the controversies concerning the origin of the components of the evoked potential in the visual cortex (especially components 2 and 3), Widen and Ajmone-Marsan (1960a) recorded simultaneously single spikes and the evoked mass response of the striate area. They identified the recorded spikes as presynaptic or postsynaptic in nature and attempted to correlate the generation of the single spike to each of the components of the cortical evoked response. They found that: ( a ) All spikes related to the components 1 and 2 were presynaptic in nature and were recorded only from white matter; ( b ) Those spikes related to component 3 were found to be either pre- or postsynaptic and recorded from either the grey or white matter; ( c ) The spikes related to the components 4 and 5 were postsynaptic and recorded from the grey matter. From these observations it was concluded that the components 1and 2 are from the fibers, 3 is both from fibers and intracortical neurons, and 4 and 5 are true responses of the grey matter. Widen and Ajmone-Marsan also demonstrated that a positive corre-

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lation exists between the presence of spikes and the amplitude of the surface evoked potentials. The spikes related to the first and second deflections appear consistently when the deflections 1and 2 reach a critical amplitude,,and remain stable, responding to every stimulus. The postsynaptic spikes related to the components 3,4,and 5 appear also when the amplitude of these components reaches a certain critical amplitude, but they are unstable, responding only irregularly to constant stimuli. There are extensive studies of Jung and his associates (Jung et al. 1952; Jung and Baumgartner, 1955) regarding the spike activities in the visual cortex. These investigators recorded single-unit activity from the striate cortex, using “endphale isole” cat preparations. From the spike patterns which were recorded following illumination of the eyes by diffuse light, they assigned the nerve cells from which they recorded into 5 categories, as follows: ( A ) spontaneous spikes, unaltered by illumination (49%); ( B ) on-units (24%);( C ) units inhibited only by strong light (3%);( D ) off-units (6%);and ( E ) onoff-units (18%).Among these 5 types of cortical units, types B, D, and E behave similarly to those in the retina and LGB. Hubel (1958)has emphasized that the type of light stimulation is an important factor for classifying cortical units. In the unanesthetized cat, new and important discharge patterns of the cortical unit have been revealed by using both diffuse and focal illumination. It was found that in addition to such units as identified by Jung with the use of diffuse light, some units which fail to respond to diffuse light give spike discharges when a localized light stimulus is used. Stimulation by intermittent light has also been used for analyzing the spikes of the visual cortex (Grusser and Grutzner, 1958). The latter investigators compared the effectiveness of repetitive electrical stimulation of the optic nerve with that of intermittent photic stimulation of the retina. It was found that the spike response followed repetitive electric stimulation up to 15&250Jsec, while the following of photic stimulation was limited to 50-60Jsec. It was also observed that the units which had a longer latency (for example, 25-120 msec) followed photic stimulation only at much lower frequencies ( Wsec). In all of these studies discussed above, the spikes of the cortical neurons were recorded extracellularly. Tasaki et d. (1954), however, have attempted intraellular recording from nerve cells in the

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cat striate cortex. Following stimulation of the radiation fibers, they were able to record a hyperpolarizing response which was associated with inhibition of spontaneous discharges. It was suggested that this negative going response (increase in the membrane resting potential) is analogous to the inhibitory postsynaptic potential (6. Eccles, 1957). Intracellular recordings from the cat visual cortex were made also by Li et al. (1960). Several types of responses to electrical stimulation of the LGB were reported which include: ( a ) exclusively depolarizing potentials; ( b ) depolarizing potentials with spikes superimposed; and ( c ) hyperpolarization potentials with or without inhibition of spontaneous spikes, The hyperpolarizing potentials, recorded by Tasaki et d.(1954) and Li et d. (1960) may indicate that the visual cortex receives inhibitory fibers. The latter reported that such potential changes were encountered at any layer of the visual cortex. They also observed that in some cases facilitation was followed by inhibition, and that a positive correlation existed between the amplitude of the surface potential (evoked potential) and frequency of the spike discharges. VIII. Corticipetal and Corticifugal Nonspecific Effects

A. CORTICIPETAL INFLUENCE

There seems to be general agreement that following stimulation of the ascending brainstem RF, there is an activation or alerting of the animal which is reflected both in the EEG pattern and in behavioral responsiveness. With respect to the effect of R F stimulation upon evoked cortical potentials, there have been equivocal results reported. Some of these have been already discussed in the Section IV. Most investigators agree in that the amplitude of the evoked potentials is reduced following RF stimulation, but Taira and Okuda (1962) claimed that the characteristic feature of R F stimulation is a decrease in the variability, or an increase in the regularity, of the evoked potentials rather than mere attenuation of the amplitude. It is interesting to compare the observation of Taka and Okuda with that of Fuster (1958), who reported that following brainstem RF stimulation, the tachistoscopic perception of the animal becomes more correct and the reaction time is shortened. Hernhdez-Pe6n et al. (1957) stated that the primary and

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secondary waves of photically evoked potentials of the visual cortex are influenced dgerently during alertness. The repetitive secondary waves were more easily attenuated than the primary wave. Horn (1960) showed that the first two components of the evoked response elicited by a flash of light are independent of the EEG pattern, and only the last component behaves in a manner similar to the EEG pattern. Arduini and Goldstein (1961) found that deafferentation, produced by raising the intraocular pressure, caused a marked enhancement of the evoked potential in the visual cortex elicited by electrical stimulation of the optic nerve. No such augmentation of the evoked potential was observed in the auditory cortex when a stimulus was administered to the medial geniculate body, A similar augmentation of electrically evoked potentials was also observed following general illumination of the retina, This strange finding may be due to the use of an unusual preparation (midpontine pretrigeminal transection of the brainstem). In this preparation, an activation pattern of the EEG is produced in darkness, while a general illumination causes a sleeping pattern (Arduini and Hirao, 1959). These authors suggested that general illumination suppresses the dark discharges of the retina; this decreases the sensory input to the RF and, consequently, the RF is released from the tonic activity. This effect must be quite striking in the midpontine pretrigeminal preparation because in this preparation the olfactory and visual pathways account for the entire sensory input into the RF. For this reason, it may be concluded that the increment of the cortical evoked potential produced by deafferentation or general illumination can be attributed to the decrease in the tonic activity of the RF. In the same Preparation, Hirao (1962) found recently that a close correlation exists between the evoked potential in the visual cortex and the EEG of the hippocampus; the second slow negative component of the cortical evoked potential may be correlated with the slow rhythms of 3-8/sec in the hippocampal EEG, while the first component is related to the rhythms of 13-/sec. Akimoto et al. (19f3l), recording from the cortex of the unanesthetized cat, studied the effects of peripheral and central arousal stimuli upon the evoked potentials and spike discharges elicited by either a flash of light or electrical stimulation (applied to the LGB or radiation fibers). Few consistent patterns were reported, but their

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most common observation was the facilitation of both mass and unitary responses. With respect to the unitary discharges, the facilitatory effect of arousal stimuli was seen more clearly in the responses to electrical stimulation than in the responses to photic stimulation. The effect of stimulating the nonspecific thalamic nuclei upon the unitary response in the cat visual cortex has been studied by Creutzfeldt and Akimoto ( 1958). Two-thirds of the cortical neurons sampled in the cat responded to both electric and photic stimuli, and the effects of both stimuli were found to be either inhibitory or excitatory. Most of the units which did not respond to the photic stimulation ( A-neurons) could be activated by stimulation of the nonspecific thalamic nuclei. Latency for the nonspecific thalamic stimulation was variable and rather long, and the spike failed to follow repetitive stimulation of more than 15-25/sec. Thus, it will be suggested that the function of these thalamic nuclei may be to modify the cortical neuron activity through multineuronal pathways. Okuda (1962) reported that the evoked response at the LGB was facilitated by stimulation of the intralaminar nuclei but inhibited by stimulation of the Nucleus ventralis anterior, which is located anterior to the intralaminar nucleus. Thus, the effects of thalamic stimulation upon the cortical responses might also be expected to depend upon the site of stimulation, Akimoto and Creutzfeldt ( 1958), however, could not demonstrate such regional differences in the cortical responses. As mentioned above, the influence of stimulation of the nonspecific thalamic nuclei upon cortical neurons appears inconsistent. Some investigators ( Creutzfeldt and Akimoto, 1958) have reported that the effect of nonspecific stimulation is either facilitatory or inhibitory, but that it is primarily independent of the neuron’s behavior to photic stimulation. On the other hand the results of Fuster (1961) suggest-in the rabbit, at least-a parallelism between the response of a cortical unit to photic stimulation and the effect of R F stimulation upon that unit. Units which responded with spike discharges to photic stimulation were found to be facilitated by R F stimulation, while RF stimulation was found to inhibit those units in which spontaneous discharge was inhibited by photic stimulation. Thus, nonspecific stimulation and photic stimulation appear to act synergically with respect to the net output of the cortical neurons.

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B. CORTICIFUGAL INFLUENCE According to Ajmone-Marsan and Morillo (1961), some of the LGB neurons respond to the stimulation of both the optic tract and contralateral visual cortex. Since the latency for cortical stimulation ranged between 1 and 10 msec, it is evident that the LGB neurons were not stimulated antidromically, but probably activated through a multisynaptic pathway. These authors have also found that if the optic tract and the contralateral visual cortex were stimulated to- . gether, either facilitation or inhibition occurred. Facilitation occurred if the optic tract stimulus (the conditioning stimulus) was applied prior to the callosal stimulus (the test stimulus). Inhibition was observed by stimulation of the reverse combination. According to Wid& and Ajmone-Marsan (19eOb), this interaction could be seen with fairly long interstimulus delays, and also even when the conditioning cortical stimulus produced no sizable spikes. It was also reported by these authors that the stimulation of the visual area I1 was much more effective than that of area I. From these findings it may be concluded that this corticifugal influence is neither a direct antidromic effect nor an antidromic effect mediated through recurrent collaterals. One may suppose that this corticogeniculate effect is mediated indirectly through the RF to the geniculate neurons, but at least the bulbar and mesencephalic RF's may be excluded since "cerveau isolk" preparations were used in these experiments. All of these facts are sufficient to demonstrate the existence of a subcortical control exerted upon the visual cortex, but none of them suggest any physiological or psychological significance for this subcortical control. Further experiments, neurophysiological as well as psychological, are needed to solve such problems. IX. Color Vision

Since Granit ( 1950, 1955b) proposed the modulator-dominator theory, the physiology of color vision has not been much advanced by studies at the level of the retinal ganglion cells. Recent progress in this field was made by Motokawa et al. (1960), who reported a complementary organization found in the receptive field of some of the carp's retinal ganglion cells. The receptive fields of such ganglion

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cells are organized so as to generate either off-discharge by stimulation of the center and on-discharge by stimulation of the periphery of the receptive field, or vice versa. I t was found that the type of the receptive fields, off-center and on-periphery, or vice versa, is not a fixed characteristic of the individual unit but that it varies with the wave length of the stimulating light (Fig. 13). For example, a unit

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FIG.13. Dependence of discharge pattern in fish retinal ganglion cell upon wave lengths of stimulus lights. Numerals indicate wave lengths (mp). W stands for white light. Stimulating light spot (0.4 x 0.4 mm in size) was centered on tip of microelectrode. Height of spike was about 200 pv. Time mark: 0.1 sec. (From Motokawa d al., 1980.)

which, when stimulated with blue-green light, shows an off -center pattern, may, when stimulated with complementary red light, change to an on-center unit. Motokawa et at. (1960)suggested that such a functional organization of the receptive field could provide a basis for color contrast (either temporal or spatial). A large number of units, whose type of discharge pattern was independent of wave length, were also found. It was suggested that the latter units contribute to luminosity contrast and other functions. Working independently, Wagner et a2. (1960) and Wolbarsht et al. (1960a,b) obtained similar results, while working on the receptive fields of the ganglion cells in the goldfish retina. They reported an interesting observation concerning selective light adaptation of on- and off-units. A steady background illumination of red light raised the threshold

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of the off-responses, while the threshold of the on-response was slightly decreased, A blue-adapting light on the other hand made the on-responding units less sensitive and the off-responding more sensitive. Using a different approach, Lennox (1958) also found wavelength-dependent differences in response at the ganglion cell level. She recorded single-fiber discharges from the optic tract of the cat while stimulating its eye with diffuse red or blue light which had been adjusted in intensity to give equal amplitude ERG responses. Latencies were also measured to determine the conduction velocity of the fibers, from which records were taken. It was found that slowly conducting fibers responded with more spikes of higher frequency to the blue light than to the red. On the other hand, the more rapidly conducting fibers responded with more spikes to red light than to blue. The cortical potentials evoked by stimulation of the eye with colored light were also studied (Lennox and Madsen, 1955; Madsen and Lennox, 1955). It was found that the latency of the cortical response was the shortest to the red flash and longest to the blue one when the intensity of these colored flashes was adjusted so that the LGB responses appeared after the same latencies. These findings seem to provide neurophysiological evidence to support the concept that information concerning different color is transmitted from the retina to the visual cortex at different velocities. However, while investigating the latency of spikes in single units in the LGB, Cohn (1956) was unable to confirm the results of Lennox. By indirect means, Motokawa and Ebe (1954) demonstrated that retinal processes could occur along different time courses depending upon the wave length used for stimulation. Recording ganglion-cell mass discharges as an indicator, they demonstrated that antidromic stimulation of the optic nerve influenced the photosensitivity of the retina. The change of the retinal sensitivity following the optic nerve stimulation was most rapid, intermediate and the slowest, respectively, when the photosensitivity was tested with red, green, and blue lights, The well-known theory of Le Gros Clark (1949) in which the basis of trichromatic vision is correlated with the 6 layers of the LGB was based on the following several observations: (a) Six welldeveloped laminae are found only in animals which can discriminate

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the colors; ( b ) Four laminae are seen in the dichromatic monkey; ( c ) Those regions of the LGB in which the laminae are not clearly distinct correspond to the peripheral retina which lacks color vision; ( d ) The central fovea, in which physiological color-blindness exists, projects into an LGB region in which one lamina is missing; and (e) A certain layer in the LGB appeared undeveloped in the monkey raised under the light which lacked some regions of spectrum. This last finding of Le Gros Clark was, however, not confirmed by Chow ( 1955). Recording from the pre- and postgeniculate single-units in the cat, Suzuki et al. (1960) studied the responses to flashes of equal physical energy, and found that the majority of the units in the darkadapted state had a sensitivity maximum which, in comparison to the absorption of rhodopsin maximum, was shifted slightly toward shorter wave lengths. This could well correspond to the “scotopic blue shift” described by Granit and Wirth (1953). In general, the spectral response curve of the LGB units was found to have main peaks at the blue and yellow regions of the spectrum, A few other humps were noticed, but these are difficult to interpret without further analysis. That some color re-coding occurs in the LGB is suggested by finding that the units which showed the twin peaks in their response curves were encountered more frequently in postgeniculate units than in pregeniculate units. De Valois and his associates (1958a, by1959, 1960) did find that the different patterns of single unit discharge were obtained while recording from different layers in the monkey LGB. From the dorsal layers only on-type units could be recorded. These responded to rather narrow bands of the spectrum: The maximum responses appeared at about 450, 510, 550, 580, and 620 mp. The units corresponding to the retinal fovea had a single maximum in their response curve, while units corresponding to the peripheral retina had a few peaks which could be shifted by light adaptation. In the intermediate layers on-off -units were recorded, and some units responded with on-discharges to one region of the spectrum, and with off-discharges to another spectral region (for instance, on- to the red, offto the blue-green light). Off-units were generally recorded from the ventral layers, and seem to be independent of color vision. It may be concluded from these observations that the laminar structure of the LGB does not represent a true trichromatic organization, but the

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dorsal, intermediate, and ventral layers do seem to be organized for hue, contrast, and luminosity, respectively. Antedating the experiments of Lennox quoted above, Motokawa et al. (1953) investigated the effects of colored lights on potentials evoked in the visual cortex of the cat. In this experiment, evoked potentials produced by diffuse illumination with flickering spectral lights were recorded, By plotting response amplitude against wave length, a maximum was found at about 500 mp. It was concluded that the evoked potential of the cat visual cortex was predominantly scotopic in nature. Also working with cats, Ingvar (1959) recorded the potentials evoked at the cortex by flickering spectral lights. He compared the cortical response with the ERG. The spectral sensitivity curve for the ERG was found to have a maximum in the region slightly below 500 mp (blue shift). When the light intensity was adjusted so as to equate the ERG amplitude, only a slight difference in amplitude was observed at the cortex with respect to wave length. This fact may indicate that, so far as scotopic vision is concerned, the retinal information is roughly conveyed to the cerebral cortex without much additional elaboration. The response-amplitude obtained for either high intensity flickering lights or under conditions of moderate light adaptation, showed a prominent maximum at 550 mp, resembling the photopic dominator curve of Granit. In addition to this main peak, a rather distinct hump appeared frequently at 430 mp, and also a small hump was seen at 610 mp. These findings indicate the possibility that the cat has a rudimentary color vision, even though behavioral studies have failed to reveal color discrimination in the cat. Motokawa et al. (1962) have recorded spike discharge of single units from the visual cortex in monkeys (Macaca ynornolgus irus, M. fuscata yukui). Spikes of radiation fibers and cortical neurons were distinguished by their configuration and further based on the properties than the radiation fibers consistently respond with many more spikes than the cortical neurons. The macula and its surrounding area were illuminated with colored lights from interference filters adjusted to equal energy. The stimulus size was 0.5" in visual angle, and its duration 0.5 sec. For each unit the receptive field was roughly determined and the stimulus spot was projected onto the center of the respective field. So far as the effect of wave length is concerned, at least 2 types

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of units could be distinguished: The first type maintained the same discharge pattern, either on, off or on-off, throughout the whole range of the spectrum; the second type changed its discharge pattern according to wave length. An example of the first type, a pure on-unit in this case, is shown in Fig. 14, in which a sensitivity maxi-

FIG.14. Spectral sensitivity curve of an on-cortical neuron of monkey. Ordinate: Wave length in mp. Abscissa: Intensities expressed in terms of transmission factors of neutral tint filters. Uppennost records represent responses to white lights at respective intensity levels. (From Motokawa et al., 1982.)

mum is found at about 480 mp and a submaximum at about 610 mp. If the total number of impulses within 0.5 sec is plotted against the wave length in such pure on-units, dominant peaks appear in the regions of red (620 mp), green (530 mp), and blue (460 mp). The response curves of some such units were single peaked, but others showed 1 or 2 submaxima (Fig. 15). In some of these units the relative heights of the dominant peak and submaxima remained unaltered over a wide range of intensities, but in others, especially those having a submaximum around 500 mp, it became progressively dominant as the stimulus intensity was reduced.

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Many of pure off-units responded to a narrow band of the spectrum in the same way as the pure on-units illustrated in Fig. 15. It is to be noted that there were many cortical neurons which responded to a very wide range of the spectrum, showing no conspicuous maximum of responsiveness.

Wavelength ( m p )

FIG.15. Spectral response curves of on-units in monkey’s visual cortex. Ordinates: Impulse number over 0.5 sec (percentage of dominant maximum). Abscissa: Wave length in mp (equal energy), Vertical bars under each curve represent 5 impulses per 0.5 sec. Curve marked by dots refers to a radiation fiber and the other curves to single cortical neurons. (From Motokawa et al., 1902.) Two examples of the second type of unit are shown in Fig. 16. The unit illustrated in A responded with on-discharge to blue-green light, but with off-discharge to red light. The example B is the reverse with respect to the discharge type. In any case, the complementary relation such as red-on and blue-green-off, or vice versa can be seen; if the spatial organization within the receptive field were studied in such units, similar complementary relation would be found between the center and periphery of the receptive field. Thus, it is apparent that these units contribute to color contrast. Some units showed a consistent maximum at about 490 mp. An example is shown in Fig. 17. This unit was of on-off type, as can be seen in the records, but units of any other types were found to have a sensitivity maximum at about 490 mp. Judging from the spectral property, they are probably scotopic units. The wave-Iength dependence of cortical neurons which was observed by Motokawa et al. (1962) generally resembles the results ob-

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FIG.17. Spectral sensitivity curve of a cortical neuron of monkey. See explanation in Fig. 14. (From Motokawa d al., 1962.)

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tained by De Valois (1960) in the LGB neurons of the monkey and by Hubel and Wiesel (1960) at the ganglion cell level of the spider monkey, and seems also to be in line with the results obtained by Lennox-Buchtal ( 1961), suggesting narrow bands of the spectral responsiveness in the monkey cortex. Although Lennox-Buchtal has announced the publication of an original paper (1962), this was not available at the time this review was being prepared. From these observations it is clear that the fundamental mechanism for color vision lies at the retinal level, the role of the visual cortex being the further elaboration of the color information with which it is supplied. The fact that the response curves of many cortical neurons have multiple peaks does not support the hypothesis that color information is conveyed through separated channels. X. Pattern Vision

One of the major visual mechanisms relating to space perception is the receptive field. The receptive field was first discovered in the frog optic nerve fiber by Hartline (1940). The fundamental organization within the receptive field of a single ganglion cell of the cat was studied extensively by Kuffler (1953). K d e r found that stimulation of the center of the receptive field of a single ganglion cell produces either on-discharges, whereas off -discharges are produced by stimulation of the peripheral zone of the receptive field, or vice versa, and on-off -discharges are recorded by illuminating the intermediate zone. The units which produce on-discharges in the center of the receptive field are called on-center units and those showing off-discharges in the center are called off-center units. The shape and size of a receptive field has been found to vary with the state of adaptation (Barlow et al., 1957). It has even been reported that on-center or off-center characteristics of a unit may change with the wave length of the stimulating light (Motokawa et al., 1960; Wagner et al., 1960). In the studies of Hubel and Wiesel (1960), the receptive field of the ganglion cell in the spider monkey was found to be larger at the peripheral retina and smaller at the fovea, and the size of the receptive field of some units being as small as 4 min of arc (20 p at the retina) in diameter. It has also shown that the receptive fields of the most units recorded in the retina and LGB are roughIy circuIar or oval in shape (Hubel and Wiesel, 1960). Suzuki et al. (1960) mapped the receptive fields of the pre- and

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postgelniculate fibers by using an automatic scanning device. The light stimulus was a flash of 30 msec in duration, thus on- and offdischarges were not distinguished. In agreement with Barlow et al. ( 1957) the receptive fields of the pre- and postgeniculate fibers were found to be approximately circular, and in most of the units a contraction of the receptive field was observed upon increasing the intensity of the adapting light. Some of the on-off-units showed rather unusual behavior during changes in the intensity of the adapting light. Increasing the intensity of the adapting light above the certain level caused a contraction of the receptive field as usual, but a further increase in the intensity of adapting light resulted in an expansion of the receptive field. Constriction of the receptive field may be explained as an attenuation of the effect of scattered light due to the moderate intensity of background illumination and also as an increase in inhibition from the peripheral zone of the receptive field. As to the expansion of the receptive field following further elevation of the adapting level, Suzuki et d.(1960) stated that under moderate adaptation the central area produces an on-response, but no offresponse appears in the peripheral zone. By raising the intensity of the adapting light, an off-response begins to appear in the peripheral zone, and these off-discharges contribute to the expansion of the receptive field. Hubel and Wiesel (1959) have shown that the receptive field of the cat’s cortical neuron differs greatly in size and shape from that of the retina. Most of the cortical cells do not respond to even illumination, but respond to a small restricted stimulus. A moving spot is more effective than a stationary one, and the effect of a moving spot of light varies depending upon the direction of movement. The concentric circular arrangement of on- and off-areas in the receptive field (commonly seen in the retina) is not found in the cortex, but one finds oblong shaped on- and off-areas which are arranged sideby-side. In those units which respond to illumination of either eye, the receptive field with respect to either eye appears identical in shape and size. Light spots which fall on the corresponding points of the either retina act synergically, but antagonistic interaction is observed following the simultaneous stimulation of the on-area in one eye and off-area in the other. These workers emphasized that, if the cortical units respond to the diffuse illumination, one must consider the possibility that such units belong to the radiation fibers.

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Baumgartner and Hakas (1962)studied the receptive fields at various levels in visual pathways of the cat. Using a stimulus pattern of vertical striations, they found that the size of the receptive fields were 0.52-0.85 mm when recorded in the optic tract units, 0.63-0.97 mm in the LGB neurons and 0.18-0.68 mm in the cortical neurons. This certainly suggests that the receptive fields of tract fibers and LGB neurons are about the same size, while the receptive field of cortical units is much smaller. The fact that the receptive field of the cortex is smaller than those of the relay nuclei seems to provide important data concerning the central mechanism for pattern discrimination. In another experiment, the same contrast pattern of black and white stripes was moved in small steps horizontally across the receptive field. Baumgartner (1981)reported that the contrast grid pattern used as a stimulus is much more effective in eliciting spike discharges than even illumination. Furthermore, as is shown in Figs, 18 and 19, the impulse number is the highest at the light-dark boundary, but not at the center of the illuminated region. The fact that the human visual sensation is highest at the light-dark boundary, is known as the border contrast; Baumgartner's observation seems to provide the neurophysiological basis for this phenomenon. If the visual angle subtended by the individual stripes in the grid pattern is less than 2"50', no border effect was observed; the spike number was the highest in the center of the illuminated area under such conditions. The cortical neurons behave similarly to the tract fibers and LGB neurons, but at the cortical neurons the border effect is seen with much narrower striations than is the case at the optic tract or LGB. It may also be noted that the border contrast effect does not appear during the first 100 msec of the train of spike discharges. This suggests that it requires at least 100 msec to produce clear border contrast. This agrees well with the observation of Katayama and Aizawa (1956), in which they demonstrate that retinal induction (cf. Motokawa, 1949) appeared after latency of 20 msec, and that 100 msec were required for the retinal induction to develop to a full size. One might suppose that such an elaborate function as border contrast would be attributable to some complicated neuronal network located distal to the retinal ganglion cells. However, Ratliff and Hartline (1959), recording from the optic nerve fiber of the Lhulus o m m t a b , demonstrated a similar phenomenon of border

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FIG.18. Contrast phenomenon observable in responses of an on-center unit of cat's optic tract. Stimulus pattern of vertical striations was moved horizontally step by step across receptive field. A part of stimulus pattern is shown on left relative to center of receptive field marked by its horizontal diameter. Response at each position of stimulus is shown on right. (From Bamgartner, 1961.)

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contrast in this species which lacks any such elaborate distal neuronal network. In an earlier experiment (Hartline et al., 1956), the discharge of impulses in a single optic nerve fiber, originating from an ommatidium of the lateral eye of Limulw, was shown to be inhibited by illumination of neighboring ommatidia. The decrease in discharge frequency (inhibition) due to illumination of the surrounding area was found to depend upon ( a ) the intensity of light SO)

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falling upon the neighboring area (roughly proportional to the logarithm of its intensity), ( b ) the area (the larger the area the greater the inhibition), and ( c ) the distance from the recording site. The distance at which the effect could be observed extended for several millimeters. It was emphasized that lateral inhibition alone can account for the border contrast effect which was found in the Lzlmulus eye. Recording the slow potentials from an inverted fish retina, Motokawa et al. (1959a), and Motokawa et al. (1959b) found that a negative slow potential was recorded outside the illuminated area

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whereas a positive potential was obtained within the illuminated part (see Section I1 on the retina). It was demonstrated that this negative slow potential possesses all the characteristics (for example, intensity dependence, area dependence, spatial distribution, etc. ) which had been reported by Hartline et a2. (1956) concerning lateral inhibition in the Limulus eye. Motokawa et uZ. (1961a, b ) have also shown a rather close correlation between the slow potential and the spike discharge of those ganglion cells which show contrast organization (cf. Figs. 1and 2). An example of such correlation is shown in Fig, 20 in which spike

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FIG.20. Simultaneous recordings of slow electric responses (lower beam in inset) and unitary discharge of fish ganglion cell (upper beam in inset). Magnitudes of slow responses and relative numbers of impulses (RNI)are plotted as il function of distances between electrode tip and center of light spot. Open and filled circles connected with solid line represent on- and off-discharges, respectively, and filled triangles connected with broken line represent slow responses. Size and position of stimulus are shown by shaded square. Intensity and duration of stimulus were 1250 lux and 0.4 sec, respectively. (From Motokawa et al., 1961b.)

number and the amplitude of the slow potential (negative and positive) are plotted against common abscissa (distance from the center of illuminated area). By depth measurement, these workers concluded that the negative slow potentials outside the illuminated part are probably produced in the secondary neuronal layer which involves bipolar, horizontal, amacrine, and Miiller cells, and that the

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generation of ganglion cell discharges may possibly be modified by these slow potentials. By recording single unit discharge at the various levels along the visual pathway, studies on pattern vision will be possible when simple stimulus patterns are used. Complex patterns have not yet proved practical for such use. Motokawa and Ogawa (1962), in a simple approach to this problem, have mapped the retinal responses

FIG.21. Distribution of positive (upward deflection) and negative (downward deflection) potentials within and outside retinal area illuminated by circuIar, triangular, or square stimulus (experiments on isolated fish retina). Illuminated parts are dotted. Solid line contour outside illuminated area represents zero-potential line which separates positive potential field from negative potential field. For mapping such distribution, stimulus pattern was moved in small steps along scanning lines, while the microelectmde was fixed at a point of retina, to make short illumination at each position, (From Motokawa and Ogawa, 1962.)

elicited by light stimuli of various patterns on the receptor surface of the carp’s retina. By use of an automatic scanning device, adjacent portions of the retina were represented on adjacent portions of the oscilloscope screen. Thus, when simple patterns were projected on the retina the distribution of local responses was shown in a topographical manner on the oscilloscope screen. Figure 21 shows the spatial distribution of responses obtained by projection of a circle, triangle, and square on the retina. The illuminated part, in which

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the positive slow potentials (upward deflection) were recorded, is indicated by dots. The positive responses were recorded slightly outside the dotted area, and still further outside, negative responses were recorded. The continuous lines surrounding the dotted area were drawn by connecting the zero-potential points where the slow potentials changed their polarity. The zero-potential lines are almost identical in shape to the stimulus pattern in case of a circle or triangle but it should be noted that the zero-potential line appeared to be cruciform for the square pattern. This result agrees well with the results of experiments on the human retinal induction (Motokawa, 1949). When equi-induction lines were constructed for circular, triangular, and square stimuli, they appeared about the same contour as shown in Fig. 21. By using the same recording procedure and the figures of the Miiller-Lyer illusion as stimulus patterns, it was found that the horizontal extent of the zero-potential line obtained from the two different stimulus patterns are greatly different in length (Fig. 22). The differences in the length of the zero-potential line do

FIG. 22. Retinal fields of slow potentials mapped in the same way as in Fig. 21 by using Miiller-Lyer figures as stimulus pattern. Illuminated parts are dotted. Positive potential is directed upwards and negative one downwards. (From Motokawa and Ogawa, 1982.)

seem to provide a peripheral basis for this well-known illusion. It is perhaps premature to offer this as the sole basis of length judgements, but it is obvious that the necessary spatial distribution of information is available at this peripheral level. Still another phenomenon related to pattern vision may be demonstrated at the retinal level. Motokawa d al. (1961a) found

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that the regions surrounding the illuminated area from which negative slow potentials are recorded become much narrower when the intensity gradients due to stray light are reduced (such as under conditions of strong background illumination ) . This certainly suggests that these negative potentials may act to counteract the effect of stray light and, thus, increase the border contrast information which is transmitted centrally. It is evident that “contrast” is one of the major factors in pattern vision, but some other factors which may play an important role for pattern vision should aIso be considered. We have fulIy discussed an on-center or off-center unit which contributes to contrast effect, but we have also encountered a pure on- or pure off-unit which is considered to be independent of contrast. Lettvin et ul. (1959) and Maturana et ul. (1960) have distinguished 5 different types of the optic nerve fibers in the frog: (1) sustained contrast detectors which discharge promptly and continue discharging if the sharp edge of any shape of an object either lighter or darker than the background moves into their field and stops; ( 2 ) net convexity detectors which respond to a stimulus with a convex edge but not to a stimulus with a straight edge. Such units have informally been designated as “bug detectors,” and their function in the economy of the frog is quite obvious; ( 3 ) moving edge detectors which respond to an edge only if that edge moves but not otherwise; ( 4 ) net dimming detectors which respond to a sudden reduction of illumination; and (5) others which respond to darkness over a wide area and for a long time, and thus do not seem to have distinct receptive fields. The units of class 1 correspond to the on-units designated by Hartline, those of class 4 to the off-units, and class 3 to the on-offunits. The unique categories in the classification of Lettvin et al. (1959) appear to be classes 2 and 5, the former being dependent upon a particular spatial characteristic of the stimulus and the latter dependent upon darkness independent of shape, Another aspect of this work which merits special comment is the form in which pattern information is transmitted from the retina. For the edge detectors and convexity detectors, in particular, it appears that the message from individual ganglion cells include information concerning the presence of edge or angles. Since the only information present at the receptors is the relative presence or absence of illumination, the re-

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sponse pattern recorded certainly implies that some meaningful coding of visual information occurs between the receptor and ganglion cells in the retina. The question, then, is the significance of the highly ordered topological projection of the retinal surface upon the cortical visual center (see Sections V and VII, on lateral geniculate body and visual cortex) in pattern vision, for it has long been assumed that pattern vision is ultimately dependent upon the topological relations maintained within the central visual projections and that the information carried along these projections, although perhaps coded in some simple form, is nearly the same as that present at the receptor cells. The conclusion that all coding of pattern information occurs in the retina is obviously extreme and incorrect. It should be pointed out first that the results of Lettvin et al. (1959) were obtained from the frog. In the frog almost all optic fibers terminate in the optic lobe (homologous to the Superior colliculus in mammals) and there is no visual cortex in the amphibia; it might well be expected that more complex coding in peripheral structures is necessary in this species which lacks the higher integrative function of the neocortex. In the second place, the receptive field of the convexity and edge detectors is limited to only a small portion of the visual field and, although a particular pattern characteristic may be conveyed by the units in question, the topological arrangements of the projection system are required to provide information concerning the parts of the visual field in which such particular patterns occur. Finally, it must be recalled that there is abundant evidence indicating that, for species which have a visual cortex, the visual processes appear at the cortex to be much finer and more delicate than at more peripheral loci in the visual pathway. It is hoped, however, that future research techniques will clarify the coding and transmission functions performed at both central and peripheral portions in the visual system. XI. Summary

1. It is generally believed that, following a photochemical reaction, a conductive nerve impulse is created in a secondary neuronprobably a bipolar cell-although the intervening processes are not known. Several investigators have designated the potentials generated in or near the receptor cell layer as “receptor potentials”; such

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potentials may partially reflect the photochemical event and the propagated impulse. Spike discharge recorded at the inner nuclear layer has been discussed with respect to its origin. 2. Lateral conduction observed at the retinal level and its physiological significance for the visual function such as contrast have been discussed. 3. The fiber analysis of the optic nerve has been compared with recorded action potentials. It has also been shown that recent data suggest a wave length dependence of the discharge pattern. 4. Evidence has been presented for the existence of the centrifugal fibers which carry impulses toward the retina, but the functional role of such fibers is still unknown. 5. The anatomy and the electrical activity of the lateral geniculate body have been described and the pre- and postgeniculate spikes were also mentioned. 6. Nonspecific effects, particularly the effect of stimulation of the reticular formation, upon the specific responses were described. 7. The evoked potential and spike activity in the visual cortex, corticipetal control from the subcortical nuclei and corticifugal control over the lateral geniculate body were mentioned. 8. Describing the responses to colored stimuli at various levels in the visual pathways, an attempt has been made to provide a neurophysiological basis for color vision, but no indisputable conclusion can be drawn from our present knowledge. 9. The receptive fields with respect to spike discharges at each level in the visual pathways were compared and their functional organization was described, aiming to furnish the neurophysiological basis for pattern vision. The electrical fields of the slow potentials in the retina also have been presented to provide a functional significance for pattern vision. REFERENCES

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ION FLUXES IN THE CENTRAL NERVQUS SYSTEM'

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By F J . Brinley. Jr

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Department of Physiology. Johns Hopkinr School of Medicine Baltimore. Maryland

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I Theoretical Section . . . . . . . . . . . . . A. Assumptions Underlying Tracer Experiments . . . . . B. Radiation Effects . . . . . . . . . . . . . C. Kinetics of Ion Movement . . . . . . . . . . D . Summary . . . . . . . . . . . . . . . I1 Extracellular Space and the Ionic Composition of Brain . . . A. Extracellular Space . . . . . . . . . . . . B. Ionic Composition of Neurons in Brain . . . . . . . I11 Exchangeable and Nonexchangeable Ions . . . . . . . IV. Spreading Cortical Depression . . . . . . . . . . A. Potassium Release during Spreading Depression . . . . B. Source of Potassium Release during Spreading Depression . C. Postmortem Potassium Fluxes . . . . . . . . . D. Sodium and Chloride Movements . . . . . . . . E. Modification or Prevention of Spreading Depression by Divalent Ions . . . . . . . . . . . . . . F. Mechanism of Production and Propagation of Spreading Depression . . . . . . . . . . . . . . . G. Recovery Processes . . . . . . . . . . . . V Effects of Drugs on Membrane Permeability of Central Nervous System Cells . . . . . . . . . . . . . . A . Local Anesthetics . . . . . . . . . . . . . B. y-Amino-N-butyric acid . . . . . . . . . . . C. Metabolic Inhibitors . . . . . . . . . . . . VI Summary . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . .

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The purpose of this article is to present a critical review of recent studies on ion fluxes in brain tissue. Because the interpretation and significance of data obtained from such experiments depend heavily upon the experimental techniques and preparations used. an attempt will be made to discuss the advantages and limitations of the experimental methods as well as the results obtained

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Data obtained from experimental studies of ionic fluxes can be most easily understood in terms of the theories of ion transport derived from studies on peripheral nerve and other tissues. For a detailed account of these theories and the experimental work upon which they are based, the reader is referred to other reviews (e.g., Hodgkin, 1951, 1958; Shanes, 1958a,b). The discussion of transport phenomena, both active and passive, presented here will be largely limited to those aspects peculiar to the central nervous system and particularly the cortex. Because of the frequent use of radioisotopes in studying ion movements, the assumptions underlying tracer experiments are discussed. The basic mathematical relations governing ion movements across single cell boundaries are presented and several simple examples of ion fluxes in multicellular tissues are considered. A brief discussion of the concept of cell permeability is also included. Various anatomical and histological studies of the extracellular space in brain are considered. An attempt is made to correlate the data and explain some apparent discrepancies. A knowledge of the amount of an intracellular ion which can exchange with extracellular ions of the same species is necessary in order to calculate membrane potentials and ionic fluxes, both of which depend upon the concentration of freely exchangeable ions inside the cell. Such data also give information concerning the extent to which intracellular ions may be held in some sort of a chemical bond. Several laboratories have reported data which indicates considerable variation in the amount of potassium (and other ions) which can be exchanged in different nervous tissues, and these experiments are considered. Most of the experimental studies of ion fluxes in the brain have been carried out in an effort to explain the phenomenon of spreading cortical depression. These experiments are considered in relation to In preparing this article the author has had the advantage of constructive criticism from a number of individuat, particularly Drs. P. W. Davies, A. J. de Lorenzo, W. H. Marshall, L. J. Mullins, R. A. Sjodin, and K. L. Zierler. He is particularly grateful to Dr. H. Pappius for providing him with some data prior to its pubIication. Preparation of the manuscript was expertly done by Miss P. Dumschott. The costs of preparing this article have been partially met by U. S. Public Health Service Grant B-4927.

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possible mechanisms for the propagation of spreading depression and the recovery of the cortex from it. Many studies of drug action on central nervous system activity have considered chiefly changes in spontaneous or evoked electrical activity. The study of drug induced changes in membrane permeability is an additional, useful tool in analyzing the effects of drugs on nervous activity. A few examples of such experiments and the preliminary conclusions drawn from the results are presented. I. Theoretical Section

Before the availability of artificial radioactive isotopes, studies on cell permeability usually necessitated experimental procedures which caused a net gain or loss by the cell of the material under investigation. Such experiments characterize the permeabiIity of the cell membrane under the circumstances of a changing internal environment but provide only inferential information about membrane permeability of the cell in a steady state with respect to the substance being studied. The use of radioactive tracers has made possible direct observation of the transmembrane ion movement when there is no net particle movement and has also greatly increased the accuracy and ease with which the nonsteady state can be studied. A. ASSUMPTIONS UNDERLYING TRACER EXPERIMENTS

1. M a s Efect One of the most important assumptions underlying all tracer experiments is that the experimental tissue cannot distinguish the radioactive from the stable isotopes and that all isotopes of a given element are treated in exactly the same way, This crucial assumption has never been adequately tested but it has a plausible physical basis, at least for isotopes of fairly high atomic number. Since radioactive elements differ from stable isotopes of the same atomic number only in the number of uncharged neutrons contained in the nucleus, the valence orbital electrons are essentially unperturbed by the presence of an unstable nucleus and hence the van der Waals’ and electrostatic interaction energies of the radioactive species with other ions are virtually the same as the interaction energies of the stable isotope. There is, of course, a mass difference but this is usually small (X total mass) for most isotopes used in studying mem-

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brane transport phenomena. An important exception is tritium which has 3 times the mass of the hydrogen atom. The use of tritiumlabelled water in diffusion experiments has revealed an “isotope effect” due presumably to the mass difference (Wang et aZ., 1953). 2. Concentration Profile Efect It should be pointed out that the data derived from tracer experiments are obtained by observing the exchange of radioactive ions and molecules with the nonradioactive species. It has been widely assumed that the conclusions drawn from such experiments also apply to the more physiological situation, i.e. exchange of nonradioactive material on one side of a membrane with nonradioactive material on the other side. Recently, however, the use of data obtained from tracer experiments to calculate nonradioactive fluxes has been questioned in several papers (Nims, 1959; Tasaki, 1960; Yeandle, 1961). The objections appear not to be related to a possible isotope effect (discussed in the first paragraph of Section LA) but rather to the fact that the concentration profile for radioactive and nonradioactive species inside the membrane may not be identical in certain experimental situations. The validity of these objections and the extent to which present conclusions concerning unidirectional membrane fluxes may require modification, has not yet been settled. It seems unlikely, however, that any of the qualitative inferences concerning ion behavior drawn from tracer experiments presented in this article will be invalidated.

B. RADIATIONEFFECH 1. Tissue Radiution There appear to be only a few estimates of the tissue dose of radiation delivered to the usual isolated preparations during tracer experiments. The exact dose, of course, depends upon the isotope used, the length of exposure, specific activity of the radioactive solution, etc.; however, calculations of tissue dose, made by Keynes and Lewis (1951) and Brinley and Larrabee (196l), suggest that the total tissue dose may be of the order of a few tens of rads to several hundred rads. Such doses are much less than those required to produce acute changes in function. About 100 krads of X-rays are re-

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quired to produce acute changes in membrane potential in frog sartorius muscle (Darden, 1960); at least 25 h a d are required to affect NaZ4exchange in squid nerve (Rothenberg, 1950) (1 krad equals lo5 ergs absorbed per gm tissue). It is doubtful, therefore, if the usual doses of radiation delivered during tracer experiments are sufficient to produce rapid alterations in those physiological functions generally considered as evidence of viability.

2. Changes in Composition by Transmutationof E E m n t s Since a radioactive isotope may be transmuted into an entirely digerent element as it decays, it is theoretically possible for the concentration of a particular element either in the bathing solution or in the tissue to change with time during a tracer experiment (considering all of the isotopes of an element as one chemical species). However, the molecular specific activity (i.e., ratio of radioactive atoms to total atoms of a given element) in biological experiments is usually so small that possible transmutation effects can be ignored.

C. FORMULATION OF THE KINETICSOF IONMOVEMENT AND INTERPRETATION OF FLUXDATA A number of comprehensive articles and reviews have been published which deal with the mathematical methods necessary for a general analysis of tracer movements in multicompartment systems (Robertson, 1957; Solomon, 1960). For this reason, the discussion in the following section will be limited to a consideration of the model systems which have been used to interpret tracer fluxes in various nerve tissues. The only initial or boundary conditions considered will be those usually occurring in such experiments. A further restriction, not always realized in practice, will be the assumption that the system is in a steady state and that there is no net gain or loss of the material under study by the tissue. A more general discussion of the formulation of the differential equations used in tracer experiments can be found in the reviews by Robertson ( 1957) or Solomon (1960) and an article by Keynes and Lewis ( 1951) . 1. Single Isolated Cell a. Outflux. The equations necessary to interpret tracer flux data can be introduced by considering an idealized situation: A single isolated cell containing radioactive tracer is placed in a very large

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reservoir containing nonradioactive bathing solution (Fig. 1A) , The specific activity of the radioactive material (i.e., counts per minute per mole) inside the cell is considered to be uniform throughout the interior, (The validity of this assumption is not known for most cells; however, in the case of the giant axons of the squid, Hodgkin and Keynes (1953)have shown that the diffusion constant for sodium A

k23

FIG.1. Three simple kinetic systems ( illustrated schematically) frequently used in analyzing the results of tracer experiments in biological tissue. Each of the systems illustrated in A, B, and C is considered to exchange with a well stirred reservoir so large that the concentration of tracer on the outside surface of the compartment is constant. T h e symbol Y J ( 0 ) refers to the initial amount of tracer within the jth compartment; the symbol kl or kt,i refers to the rate constants into or out of the appropriate compartments i and j. In B and C, compartment 1represents the intracellular space and compartment 2, the extracellular space.

and potassium in axoplasm is essentially the same as for diffusion in sea water. If their conclusions apply also to mammalian tissues, and particularly to cells with small dimensions such as occur in the central nervous system, then any local excess of ion which occurred inside a cell would become uniformly redistributed within milliseconds.) During any time interval a certain number of radioactive particles will strike the inside of the membrane. This number will depend, among other variables, upon the concentration of internal radioactive ion, C , , . The number which leave the cell per unit area will be a certain fraction of those which strike it. Lumping all of the other variables except concentration into one constant, P, we can write for the outflux of radioactive particles per unit area: m, = PCi,

(1) The rate at which radioactive particles can leave a cell of surface area, A, is then:

ION FLUXES IN THE CNS

-=

dt

189

-Am, = -APCi.

Yin is the intracellular radioactivity. Since C,, = Y i , / V where V is the cell volume, Eq. (2) may be rewritten as:

(The bath in which the cell is immersed is considered so large that no radioactive ions ever re-enter the cell.) If the experimental conditions are such that A, V, and P remain constant during the experiment, then Eq. (3) can be integrated directly to give: Yin = Yin(0)e-k' (4) where k = A P N , and Y , , ( O ) is the initial intracellular radioactivity. Thus, the amount of radioactivity remaining inside the cell is a single decreasing expotential function of time. By measuring the amount of radioactivity inside the cell as a function of time after it has been placed in a nonradioactive medium, one can calculate k, the rate constant for loss of radioactivity and, hence, evaluate the permeability coefficient. b. Influx. The equations for gain of radioactivity by a single cell, initially containing no tracer, placed in a radioactive media can be obtained by arguments similar to those presented for outflux. In the case of outflux, it was possible to keep the concentration of tracer on the outside of the membrane at a negligible value by using a very large reservoir of bathing solution. A comparable simplification is not possible for influx so that one must consider the net change in tissue radioactivity to be the difference between the amount entering and the amount leaving. The amount entering can be taken to be a constant, X, if the external specific activity remains constant. The amount of tracer leaving is proportional to the concentration of tracer already inside. Thus, we can write:

The solution of this equation for an initial intracellular radioactivity equal to zero [i.e., Yi.( 0) = 01 is:

Y*(t) = Y h ( a J ) ( l - e - k l )

(6)

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where k = A P N . One notes that the regression of In (1-[ Yi,( t ) / Yin( ) 1) on t is linear with a slope of 4. However, the use of this logarithmic function requires a knowledge of the steady-state level of intracellular radioactivity, Y,,( 00 ). In some experimental situations, it may not be possible to obtain t h i s value. In such a case, Eq. ( 6 ) cannot be used to determine k. However, the rate constant can s t i l l be obtained in the following manner. Differentiating Eq. ( 6 ) we have: dYk/dt = - k Y i n ( m ) € - k ~ (7) In (--dYin/dt) = In [-IcYi,(.a)] - Ict (8) Thus, by plotting the negative logarithm of the slope of the uptake curve for tissue radioactivity against time of uptake, one can still determine k, the rate constant. Such measurements of k are usually not very accurate because of experimental inaccuracy in determining the slope. In the steady state, the rate constant for radioactive ion accumulation should be the same as that for tracer loss as can be seen by comparing Eq. ( 4 ) and ( 6 ) . c. The Pemability C o e . The constant P has the units “cdsec” and can be regarded as a measure of the ease with which a particle can move through the membrane. The magnitude of this permeability coefficient depends upon such factors as temperature and the composition of the membrane, (i.e., amount of fixed charge, size of aqueous channels, etc.,). In the case of ions, the apparent permeability coefficient, as defined above, depends heavily upon the transmembrane potential. A more satisfactory formulation in this situation is to relate the permeability coef6cient ( P ) to the electrochemical mobility of the ion ( p ) and to make the potential dependence explicit in the flux equation. In such a case, we have: exp (-Fu/RT) in which p - R T p P (9) 1 - exp (-F’U/RT) F a where F is the Faraday constant, R, the gas constant, 21, the transmembrane potential, and T the absolute temperature. P (in “cm/ sec”) is related, as shown, to the mobility, p, of the ion, the partition coefficient, p, for the ion between the solution and the membrane, and the thickness of the membrane, a, through which permeation occurs. A similar equation obtains for influx. For a further discussion

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of these equations and their derivation, the reader is referred to articles by Hodgkin and Katz (1949) and Keynes ( 1951).

2. Multicompartment Systems The above section was concerned with presenting some of the important equations relating the exchange of radioactive tracer between a cell and its environment. The idealized case considered above of a single cell surrounded by a large reservoir is rarely met in practical experimental work. In most excised tissues, the cells are surrounded by a variable amount of extracellular space through which ions must pass to reach the bathing solution. Even in so-called single-cell preparations, such as lobster or squid axons, the cell is surrounded by a thin layer of Schwann cells and possibly some connective tissue. The interpretation of experimental flux data obtained from systems which contain an extracellular space will be considered below. a. Parallel Compartments. Consider the cell shown in Fig. 1B. In this example, the cell and its extracellular space are arranged in parallel, i.e., each compartment can exchange directly and independently with the reservoir. The total radioactivity of the system is simply the sum of that contained in each compartment, and the equation analogous to Eq. (4), describing tracer loss from the system, is just: Yi,(t) = Y1(0)E+f

+ Yz(O)E-kgt

(10)

where Y l (0) and Y , ( 0 ) are the initial amounts of tracer in compartments 1 (intracellular ) and 2 (extracellular) . The rate constants for the respective compartments are k , and k,. A semilogarithmic plot of such a function is shown in Fig. 2A using values for the various parameters that one might expect to find for sodium exchange from a neuron surrounded by a glial cell layer acting as an extracellular space. Since k , is assumed larger than k,, the second term of Eq. (10) approaches zero more rapidly than the first and for sufficiently large values of t the time course of outflux is described by only the first term of Eq. (lo), i.e.:

Yi, = YI(O)€+'C

(11)

for large t. Therefore, the slope of the terminal linear portion of the curve shown in Fig. 2A gives the rate constant 12,. Extrapolation of

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t B

lot "0

I0

20

...

30

MI N I I T E S I I._

I _ -

40

50 0

10

20 30 MlNllTFS

...,..-. --

40

FIG. 2. A. Semilogarithmic plot of loss of tracer ions from a parallel compartment system. B. Semilogarithmic plot of loss of tracer ion from a series compartment system. Parameters used in calculating the equations plotted in Fig. 2 are as follows: The intracellular ion [ Y , , ( O ) ] constitutes 56.5% of total ion in the system (corresponds to the relative amount of intracellular sodium contained in a spherical neuron, 80 fi in diameter, surrounded by a glial cell layer, considered as an extracellular phase, 1 p thick. The internal sodium concentration is 15 mmolesAiter, the extracellular concentration is 150 mmoles/fiter. The time constant for exchange of the intracellular compartment, operating independently, is 30 min. The time constant for exchange of the intracellular compartment, operating independently, is 3 min. At time zero, the specific activity of tracer is the same in both compartments.

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the terminal linear portion of Eq. (10) back to zero-time gives Y,(O) as shown by the dotted lines in Fig, 2A. Subtraction of the first term from the original curve and replotting on logarithmic coordinates yields a straight line representing the second term in Eq. (10). The slope and intercept of this derived straight line yield Icn and Y , (0), respectively. In this way, a purely graphic analysis of flux data from a parallel two-compartment system permits unambiguous identification of the rate constants and size of the intracellular and extracellular compartments. Much of the early data on ion fluxes in tissue were analyzed as outlined in the preceding paragraphs, tacitly assuming that the extracellular space was in parallel with the intracellular space. In many cases, however, the extracellular space is more adequately represented as a compartment in series with the intracellular space because material leaving an excised axon, for example, must first traverse the surrounding Schwann cells and connective tissues before it reaches the external bathing medium. This kinetic situation is illustrated in Fig. 1C. In this case the differential equations describing tracer movement in the system are considerably more complicated than the parallel compartment case.

dx2 dt- -

+kl,2X1

-

(k2J

+ k2,s)Xz

The k i , j refer to the rate constants for the compartments illustrated in Fig. 1C. The symbol, Xj refers to the specific activity of the isotope in the jth compartment. The total amount of tracer in compartment j is given by Y,= X,V& (I-', the compartment volume, and Gin,, the concentration of chemical species within the compartment, remain constant during the experiment). The solutions of this system of equations can be obtained by straight forward mathematical techniques (Robertson, 1957). For the case of a tissue previously soaked in radioactive media until both the intra- and extracellular compartments are equilibrated with tracer and then transferred to a nonradioactive media, the total radioactivity is given by:

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where

and

Yini( 0 ) is the initial radioactivity in the jth compartment. This function is plotted in Fig. 2B. Although the solution looks similar to that obtained for the parallel compartment case in that it consists of two exponential terms, the coefficients of the exponential terms and the lambda’s are functions of the rate constants of both the intra- and extracellular Compartments. When a graphical analysis, of the sort described above for the parallel compartment case, is applied to Eq. ( 13), the right hand side of the equation can be separated into “fast” and “slow” compartments. The rate constants and relative sizes of the fast and slow compartments can be determined as already described. However, these quantities are not equal to the corresponding parameters for the intra- and extracellular compartments of the model. In principle, the rate constants for the individual compartments can be determined from Eq. (14)if XI and A, have been determined graphically and if /3 is known. The constant, /3 can be evaluated provided the relative sizes of the extra- and intracellular spaces are known (see Section 11). In practice, however, it is frequently difficult to measure the rate constant of the fast component accurately, because of the rapidity with which the fast component exchanges. Equation (13) is plotted in Fig, 2B for the same parameters as used in Fig. 2A. As can be seen from the figure, the assumption that the size of the extracellular space (which is the same as that for the parallel compartment case) is equal to the value of the slow compartment extrapolated to zero time can lead to serious errors. This

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situation is also discussed by Huxley (see Solomon, 1960). He gives a formula for calculating the size of the extracellular compartment, given the size of the fast ( f ) and slow ( s ) compartments and the XI and XB. Using the present notation, his equation is:

Although the arithmetical complexity of Eq. (13) is already rather formidable, it may not in all cases adequately describe the loss of tracer from an equilibrated tissue. This equation was obtained assuming that the extracellular space (compartment 2) was a well-mixed compartment whose exchange with the reservoir could be described by a single rate constant, k2,3.Actually, material passes through the extracellular space by diffusion, and significant concentration gradients may occur within the extracellular space, especially if the rate at which the tracer can exchange with the intracellular space is relatively rapid compared with diffusion in the extracellular space. In such a case, the time-course for tracer movement into or out of a tissue must be obtained by a solution of the diffusion equation:

(in which D is the diffusion coefficient) with the appropriate boundary conditions, Some solutions of analagous heat flow problems in

composite cylinders have been given by Jaeger (1941). However, the numerical evaluation of the parameters involved is extremely tedious and it is doubtful if the accuracy of most experimental kinetic data is such as to justify the additional labor of evaluating the coefficients. Moreover, it is a moot point as to whether the simplified diffusion model used in order to obtain a tractable mathematical solution offers a significantly better fit to the kinetic data than does the series compartment model.

3. Multicellular System When the time-course of tracer outflux from a whole tissue has been examined, it frequently appears to approximate the theoretical curves shown in Fig. 2B, e.g., rat muscle (“extensor digitorum longus,” Zierler, 1960), rat sympathetic ganglion (Brinley and Larrabee, 1961), frog sciatic nerve (Shanes and Berman, 1955b). It has

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sometimes been assumed that the tracer outflux from each cellular element is identical and that the observed outflux from the whole tissue is simply the sum of the contribution from the individual cells. Such an assumption may lead to unjustified conclusions if the cell population is heterogeneous either with respect to size or membrane properties. The total outflux of tracer from a population of elements such as those considered in Section C.1.a is, by extension of Eq. ( 4 ) simply:

(17) where N , is the number of elements with volume V,.If the cellular elements are considered spherical for mathematical simplicity and if the intracellular concentration C1,(0) is the same for all elements, then:

i

It can be seen that if the elements contributing to the outflux differ either in size (variable rl ) or membrane permeability (variable Pj ) , then the outflux will not follow a single exponential time-course. However, in order to observe definite nonlinear behavior in a semilogarithmic plot of kinetic data, it is necessary to follow the course of tissue tracer loss (or gain) for a time interval sufficient to allow the tissue radioactivity to change by at least one order of magnitude when the time constants for the compartments differ by a factor of only two or three. Such precision is usually not obtainable in biological tracer experiments and hence the outflux curves may appear to be linear when they really are not. One can, of course, always calculate a rate constant for ion exchange which characterizes the tissue as a whole using the formalism of Section C.2. However, the relation of this constant to tracer movement across any specific cell membrane need not be obvious. The presence of an extracellular space complicates the situation still further. An attempt has been made by Harris and Burn (1949) and Keynes (1954) to handle the problem of dif€usion in the extra-

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cellular space by considering the intracellular space as a source (or sink) distributed uniformly throughout the tissue and solving the following set of equations:

aCin - - aCex - @’in

09b) at where C,, and Cin are the concentrations of tracer in the extra- and intracellular spaces, c is the volume fraction of the extracellular space, a and p are rate constants for tracer movement across the cell membrane. A complete discussion of the solution of this system of digerential equations has been given by Crank ( 1956). The formulas obtained by solving Eq. (19) with the appropriate boundary conditions are extremely cumbersome and the experimental flux data are usually not sufficiently precise to justify fitting to the solutions of Eq. (19). However, the formulas can be used to correct rate constants calculated from the slope of outflux (or influx) curves for slowness of diffusion in the extracellular space. Keynes (1954) has used a simplified form of the solution to estimate the effect of diffusion in the extracellular space on the rate constant for sodium exchange from muscle fibers contained in a small toe-muscle of the frog. He found that the rate constant for sodium outflux calculated from an equation equivalent to Eq. (10) was about lm lower than the “true” rate constant calculated from solutions to Eqs. ( 19). Under the conditions prevailing in the central nervous system, i.e., very small extracellular space and rapid tracer exchange, the rate constants for the intracellular compartment calculated from Eq. ( l o ) , or even Eq. (13), will probably be considerably less than the true rate constants for this compartment. It also follows from a consideration of these models (and can be seen even for the simple series compartment model. Fig. 2C) that changes in the ratio of extracellular to intracellular space can change the observed rates constants for the system, even when the rate Constants for the individual compartments themselves do not vary [see Eqs. (13) and (14)]. This suggests that experimental treatments (such as the application of metabolic inhibitors to nervous tissues) which alter the “whole tissue” rate constants should be evaluated for possible effects on the ratio of intra- to extracellular space before the

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results can be ascribed unambiguously to changes in the transport of material into or out of the intracellular mass.

D. SUMMARY The histological complexity of brain tissue precludes an accurate calculation of transmembrane fluxes from kinetic data obtainable with current experimental techniques on whole brain or tissue slices. Nevertheless such data provide qualitative information (considered in Sections 111, IV, and V of this review) which is generally consistent with the conclusions reached from studies of simpler preparations such as the squid axon, II. Extracellular Space and the Ionic Composition of Brain

A. EXTRACELLULAR SPACE In order to calculate transmembrane ion fluxes from the observed rate constants [see Eqs. (4) and (13)],one must know the intracellular ionic composition. In tissues which possess both intracellular and extracellular spaces, the determination of the total amount of ion in the tissue does not suffice to give the intracellular concentration because of the contribution of the extracellular ions to the total. The size of the extracellular space has been determined for many tissues by soaking an excised preparation in a bathing solution to which has been added-in amounts too small to affect electrolyte concentrations either by osmotic imbalance or pharmacological action-various substances which ideally are assumed to diffuse through the extracellular space but not across cell boundaries into the intracellular space. The extracellular space can also be determinded in v i m by injection of the test substance into an animal, usually by the intravenous or intraperitoneal route. After a suitable interval, the animal is sacrificed and the tissues analyzed for the test substance. If one assumes that the concentration of test substance in the extracellular space inside the tissue is the same as the concentration in the bathing solution, then one can calculate the volume of the extracellular space (ml/gm of tissue) from the known bathing-sohtion concentration and the amount of material contained in the tissue. Since different substances appear to penetrate tissues to varying

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degrees, Shanes and Berman (1955a) have made the reasonable suggestion that the volume accessible to a particular molecule be designated in terms of the substances used rather than be referred to as the “extracellular space.” Thus, the volume of tissue into which the inulin molecule can diffuse would be called the “inulin space.” 1. Definition of Extracellular Space

The expression “extracellular space” has been used in various ways by different authors. For example, some authors have considered glial cells to be part of the extracellular space (Woodbury d al., 1956; Woodbury, 1958). Manery considers the connective tissue to be part of the extracellular space and both Manery (1952) and Shanes and Berman (1955a) also consider myelin to be in this category. The electron microscopists have frequently used the expression “extracellular space,” in its strictest sense, to refer to the volume of brain lying exclusively outside of cell membranes. Horstmann and Meves ( 1959), De Robertis and Gerschenfeld ( 1961) , De Lorenzo (1961) and others have reported that the extracellular space, defined in this way, comprises only a narrow cleft of the order of loo300 A thick between cells in the cortex. Horstmann and Meves (1959) measured the total area of clefts in a number of electron micrographs of the brain of a species of shark, Scylliorhinus canicula, and compared it with the total area of the micrograph. In this way they calculated the extracellular space to be 4.6% (range: 2.5-10.6%). As these authors have pointed out (see their Fig. 7 ) , the amount of extracellular space as measured in this way will be relatively larger in sections containing fine dendritic and glial processes and smaller in sections with large cell bodies. Despite this source of uncertainty, the results of most electron microscopic studies would indicate that the size of the extracellular space is probably less than 10%(i.e., less than 0.1 mVgm wet brain). 2. Selection of Proper Test Molecules Some of the work on the extracellular space in brain has utilized radioactive sodium or chloride as tracers which could diffuse into the extracellular space (Woodbury, 1958; Woodbury, et al., 1956). Since these tracers exchange at a significant rate with the intracellular sodium and chloride, the extracellular space cannot be calculated simply from a knowledge of the total amount of tracer taken up by

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the tissue and the tracer concentration in the external medium, because some of the tracer will be inside of cells. Consequently, one must resort to a kinetic analysis of the sort described in Section 1.2. It was pointed out in that section, and has been discussed in more detail in other articles (Robertson, 1957; Solomon, 1960), that it is extremely diflicult to relate “compartments” determined solely from kinetic data to any specific anatomical region within a complicated tissue. This is particularly true in the cortex where the extracellular space, microscopically, appears to be a very complicated interweaving of narrow clefts without any simple geometrical representation that can be used as a model for kinetic analysis. For these reasons, it is this author’s opinion that no definite conclusions can be reached concerning the size of the extracellular space of the sort envisaged by electron microscopists from experiments using ions such as sodium or chloride which cross the nerve membrane readily. The objections to the use of these ions for extracellular space measurement do not apply to experiments which utilize a nonpenetrating substance as the test material, provided the experiments last long enough for the test molecule to equilibrate with extracellular water. In such experiments the extracellular space can be calculated from the steady-state distribution of the test substance between brain and bathing medium. If, furthermore, such experiments can be done in the intact animal and not ha Vwro, then uncertainties due to possible tissue swelling can be avoided (see below), Several experiments of this sort have been performed by injecting inulin, (Woodbury et al., 1956), sulfate (Woodbury et d.,1956), and thiocyanate (Streicher and Press, 1980) intravenously and allowing the material to pass into the brain tissue. The size of the space accessible to these substances (excluding the cerebral vasculature), presumably after a steady-state distribution had been reached, was 3.9, 4, and 10% of brain volume, respectively. These data probably overestimate the size of the extracellular volume occupied by these substances for two reasons: ( a ) the diBculty of washing out all of the plasma, where the test substance is in relatively high concentration, from the cerebral vasculature. (b) the possibility of slow intracellular accumulation. Very recently, Rall and Patlak (1962) perfused ventricles of dogs with solutions containing tracer amounts of C“-labelled inulin. Sections of brain at various distances from the ventricular lumen were

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removed after the perfusion and the amount of radioactive inulin contained therein was determined. From a knowledge of the experimentally determined concentration gradient, the concentration of inulin in brain tissue could be calculated. The inulin space was estimated to be about 10%. This type of experiment does not depend for its validity upon any assumptions about the extent of equilibration of the inulin molecule with the extracellular water, nor upon the ability of inulin to cross the blood-brain barrier. It does, of course, assume in common with all extracellular space measurements, that the test molecule does not cross membranes and reach the inside of cells. The various extracellular space measurements discussed in the preceding paragraphs provide qualitative support of the notion that there is a small volume of brain tissue, about 510%of the brain volume, which is accessible to substances which ordinarily cross cell membranes with diEculty. It seems reasonable to identify this volume tentatively with the small cleft between cells seen by the electron microscopists,

3. Extracellulur Space in Tissue Slices The inulin and thiocyanate spaces of excised brain tissues are considerably larger than comparable spaces determined in utuo. Several investigators have reported that excised brain tissue slices (0.35-2mm thick) swell during incubation in a variety of media (Allen, 1955; Pappius and Elliot, 1956a, b ) . This increase in fluid content has been termed "water of swelling" by Pappius and Elliot (1956a). These workers found that the volume of this water-ofswelling agreed rather well with their determination of the inulin space in tissue slices (0.32 ml/gm of tissue), indicating that only the water-of-swelling was accessible to the inulin molecule. These results suggested that the kinetics of water movement in cortical tissue slices might be rather complicated. The water-ofswelling presumably represented fluid which enters cells in the tissue slice (the volume of the water-of-swelling is too large to be ascribed simply to extracellular edema). One would have expected that once the cell membranes became permeable to inulin this molecule would have been able to equilibrate with all (or at least most) of the intracellular water. This supposition leads to the prediction that the inulin space should have been considerably larger than the

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F. J. BRINLEY,

JR.

volume of the water-of-swelling. The fact that the two spaces are nearly the same size suggests that either: ( a ) there are intracellular barriers to water diffusion or ( b ) that the water-of-swelling which occurs in tissue slices is not distributed uniformly throughout the tissue. Very recent work by Pappius and Elliot (1962)indicates that the second explanation is probably correct. They studied the distribution of serum proteins labelled with fluorescent dyes (which appear to equilibrate with the water-of-swelling in the same manner as inulin). By examining sections of tissue slices microscopically, Pappius and Elliot found that the fluorescence was limited to the marginal areas of the slices (except for some inside blood vessels). Their conclusion was that the water-of-swelling, and hence the protein and inulin spaces, were largely confined to the edges of the slices which had presumably been damaged during cutting. This interpretation, if correct, indicates that slices of cerebral cortex are rimmed by tissue which behaves quite differently from that in the interior as far as water or electrolyte movement is concerned. The possibility that tissue slices may have a “compartment” for water or electrolytes that results from the slicing procedures should be kept in mind when considering the results of tissue-slice experiments. B. IONIC COMPOSITION OF NEURONS IN BRAIN As a tentative, working hypothesis, it will be assumed that the extracellular space in whole brain is about 5%of the total wet weight. Some of the conclusions concerning the intracellular ionic composition of brain cells which follow from this hypothesis will now be considered. Table I contains data on the sodium, potassium, and chloride content of mammalian brain. From these data the intracellular concentration of the three ions can be calculated with the following assumptions: 1. The extracellular space has the same ionic composition as plasma and has a volume of 50 mVkg wet brain. 2. The internal ionic composition of all cells in the brain is the same. 3. The intracellular water (750mVkg wet brain) is equal to the total water minus the extracellular water. The intracelIuIar concentrations in mammalian brain tissue calcu-

203

ION FLUXES IN THE CNS

TABLE I IONIC COMPOSITION OF BRAIN^ Tiesue

K+

Na+

C1-

52.0

29.1 Cummins and McIlwain (1961)

(121 .O)

(73.4) Cummins and McIlwain (1961)

Source ~~

Guinea pig cortex 111.8 (fresh) Guinea pig cortex (52.6) (incubated 30 min) Cat brain 87.5 Cat brain 99 Rat brain 99.3 Rabbit brain 90.0 Dog cerebrum 95.6 Rat cortex 104 Mead 98.2 a

41.3 Yannet and Darrow (1938) - Pappius and Elliott (1954) 34.6 Lowry et al. (1946) 40.0 Ames and Nesbett (1958) 36.7 Eichelberger et al. (1949) - Pappius and Elliott (1954) 36.3

53.6 63 45.7 51 .O 51 .O 56 53.2

Amounts given in mmoles/kg wet brain. Numbers in parentheses have not been included in the averages.

lated with these assumptions are shown in Table 11. The internal ionic concentrations of some other nerve tissues are also included for comparison. It can be seen from the table that the calculated concentrations for mammalian brain tissue agree reasonably well IONICCOMPOSITION

OF

TABLE I1 NERVOUS TISSUES

AND

EXTRACELLULAR FLUIDS

Internal Composition of Some Nerve Tissues" Tissue CNS cell Cat sciatic nerve Cat spinal motoneuron Toad sciatic nerve Squid axon

K+

Na+

136

61

C1-

Source

43 Calculated (5% extracellular space) 41 0-17.5 Krnjevic (1955) 15 9 Eccles (1957, Table I) 42 27 Shanes and Berman (195513) 50 40 Hodgkin (1958)

181-183 150 135 400

Composition of Some Extracellular Fluids or Bathing Solutionsb Mammalian extracellular fluid Toad Ringer's solution Seawater a

5.5

150

125 Eccles (1957, Table I)

1.7 10

108 460

108 Shanes and Berman (195513) 540 Hodgkin (1958)

Amounts in mmoles/liter of water. Amounts in mmolee/liter.

204

F. J. BRINLEY, JR.

with those obtained for cat and frog sciatic nerve. However, all of these preparations have a much higher internal sodium relative to the extracellular sodium concentration than is found in squid axoplasm. Eccles (1957) has reported a relative intraextracellular sodium ratio for cat motoneuron much closer to that found for squid axoplasm. His method, which is based on an identification of the membrane potential at the peak of the spike with the “sodium equilibrium” potential, can also be applied to other neurons in the central nervous system. The argument for equating the membrane potential at the peak of the spike with the sodium equilibrium potential is as follows. The variation of nerve membrane potential in various experimental situations as well as during the spike is reasonably well described by the constant field equation ( Hodgkin and Katz, 1949),e.g.:

P refers to the membrane permeability to a particular ion (as subscript), and the subscripts i and o refer to ionic concentrations “inside” and “outside” of the cell. Concentrations (as Nu) are italicized. During activity, the membrane permeability to sodium ( PNs) is postulated to increase many times so that 9) is determined mainly by the “PNsNa” terms. During a spike, therefore, the membrane potential is determined largely by the ratio of the sodium concentration terms, and Eq. (20a) can be written as: WAP

= -7 1 ,

[*I

Nuo

or ~a~ = ~ a , e x p

If wAPand Nu, are known, then Nui can be calculated. Actually the other terms in Eq. (20a) are not completely negligible during the spike. The membrane potential during the peak of the spike only approaches wNa (and is always less), Therefore, the value of Nu‘ calculated under these assumptions is really a maximum value. Despite these limitations, the procedure gives an estimate of the internal sodium concentration where direct determination is not possible. Internal sodium concentrations, estimated from the spike potential, are given in Table I11 for several mammalian neurons. T h e

TABLE I11 INTERNAL SODIUM CONCENTRATIONS FOR MAMMALIAN NEURONS

cell

Va (mv)

Action potentisla (mv)

Cat motonewon Hippocampal pyramidal cell Sympathetic ganglion (rabbit) b n s h a w cells (cat)

-70 -62 - (65-80) -60 max.

96-120 70 70-96 70 max.

Pyramidal cells (cat) Glial cells, tissue culture

-60

80 max.

-40

40-70

Inside negative with respect to outside. Action potential is positive with respect to resting potential. Calculated from: Nai = 150 exp ( - V ~ ~ / 6 1 . 5 ) . d Data obtained from Eccles (1957) Table I, p. 24.

Overshoot (mv)

Naic (mmolefiter)

+(26-52) +(5-16) +10

15d 90 125-82 115

VAP

+8

+20 max.

+(0-30)

71 15042

source

Eccles (1957, pp. 68-69) Kandel et al. (1961) Eccles, R. M. (1955) Frank and Fuortes (1956) Eccles et al. (1954) Phillips (1956) Hild and Tasaki (1962)

z8

8

@

2

kl

2 n:

206

F. J, BRINLEY, JR.

range of values is considerable, as one might expect, but all cells (with the exception of cat motoneuron) have a ratio of intra: extracellular sodium that is considerably larger than would have been expected from data on squid nerve. Since the resting and spike potentials of each of the cell types listed in'Table I11 were measured with micropipette techniques, it may be argued that the cell had been injured during penetration with a consequent increase in sodium permeability permitting an inward leak of sodium and raising the internal sodium concentration. However, the maximum values for Nu, presented in Table I11 are in qualitative agreement with those presented in Table I11 for vertebrate nerve, which were obtained from experiments in which mechanical injury did not occur, although of course the possibility of injury caused by the agents used to measure the extracellular space cannot be excluded. The tentative conclusions which can be drawn from the data in Tables I1 and I11 are that the internal sodium concentration in mammalian neurons appears to be relatively higher than in invertebrate axons, and that the smaller action-potential overshoot seen in mammalian neurons may be explainable on the basis of this relatively high internaI sodium. None of these data excludes the possibility that the internal sodium concentration in the dendrites of cortical neurons may be even higher than the average values for the intracellular space given in Table I1 or the calculated values for neuron soma given in Table 111. Because of the much larger area to volume ratio of dendrites compared to cell bodies, a net influx per unit area of membrane of sodium, such as might occur following an excitatory postsynaptic potential, would be much more effective in raising the internal sodium of concentration of dendrites as compared to cell bodies. (As an illustration of the preceding statement, consider a dendrite in the shape of a right circular cone with a uniform taper such that the radius of the cone at a vertical distance, x, from the base is r = (R(L- z)/L, where (R is the radius of the base and L is the vertical distance to the apex. The increase in internal concentration, AC,, of an ion at a distance J: from the base due to a sudden influx of m mo1es/cm2 is ACs = 3nA,/V, = 2'3ZL/(R(L - z).The effect on the internal concentration of a sudden influx is more pronounced the smaller the dendrite, i.e., larger x.) It is conceivable that continued synaptic bombardment on the terminal dendritic filaments in the

ION FLUXES IN TEE CNS

207

central nervous system keeps the average internal sodium concentration sufficiently high to render them inexcitable and unable to conduct action-potentials, Ill. Exchangeable and Nonexchangeable Ions

In calculating membrane potentials from such theoretical formulations as the constant field equation [Eq. (2Oa)], one generally replaces the thermodynamic-activity terms, appearing in such equations, by the intracellular concentrations. Such a simplification is not always justified and can lead to serious error if some of the intracellular ions are bound in some manner to other ions or molecules. A similar problem arises in calculating transmembrane fluxes from tracer data. Tracer experiments themselves provide only rate constants for exchange. To obtain the flux (in units of moles/sec), one must know the intracellular concentration, not of total ionic species, but rather of that fraction that is free to exchange under the conditions of the experiment, For these reasons, it is important to have information about the exchangeability of ions in the central nervous system. In considering the evidence for exchangeable or nonexchangeable ions, one must distinguish clearly between covalent bonding, in which individual ions are firmly held, and electrostatic bonds, where the presence of a stoichiometric number of oppositely charged ions is required for electroneutrality. This important distinction can be made clear by considering a simple dialysis experiment, in which the dialyzing membrane is impermeable to protein. Assume that a solution of potassium chloride, containing in addition a potassium salt of a negatively charged protein, is placed on one side of the membrane (side A ) and distilled water on the other ( side B ) . Potassium chloride will pass freely through the membrane but side A will always contain some potassium ions which are required to neutralize the negative charge on the protein molecules. Inasmuch as a fraction of potassium ions fails to cross a membrane known to be potassium permeable, it might be concluded that these ions were “bound” or firmly attached to the protein molecule. That this is not so, can easily be shown in principle by adding to side A carrier-free K4*Cl.Nonradioactive potassium ions will now appear on side B, indicating that the individual nonradioactive ions were not bound firmly to the protein and could easily be replaced by

208

F. J. BRINLEY, JR.

other ions. Such potassium ions would appear to be freely exchangeable in the usual tracer experiments. A. POTASSIUM IN Nmvous TISSUE The possible occurrence of potassium, bound in the covalent sense, has been extensively investigated in nervous tissue. The available information is summarized below. 1. Crab Nerve

Keynes and Lewis (1951) investigated potassium exchange in the leg nerve of the crab, Carcinus, The exchange of potassium was measured with tracer techniques and the total potassium by activation analysis. It was concluded that at least 90% of the internal potassium had exchanged after prolonged soaking in various artificial sea waters containing 1 5 times the normal potassium concentration. Axons soaked in normal seawater had Iost almost two-thirds of their original potassium content by the end of the soaking period (9.3 hrs); however, preparations soaked in 2 and 5 times the usual potassium concentration exchanged essentially all of their internal potassium with much less net loss of internal potassium. 2. Cephalopod Axons Practically complete exchange of the internal potassium in Sepia axons has been demonstrated by Hodgkin and Keynes (1953),using tracer techniques similar to those described above for crab nerve. In another set of experiments using Loligo axons, these authors (Hodgkin and Keynes, 1953) measured the mobility of sodium and potassium ions by measuring the velocity of a patch of radioactive ions during a longitudinally applied potential gradient. Their calculations showed that the mobility of sodium and potassium in axoplasm was the same as in seawater indicating that no significant binding of either ion had occurred. 3. Toad Nerve Shanes (1957)has reported that in the desheathed toad nerve (which is in a steady state with respect to internal potassium), potassium influx is not significantly different from the potassium outflux calculated from the time constant of tracer exchange and the total internal potassium. This equality of influx and outflux in the

ION FLUXES IN THE C N S

209

steady state indicates that there can be no large amount of nonexchangeable potassium or else the outflux would appear to be larger than the influx. 4. Sympathetic Ganglion

In this preparation, the time constant for exchange of tissue potassium is short compared to the length of the time interval in which the excised preparation can be maintained in a steady state (influx can be followed for about 16 time-constants before the preparation begins to lose internal potassium), Thus, the experimenter can determine the amount of exchanged potassium directly from the accumulated tissue radioactivity and the specific activity of the bathing medium. Harris and McLennan (1953) reported an exchangeable potassium of about 46%for isolated sympathetic ganglion. Brinley and Larrabee (1961), using a different species of rat, found that the exchangeable potassium was about 80%of the total potassium in ganglia from which the connective tissue sheath had been removed and about 60%for sheathed ganglia. These latter investigators have concluded that connective-tissue potassium, as exemplified by the ganglion sheath, exchanges rather slowly, and suggested that the 20% residual nonexchangeable potassium observed in desheathed ganglia may have been due to interstitial connective tissue within the ganglion. 5. Cerebral Tissue

Katzman and Leiderman (1953) studied whole brain potassium exchange in intact rats in the following manner. The specific activities of blood and brain tissue were followed over a period of 72 hr in a group of rats given simultaneous intraperitoneal injections of K4Cl. It was found that the potassium specific activity of both brain and plasma reached relatively constant values about 48 hours after the intraperitoneal injection. However, the brain specific activity remained about 20%lower than the plasma spec& activity. These workers concluded, therefore, that about 2M of the whole brain potassium had failed to exchange significantly with the plasma K42during the 48-72 hr experimental period. Experiments performed on slices of isolated guinea pig cortex also suggest that there may be a small fraction of brain potassium

210

F. J. BRINLEY, JR.

that does not freely exchange with the potassium in the environment. Krebs e# a2. (1951) reported that about W Q W of the tissue potassium was rapidly lost to potassium-free media when the slices were made anoxic. From the earlier discussion in this section it will be realized that the figure of 8040%loss represents a lower limit to the amount of exchangeable potassium because some cations must remain in the tissue to neutralize the charge on nondihsible anions. On the other hand, the anoxia to which the slices were subjected may well have altered substantially the amount of normally nonexchangeable potassium. 6. Location of Bound Potassium in Brain: Summry

The data obtained from brain and sympathetic ganglion indicate that in complicated nervous tissue containing perikarya and cell processes in addition to axons, there may be a small amount of potassium that does not exchange with potassium in the environment under the usual experimental conditions, The tracer techniques used in these experiments show only that there was a certain fraction of total ion that appeared to exchange with a much slower time-constant than the rest of the tissue potassium. The term “compartment” which is frequently used to describe the fractions of material which exchange with different time-constants is unfortunate because the term implies the existence of discrete anatomical locations for the substance. The assignment of these fractions to spec& anatomical locations results from inferences made by the experimenter. Conclusions as to the location of any fraction cannot be obtained solely from tracer experiments since these techniques cannot distinguish between: ( a ) an ion fraction held in a nonionic bond; and ( b ) a fraction potentially freely exchangeable but sequestered behind a potassium-impermeable barrier. It is not possible at present to decide which of these two possibilities (or perhaps both) is the correct explanation for the nonexchangeable potassium in brain. There is some evidence that the cephalin fraction of cerebral lipids forms nonionizable salts with sodium and potassium (Folch d al., 1957). However, such bound ions, e.g., potassium, can be reversibly displaced by other ions, e.g,, sodium, magnesium, and calcium. It seems unlikely, therefore, that any potassium ions “bound” in this sense would fail to exchange with radioactive ions of the same species.

ION FLUXES IN THE CNS

211

On the other hand, it is not obvious what anatomical class of cells in the brain could be acting as a compartment with a potassiumimpermeable boundary. It is customary to extricate oneself from this impasse by invoking the “blood-brain barrier.” Judging from the data of Katzmann and Leiderman (1953),the barrier must exist in two places: ( a ) at or near to capillary endothelium, to explain the slow rate of exchange of potassium in brain compared with other organs; and ( b ) around certain cells containing 20% of the brain potassium, to explain the failure of this amount of potassium to exchange at all. One could even speculate that since the neurons comprise about 20%of the brain mass, and if the neurons and glial elements have about the same internal potassium, neurons are the cells comprising the nonexchangeable compartment. This explanation of the nonexchangeable compartment is in general accord with a recent concept of the blood-brain barrier formulated by De Robertis and Gerschenfeld (1!361), but there is no direct evidence to support it. IV. Spreading Cortical Depression

Spreading cortical depression (SD) is characterized by a reversible loss of electrocortical activity lasting about 1 min associated with a slow negative potential change (SPC) of 5-15 mv with respect to an indifferent electrode. The depression of cortical activity and the SPC typically propagate together across the cortical mantle in a radial direction outward from the locus of stimulation with a velocity of 23 mm/min. A spreading depression can be elicited in a variety of ways such as topical application of high potassium solutions or certain drugs, local mechanical trauma, or repetitive electrical stimulation. A more complete account of this phenomenon is contained in a recent review article by Marshall (1959). Only those aspects of spreading depression pertaining to ionic movements will be discussed below.

A. POTASSIUM RELEASEDURING SPREADING DEPRESSION The loss of evoked and spontaneous electrical activity from the cortex plus the negative SPC suggests that the phenomenon of spreading depression is associated with depolarization of the cortical mantle at the site of SD. If this interpretation of the electrophysiological data is correct, then potassium ions should be lost from a localized region of cortex as a wave of SD passes through the area.

212

F. J. BRINLEY, JFL

The expected potassium release from the cortex during spreading depression has been found independently by two laboratories. Brinley et a2. (196Ob) perfused a localized region of brain by placing a small polyethylene chamber on the cortical surface. The principle of the method is illustrated schematically in Fig. 3A. In

t

UPPER RESERVOIR

FIOW rate

I.occ/min

(20vols /min)

LOWER RESERVOIR

FIG. 3A. Diagram of perfusion chamber used by Brinley et ul. (1960) for efflux studies. The chamber rests directly upon the cortex. A silver-silverchloride electrode is used for recording cortical electrical activity and slow potentiaI changes. 3 1

2 4

5 6

FIG.3B. Diagram of apparatus used by KPivLnek and BureE (1960) for outflux measurements. The chamber is attached to the skull ( 4 ) with cement (2). The dura ( 5 ) is left intact. (Reprinted courtesy of Physiobgiu Bohernoslovenica. )

order to avoid damage to brain tissue, the chamber must rest gently on the cortex. However, there must be no leak of fluid around the edges of the chamber if the perfusate is to be collected quantitatively. These two requirements were met simultaneously by making the chamber part of a siphon system. Fluid was siphoned out of an

ION FLUXES IN THE CNS

213

upper reservoir, through the chamber, and into a lower, collecting reservoir. The heights of the reservoirs were adjusted so that the pressure head inside the reservoir was nearly zero. This permitted a water-tight seal to be maintained between the edge of the chamber and the cortical surface while fluid passed through the chamber at the rate of 1-2 ml/min. There was usually sufficient activity in the perfusate for samples of the wash fluid to be taken at 15-sec intervals. By placing the chamber over a region of cortex previously loaded with K42, it was possible to measure the rate of tracer outflux before, during, and after, the passage of a wave of spreading depression through the area perfused by the chamber. The results showed that the K42outflux not only increased by several times during a wave of spreading depression, but that the time course of the extra potassium outflux followed very closely the time course of the SPC (see Fig. 4). The amount of radioactivity in the wash fluid (measured as counts per minute per sample) can be converted to moles of potassium outflux provided the spec& activity of the potassium in the perfused region of cortex is known. But, the calculation of this tissue specific activity depends upon assumptions about the extent of radioactive potassium exchange in the cortex during the loading period. For this reason, tracer data at present can give only rather crude estimates of the absolute size of the potassium efflux. The figure reported by Brinley et csl. (196Ob) was 6 X 10-lomo1es/mm2 cortical surface for one spreading depression. This value does not represent the net amount of potassium lost by the cortical surface during SD. As can be seen from Eq. ( 5 ) , the net change in tissue potassium is the difference between the influx and the outflux. Since no measurement of influx of radioactive potassium was made during these experiments, the figure of 6 X 10-lomoles/mm2 represents an upper limit to the amount of potassium actually lost by the cortex during one spreading depression. Uiv6nek and Bureg (1960) studied ion movements during spreading depression in anesthetized rats by a similar technique. These investigators used a chamber mounted epidurally (Fig. 3B) and washed the dural surface with potassium-free solutions, such as isotonic sucrose or sodium chloride. The concentration of potassium ion in the wash fluid after it had passed over the dura was determined with a flame photometer and taken as a measure of the amount of potassium lost from the cortical surface. Because of the

214

F. J. BFUNLEY, JR.

A

I

I

0.5'

I'

A I I 15' 2' 2.5' TIME IN MINUTES

I 3'

I 3.!

FIG.4A and B. The time course of potassium outflux (upper part of figure) and the slow change in potential (lower part) associated with a propagated spreading depression. (Fig. 4B, courtesy of the J . of NeurophysioL)

minute amounts of potassium ion lost from the surface in these experiments, it was necessary (in order to obtain detectable amounts of potassium) to collect samples for not less than 2 min and to perfuse a somewhat larger region than was necessary in the tracer experiments. For these reasons, the spatial and temporal resolution of potassium outflux during spreading depression afforded by the flame photometric method at present is not as great as that of the tracer experiments. However, the method used by nivlnek and Burd has the advantage that it permits direct measurement of the total potassium ion washed off of the d u a l surface and, by inference, from the cortical surface during spreading depression. The amount of potassium lost by the cortical surface during one

ION FLUXES IN THE CNS

. . . . .

-

L X

215

x+

t-

3 0

:;I ---/TI -6 -8 10

0

B

60 120 160 240 300 360 420 480 540

TIME IN SECONDS

spreading depression in the experiments of KiMnek and Bureg can be calculated from their data to be 31 x mo1es/mm2 per spreading depression [a recent personal communication (Bur&, 1962) indicates that a more accurate figure is 16 X 1P0moles/mm* per spreading depression]. This figure is somewhat larger than the rough estimate of 6 x mo1es/mm2 obtained by Brinley et ul. However, no strict comparison of the data is possible because, in addition to the fact that the experiments were performed on different species, Kfivhek and Bur& measured potassium lost into potassium-free solutions while Brinley et al. (196Ob) measured tracer outflux into solutions containing the usual amount of potassium (25mM). Regardless of quantitative differences in the results obtained by the two laboratories, the data provide clear evidence that the slow potential change associated with spreading depression is associated with a large increase in potassium outflux from the cortical tissue and hence presumably with depolarization of cortical cells. Neither

216

F. J. BRINLEY,

JR.

technique at present provides any information regarding the extent to which glial cells contribute to the increased potassium outflux which occurs during spreading depression. In view of data that glial cells may constitute the major fraction of brain mass (Scholl, 195s), it seems reasonable to think that a substantial portion of the observed increase in potassium outfiux is due to depolarization of glial cells. The data presented here can also be used to provide a rough estimate of the extracellular concentration of potassium present during spreading depression, A maximum increase in outflux of fourfold-as measured at the surface of the cortex by the perfusion technique-indicates about a fourfold increase in the extracellular potassium concentration at the surface, i.e., about 20 mM. The maximum increase in the extracellular concentration beneath the surface is undoubtedly higher.

B. SOURCEOF POTASSIUM RELEASED DURING SPREADING DEPRESSION In view of a recent opinion (Tschirigi et al., 1957) that the SPC may be related to a transpial potential change rather than a change in the polarization of cortical elements, the evidence that the potassium outflux determined in the above experiments originates in cortical tissue and not elsewhere, i.e., the cerebral vasculature, will be briefly discussed. U i v h e k and Bureg demonstrated that, in their experiments, the excess potassium efflux obtained during spreading depression did not come from the cerebral vasculature underlying their perfusion chamber, in the following manner: These investigators gave rats intraperitoneal injections of radioactive potassium shortly before the initiation of spreading depression. Because brain-potassium exchanges rather slowly with intravascular potassium, during the experimental period the radioactive potassium that washed off of the epidural surface came from the vasculature and not from the cortical substance. When spreading depressions were induced on the rat cortex, the per cent change in total potassium concentration in the perfusate was not significantly different from the per cent change in the specific activity. This implies that the additional potassium washed off during the spreading depression had zero specific activity and, hence, must have come from the essentially nonradioactive aortical tissue and not from the highly radioactive blood.

ION FLUXES IN THE CNS

217

The same conclusion, that blood potassium was not the source of potassium released during SD, can be drawn from the rabbit experiments of Brinley et al. In these experiments prolonged local perfusion of the cortex with radioactive Tyrode’s solution resulted in minimal blood radioactivity. Surface cortical blood flow in the perfused area would have had to increase by an impossibly large amount to explain the observed increases in radioactivity during spreading depression on the basis of loss of radioactive potassium from the blood. The observations showing an increased potassium release during SD are most easily explained by depolarization of surface cortical elements, although the present data by no means eliminate an additional explanation based on potential changes in noncortical tissue. C. POSTMORTEM POTASSIUM FLUXES Because of the complicated geometry of the histological architecture of the superficial cortex, the observed potassium effluxes cannot be related to movement across any particular cellular membrane. For this reason, it is not possible to relate potassium outflux quantitatively to a membrane potential. However, it is possible to make comparisons of percentage increases in potassium outtlux which are associated with different experimental procedures and, thus, draw qualitative conclusions about the extent of cortical depolarization. Using this approach both KiivAnek and Burei (1960) and Brinley et al. (1960b) compared the potassium outfiux obtained during spreading depression to that which occurred immediately post mortem in animals either rapidly exsanguinated or injected with large doses of intravenous nembutal. Both laboratories reported that, immediately post mortem, the cortical potassium outflux shows a percentage increase comparable to that observed during a spreading depression, suggesting that SD is associated with superficial cortical depolarization of a degree comparable to that occurring post rnortem. However, it must be recognized that the depolarization producing the postmortem SPC probably involves deeper cortical elements than those involved in SD ( LeBo, 1951) .

D. SODIUMAND CHLORIDE MOVEMENTS If the spreading depression phenomena is related to membrane depolarization, then one would expect the slow potential change to be correlated with an increase in the membrane permeability to

218

F. J. BRINLFX, JR.

other ions, particularly sodium and chloride, as well as potassium. In this section the experiments designed to demonstrate such permeability changes to sodium and chloride during spreading depression will be discussed. 1. Sodium Movements

Since the net driving force for sodium is in the inward direction, any increase in membrane permeability for sodium during spreading depression should also lead to a net uptake of sodium by the superficial cortical cells. Unfortunately, a transient uptake of sodium by the cortex is technically difficult to measure directly. E i v h e k and Burei (1960) attempted to circumvent this problem by using sodium-free solutions in their perfusion chamber. If the extracellular sodium concentration can be reduced sufficiently, then the normal electrochemical gradient will be reversed and the internal sodium will be continually lost to the environment. In this experimental situation, increases in membrane permeability due to depolarization associated with SD can be expected to result in an increased outflux of sodium. The results obtained by KEivhek and Burd (1960) showed some definite increases in per cent sodium loss during SD, but the averaged data showed no significant change. It is possible that the increment of Na in the perfusate due to release of intracortical sodium was too small to be measured accurately since these workers concluded from other experiments that most of the sodium in the control washes came from the cortical blood supply and presumably was not affected by depolarization of cortical cells. It is also possible that washing the epidurium with sodium-free solutions does not reduce the concentration of sodium in the extracellular space sufficiently to reverse the normal inward sodium electrical potential gradient. If this were true, one might not have observed an increase in net sodium outflwr during spreading depression, even though sodium permeability may have increased. 2. Chloride Movements

According to the estimates of internal chloride given by Eccles (1957), the equilibrium potential for this ion is near the resting membrane potential. Any depolarization of the membrane (i.e.,

ION FLUXES IN THE CNS

219

decrease in membrane potential) should result in a net inward driving force that would move chloride into the cell interior. Evidence for such net chloride movement into the interior of apical dendrites in the rat and rabbit during SD has been reported by Van Haareveld and Schad6 (1959), using a silver staining technique to precipitate the chloride ion. Brain sections taken from regions of cortex undergoing SD (as indicated by the appearance of the slow potential change at the site selected for removal) were found to be more deeply stained than control sections removed from areas not exhibiting an SPC at the moment of extirpation. Unfortunately, these results cannot yet be regarded as conclusively demonstrating net chloride movement into the cell interior because the silver staining technique may not be specific for chloride ion and the distribution of dark staining particles may have been altered by the fixing procedures.

E. MODIFICATION OR PREVENTION OF SPREADING DEPRE~SION BY DIVALENT IONS BureH and BureIovA ( 1956) and later Bureg (1960) have investigated the effects of calcium and magnesium on spreading depression. Their results will be discussed with respect to: 1. antagonism of potassium depolarization of the cortex by calcium or other divalent ions; 2. prevention by local application of calcium or magnesium of the propagation of spreading depression through a region remote from the initiating site.

1. Protection Against Initiation of Spreading Depression It was found in a number of experiments that calcium or magnesium ions added to a solution of potassium chloride will prevent the production of a spreading depression when the solution is applied topically even though the concentration of potassium which was used would have been adequate to cause the SPC when applied alone. This antagonism between calcium (magnesium, barium, and strontium were also effective) and potassium extended over more than a tenfold concentration change for both ions, and these authors were able to carry their observations into rather hypertonic concentration ranges (e.g., 1.35 M KCL was antagonized by 1.0 M CaCl,).

220

F. J. BRINLEY,

JR.

It might be thought surprising that any reproducible correlations could be obtained with the use of such hypertonic solutions; however, these solutions were applied epidurally, and it is probable that the actual concentrations within the cortex were considerably less than the applied concentration. Calcium and the other divalent ions should be effective in preventing SD if these ions can prevent the depolarization of nerve membrane caused by high external potassium concentrations. The ability of calcium to reduce potassium conductance (Frankenhaeuser and Hodgkin, 1957) and the potassium outflux (Shanes, 1958b) in nerve is pertinent to the question of calcium protection against the propagation of SD (see below) but does not necessarily explain the ability of this ion to prevent the initiation of SD by topically applied potassium since in this case the increased extracellular potassium concentration is not due to the leakage of potassium ions from inside the cell. Calcium has been classed as a stabilizer of excitable membrane by Brink (1954), Fleckenstein (1951) and Shanes (1958a) in the sense that an increase of external calcium has little effect on the resting membrane potential but does reduce the changes in membrane potential caused by the external application of potassium or other depolarizing agentsa2The stabilizing action of calcium could explain the ability of this ion to prevent spreading depression in the experimental situation used by BureI and Buregovh ( 1956). 2. Efect of Calcium on Propagation of Spreading Depression During the propagation of a wave of spreading depression (SD) through a localized region of cortex, only a limited amount of potassium can be released into the extracellular space because of the limited intracellular reservoir and the speed of cortical repolarization. The maximum possible extracellular concentration of potassium which can occur in a region of cortex through which SD is passing

’In peripheral nerve, there appears to be some species variation in the ability of calcium to maintain the resting potential in the presence of potassium. Guttman (1940) reported that calcium could antagonize potassium depolarization in the walking-leg nerve of the spider crab. However, Steinbach et al. (1944) found that excess calcium would not prevent depolarization of squid nerve, but it would maintain the degree of “membrane rectification” near the resting level even in the presence of 5-8 times the normal external potassium concentration.

ION FLUXES IN THE CNS

221

is, therefore, relatively small (no more than isotonic) compared to the very large concentrations of potassium which have been applied topically to the cortical surface to produce spreading depression. Therefore, one might expect that the maximum effective concentration of the divalent ions in preventing passage of SD through a region of cortex remote from the initiating site is relatively low compared to the concentrations necessary to protect the cortex against the SD-producing effects of high potassium solutions. Experimentally, BureH and BureIovP (1956) found this to be the case. Calcium ions applied locally to the cortex produced maximum protection against the propagation of spreading depression (reducing the SPC to 20%of the control value) at a concentration of 140 mM. (The authors offered the reasonable explanation that the residual, small SPC was due to electronic potential spreading from slow changes in potential in unprotected areas in the vicinity of the recording electrode. Support for this interpretation was afforded by experiments in which potassium-free calcium chloride ( 162 mM ) was used to perfuse a local region of cortex remote from the site of initiation of SD. Little or no SPC appeared in the perfused area and there was no increased potassium in the perfusate. ) The explanation offered by BureH and BureHovi (1956) for their observations follows from the work of Frankenhaeuser and Hodgkin (1957) on squid axon and Gossweiler et al. (1954) on rat diaphragm, i.e., the potassium conductance and potassium outflux are reduced in the presence of an increased external calcium concentration. Presumably, in the mammalian cortex, calcium acts in a similar manner. The slow change in potential of a wave of SD propagating into a region of cortex protected with calcium, will result in less potassium outflux into the extracellular space, thus causing less depolarization of adjacent neurons. Hence, there will be less SPC in the perfused region; the SD reaction will lose its autoregenerative properties and will not propagate through the protected region.

F. MECHANISM OF PRODUCTION AND PROPACATTON OF SPREADING DEPRESSION 1. “Trigger Substance” Hypothesis Recently BureH et al. (1960) and Van Haareveld (1960) have measured the ability of a variety of chemical compounds to produce

222

F. J. BFUNLEY, JR.

spreading depression. Considering the differences in species and technique, the results, where comparison is possible, are in essential agreement (see Table IV) . One striking fact which has emerged from these studies is that glutamine, glutamic acid, asparagine, and aspartic acid will produce spreading depression ( SD) when applied topically in relatively low concentrations (5-20 mM). The actual effective concentrations at the locus of action are undoubtedly a good deal less. In contrast to the potency of these 4 compounds in producing SD, there are several others, closely related structurally, which are quite ineffective in producing SD unless the topically applied concentrations are extremely high (see Table IV). These observations suggest that glutamic and aspartic acids, together with their minated derivatives, all of which are known to be present in cortical tissue, may have some specific effect on the membrane of cortical cells. Since these substances are present normally in cortical tissue Bure3 et al. (1960)and Van Haareveld (1960)have suggested that the release of some of these compounds into the extracellular space may be the important step in the initiation and propagation of SD. While it is well established that some compounds when applied topically to the cortex can start SD, there is as yet no direct evidence that the concentration of any of these substances in the superficial cortex changes during the slow change in potential associated with spreading depression. One very important piece of evidence for supporting the ^trigger substance” hypothesis would be the demonstration that an increased concentration of some compound appears in the extracellular space during SD and not otherwise. One way of performing such an experiment might be the following: An experimental animal is injected with C?*-labelled glucose, or some other metabolic substrate. After a time-interval sufscient to permit incorporation of this material into brain tissue, the brain is exposed and a perfusion chamber placed on the surface. The wash fluid from the chamber is collected in the usual manner and the amount of radioactivity is determined. The occurrence of greater radioactivity in samples during periods when the perfused region was undergoing SD than during control periods would unequivocally indicate the presence of some carbon-containing substance in the extracellular space that is normally not present, assuming that

223

ION FLUXES IN THE CNS

the permeabilities of the pia and the blood-brain barrier do not change during SD. Kiivhek (1961) has pointed out that some observations by Grafstein (1956) would seem to preclude the possibility that a negatively charged particle, such as the glutamate or aspartate ions, could be responsible for propagating spreading depression across the TABLE IV FOR THEIR ABILITYTO PRODUCE SPREADING DEPRESSIONO

COMPOUNDS STUDIED

substance

Rabbit, cortical surfaceb (threshold conc in mM)

Rat, epidural surfacec (EDsain m M ) d

~~

DL-Glutamic acid L-Glutamic acid D-Glutamic acid D-Glutamhe L-Glutamine L-Asparaghe L-Aspartic acid La-Aminoadipic acid Succinic acid L-Proline Glutaric acid PKetoglutaric acid L-Ornithine ma-Aminobutyrk acid 7-Aminobutyric acid 8-Alanine Isoglutamine Glycyl glutamine

-

224

15 3.6

-

80 13 17 35

200

17

77 55

89 No effect

100

No effect No effect No effect No effect No effect

No effect

-

No effect No effect No effect No effect

~

Topical application. b Van Haareveld (1960). Burei et al. (1960). d The EDsodoseproduces SD in 50% of animals.

cortex. Grafstein found that a horizontal potential gradient imposed on the cortex by passing a steady current through electrodes placed on the cortical surface, could influence the velocity of propagation of the SPC wave. The rate of propagation of SD was increased when the applied electric field was so oriented that the negative electrode was in front of the SPC wave and decreased when the positive elec-

224

F. J. BRINLEY, JR.

trode was in front, These findings are easily explained if a positive ion was responsible for causing the SPC because the velocity of such ions would be increased by the electric field. The effect is just the reverse of what would be expected if a negatively charged ion were involved in producing the SPC. 2. Potassium-Release Hypothesis This hypothesis regards an increase in the extracellular potassium concentration to a critical level as the essential requirement for producing a spreading depression. Support for this hypothesis derives from three main lines of evidence: ( a ) effect of metabolic inhibitors in producing spreading depression; ( b ) occurrence of neuronal discharge during spreading depression; ( c ) production of spreading depression by orthodromic or antidromic stimulation. These will be discussed in turn. a. Metabolic Inhibitors. It is well established that topical application of metabolic inhibitors such as 2,4-dinitrophenolYsodium cyanide, and sodium azide in concentrations of about 1 mM will produce spreading depression (Burei, 1956). Since these drugs are known to reduce potassium active transport and increase to a certain extent the potassium permeability of the membrane in frog and squid nerve ,{Shanes, 1958a; Hodgkin and Keynes, 1955), the inference has been made that these agents increase net potassium loss into a restricted extracellular space and, thus, increase the extracellular concentration of potassium. Qualitative estimates of the amount of potassium ’in the extracellular space ( Brinley et al., 1960b) during the maximum of the SPC were in general accord with estimates of threshold potassium .concentration required for producing spreading depression as determined by Burei ( 1956). Since the metabolic inhibitors which produce spreading depression (SD) may affect the membrane permeability to substances other than sodium or potassium, one cannot assume that an increase in extracellular potassium concentration is the only significant change produced during the topical application of inhibitors. Other substances allowed to escape from the cell interior, or elaborated by the cell due to its altered metabolism, may be more important in causing depolarization. Such a compound could indeed be the “trig ger substance” alluded to earlier. b. Neuronal Discharge. Grafstein (1956) observed that during

ION FLUXES IN THE C " S

22s

SD an intense neuronal discharge (recorded with intracortical electrodes) occurred concomitantly with the arrival of the SPC at the recording electrode. This finding suggested to her that the depolarization of intracortical neurons released enough potassium into the extracellular space to depolarize adjacent contiguous neurons causing them to release potassium in turn and, thus, propagate the SPC along the surface of the cortex. c. Orthodromic or Antidromic Stimuhtion. LeGo ( 1944b), LePo and Morrison (1945) and Leilo et al. (1959) reported that tetanic transcallosal stimulation produces propagated spreading depression. Van Haareveld and Stamm (1955) have concluded that stimulation of the auditory and visual systems may produce SD. In none of these experiments were topical drugs applied. The initiation of the SPC must have been related soIeIy to the depolarization of cortical cells produced either by antidromic or orthodromic stimulation. The simplest interpretation of these experiments is that sufficient potassium was released by the stimulated cells to raise the extracellular potassium concentration high enough to depolarize neighboring unstimulated cells and, thus, initiate a spreading depression. G . RECOVERYPROCESSES It has been shown in the preceding sections that the time course of the SPC is correlated with potassium outflux from the cortical surface. It has also been suggested that this increase in surface potassium outflux results from an increase in the extracellular potassium concentration, which produces depolarization of cortical cells, and accounts for the negative swing of the SPC. To account for the positive swing of the SPC on the basis of changes in the extracellular potassium concentration, it is necessary to assume that the excess potassium ions are removed in some way, Several suggestions as to how this removal might occur will be presented in the following paragraphs. There is no direct evidence to support any of these hypotheses and the discussion should be regarded as largely speculative. 1. Diffusion of Potassium from the Regions of Covtex Znvolved in Spreading Depression

The question of whether potassium can diffuse away from the region involved in spreading depression sufficiently rapidly to ac-

226

F. J. BRINLEY, JR.

count for the positive swing of the SPC,can best be answered by considering the following simplified situation. Assume that, at the peak of the SPC,a uniform distribution of potassium exists in the extracellular space of the cortical mantle. [This assumption is not quite valid since Leg0 (1951) has shown that SD propagates more rapidly in the upper layers of the cortex so that at any given moment there will be less excess potassium in the extracellular space of the deeper cortical layers.] Assume further that, at the surface of the cortex, the excess potassium concentration (ie., the concentration of potassium in excess of that normally present) is always kept zero by circulation of the cerebrospinal fluid over the brain surface. Assume also (for mathematical simplicity) that, immediately below the cortical gray layers, the excess concentration is also kept at zero. With these assumptions about the initial and boundary conditions, the problem of the recovery of the cortex from spreading depression reduces to a consideration of the diffusion of potassium out of a thin slab of thickness, L, with a uniform initial concentration of ion and both faces kept at zero concentration for t>O. The ratio of the total amount of ion, Yi,( t ) , remaining in the slab after time t to the amount initially present, Yi,(0), is (see Crank, 1956, p. 45) :

If the thickness of the cortical region involved in SD is taken as 2 mm and the diffusion coefficient for potassium as 2 x 10-~cm2/sec (the value for potassium dihsion in saline solution), then it can be calculated that it will require about 6-7 min for !30% of the excess potassium to diffuse out of the cortical layers through which SD has passed. This calculation underestimates the time required for d i h sion in two ways: a. The effective diffusion coefficient for potassium in the extracellular space is probably not more than 10-20% of its value for saline on the basis of work by Shanes and Berman (1955b) on toad sciatic nerve. b. The excess concentration of potassium at the lower surface of the cortical mantle will not remain at zero [as assumed in Eq. (Zl)] but will increase with time.

ION FZUXES IN THE C N S

227

However, even from this simplified model, one can see that the time required for potassium to difise away from the cortical regions through which a wave of SD has passed, is too slow by at least a factor of 2-3 to account for the positive swing of the SPC. 2. Removal of Potassium by the Cortical Circulation Data discussed earlier suggest that during spreading depression the extracellular potassium concentration is at least 20 mM. If the extracellular space is taken to be about 5%of the brain volume, then the maximum of potassium in the extracellular space would be 0.75 X moles/gm brain (0.05 X (20-5) X X Assuming a cortical blood flow equal to that found in man (Kety and Schmidt, 1948) of 0.54 ml/gm brain/min, the average concentration of excess potassium in blood would be 1.4 mM (0.75 X 10-8/5.4 X if all of the potassium were removed in 1min. Since the excess potassium concentration is unlikely to be more than double the figure of 20 mM, given above, and in no case more than isotonic (130 mM), it appears likely that the normal blood flow through a region depolarized by SD would be adequate to remove the potassium released by SD rapidly enough to account for the positive swing of the SPC-assuming that the capillary endothelium became readily permeable to pota~sium.~ Rapid, brief changes in vascular permeability, of the sort necessary to account for cortical recovery from SD in this way, appear not to have been studied. Until such data are available, it is impossible to assess the role of the cerebral circulation in removing potassium from the extracellular space, following release during spreading depression.

3. Active Transport It is known that transport mechanisms exist in peripheral nerve that can move sodium and probably potassium against electrochemical gradients (Hodgkin and Keynes, 1955; Shanes, 1957). In the 31t is probable that there are alterations in blood flow in the involved regions of cortex during SD. At least 6 papers have been published reporting the occurrence of vasoconstriction and/or vasodilation during SD (Leiio, 1944b; Van Haareveld and Stamm, 1952; LeZo, 1954; Bure.5ovz5, 1956; Sonnenschein and Walker, 1956; Van Haareveld and Ochs, 1957). The evidence is conflicting and no unanimity of opinion exists concerning the extent or sipificance of these changes (see Marshall, 1959, pp. 262-263, for a summary of the evidence).

228

F. J. BRINLEY,

JR.

resting nerve these ion-pumps act to prevent changes in the internal ionic composition that would otherwise result from passive leakage of sodium and potassium down their concentration gradients. These pumps must also be able to protect the cell against the changes in internal sodium and potassium which occur during an action potential. If there were similar transport mechanisms within cortical neurons which normally subserved the function of keeping the internal ionic composition constant, such pumps could act to reaccumulate potassium following its loss to the extracellular space during spreading depression. If these transport mechanisms could act rapidly before the potassium released into the extracellular space has diffused away or entered the circulation, potassium reaccumulation within the cell would tend to return the extracellular as well as the intracellular potassium concentrations toward normal, The cortical cells could then repolarize as the extracellular potassium concentration dropped, thus terminating the negative SPC. It is important for the support of t h i s active-transport hypothesis to be able to demonstrate that potassium reaccumulation in the cortex is rapid enough to account for the cortical repolarization following the negative SPC which occurs with a time constant of a few tens of seconds. Recently Cummins and McIlwain (1961) have reported data which suggest that, in fact, cortical tissue may accumulate potassium at a sufficiently rapid rate to explain cortical repolarization following SD. These workers studied potassium uptake by thin (0.35-mm) slices of guinea pig cortex following a period of electrical stimulation of the slices with condenser discharges. Their results showed that after the end of stimulation, the tissue slices regained, within 1min, an amount of potassium equal to that which had been lost during stimulation. The rate of uptake across individual cell boundaries was undoubtedly even faster because Cummins and McIlwain used tissue slices and the potassium ions had to diffuse into the extracellular space of the tissue from the bathing solution before it could be picked up by the cells. There is no direct evidence that this accumulation of potassium, described by Cummins and McIlwain, is active transport in the sense that it represents net movement of ions against an electrochemical gradient; however, it appears that the process is associated with utilization of metabolic energy because oxygen consumption and lactate production are increased not only during stim-

ION FLUXES IN THE CNS

229

ulation but also for a short period of time thereafter. Actually, as these workers point out, the increased respiration and glycolysis persist after the end of stimulation for only 5-20 sec whereas the uptake of potassium after stimulation appears to last for a maximum of 1 min. However, the absolute time differences are not large, and the time resolution of the uptake process is about 1 min so that the observed difference in duration may not be significant. V. Effects of Drugs on Membrane Permeability of Central Nervous System Cells

No attempt will be made either to review or summarize the vast literature on central nervous system pharmacology. Instead, the discussion will be limited to a few selected experiments dealing with drug-induced changes in membrane permeability of cells in the mammalian nervous system. A. PRELIMINARY COMMENTS Radioactive isotopes have been used extensively to study the effects that drugs have on membrane permeability in peripheral nerve. However, when one attempts to apply such techniques to the intact (or nearly intact) cortex, there are some consequences of the histological and functional complexity of the brain which must be kept clearly in mind. 1. Histological Considerations It has already been pointed out in the theoretical section [see

Eq. (13)] that in any complicated tissue one may have difficulty in recognizing the intracellular component of the experimental outflux curve. Tracer studies done on the cortex suffer from the additional complication that only about 10-205& of the cortical population is neuronal ( Scholl, 1956). Consequently, the tracer outflux from cortical tissue may represent mostly contributions from glial cells. Furthermore, some drugs may act upon very small and restricted areas of nerve membrane. Any flux changes produced by such drugs, acting solely at such restricted loci, would produce very minor and probably undetectable alterations in the total tissue flux although the electrical activity of the cell may be profoundly altered, Therefore, one should not necessarily expect a close correlation between

230

F. J. BRI-Y,

JR.

tracer outflux from the cortex and electrical activity, either evoked or spontaneous. Nonetheless, as will be seen, the effect of certain drugs which alter electrical responses recorded from the cortex also produce changes in tracer outflux which are most easily explained in terms of a generalized effect on cortical cell membrane of the sort produced by the drug on other nerve membranes, 2. Functional Considerations

Since the normal cortex has considerable spontaneous activity, the outffux of a radioactive tracer from cortical tissue will reflect net movement into or out of active cells as well as exchange with resting cells. The relative contributions of each to the experimental outflux should remain constant as long as the amount of nervous activity remains constant. However, it is possible that some drugs may produce flux changes by an effect on nervous activity which changes the ratio of active to resting membrane but does not alter the properties of either.

B. LOCALANESTHETICS Since the effects of local anesthetics on membrane permeability have been studied in greatest detail in squid nerve and frog nerve, the conclusions drawn from such studies will be briefly summarized before describing experiments performed on central nervous system neurons. A more complete description of the effects of local anesthetics on nervous tissue is contained in a review by Shanes (1958a, b). 1. Effects on Resting Nerve

The most striking effect of procaine on peripheral nerve is a decrease in potassium permeability, manifest by a reduction in potassium influx and outflux in both toad and squid nerve fibers (Shanes, 195813, p, 1%).In neither species is there any significant alteration of the resting potential. 2. Effects on Action Potential

Voltage clamp data, obtained independently by Taylor (1959) and Shanes et al. (1959),show that the major action of procaine is to reduce the peak sodium and potassium currents which occur

ION FLUXES IN THE C N S

231

during a voltage step to about 40 and tN!Z of the normal values, respectively. From such data one would expect that procaine would tend to slow the rate of rise of the action potential and reduce its maximum amplitude. Such alterations of the action potential have been found in the purkinje fibers of calf and sheep heart (Weidmann, 1955)and in squid axon ( Shanes et al., 1959).

3. Iontophoretic Application of Procaine to Spinal Neurons Curtis and Phillis (1960) used multibarrel microelectrodes to record intra- and extracellular potentials from spinal neurons during the introduction of procaine hydrochloride by ionophoresis out of a microelectrode located in the vicinity of the cell. These authors found that procaine abolished spike activity without significantly affecting resting membrane potential of either the excitatory or inhibitory postsynaptic membrane potential. The effects were rapidly reversible within 1 to 2 min after local application of procaine ceased. This is in contrast to the slow reversal of procaine action seen in both squid nerve or rabbit cortex. These preparations require many minutes to recover after procaine is removed. Curtis and Phillis interpreted their data in accordance with the “stabilizing” concept of procaine action upon nerve membrane developed by Shanes, concluding that the sodium conductance (at a given membrane potential) was reduced to such an extent that the membrane depolarization required to give a regenerative inward sodium current was greater than that obtainable from an excitatory postsynaptic potential.

4. Topical Application of Procaine to the Cortical Surface Brinley et al. (1960a) studied the effect of procaine on K42outflux from the cortex by perfusing Tyrode’s solution containing procaine through a chamber placed on the cortical surface. Following introduction of the test solution, the radioactive outflux promptly dropped 251%.The concentration of procaine used (36 mM ) was approximately that which has been used for studies of the effect of topically applied procaine on cortical electrical activity but was 5-10 times greater than the usual concentrations applied to nerve for voltage clamp or ion flux studies. The slow reversibility of the procaine effect on both the potassium efflux from, and electrical activity

232

F. J. BRINLEY, JR.

of, mammalian cortex may be only a consequence of the larger concentrations used, but it may also indicate a genuine species difference in the response to procaine. Subject to the restrictions on interpretation mentioned in the introduction to this section, it is concluded from the potassium outflux experiments that procaine acts on the mammalian cortex in a manner similar to the effects of local anesthetics on peripheral nerve, i.e., it tends to stabilize cell membrane, both resting and active. The tracer flux evidence that procaine depresses cortical excitability is in accord with a large body of electrophysiological data based on the studies of evoked potentials (see Goldring et al. 1958,for discussion and further references). C. 7-AMINO-N-BUTYRIC ACID

ydminobutyric acid (GABA) has been extensively studied in an attempt to identify this compound as the inhibitory transmitter in the central nervous system. However, in spite of numerous electrophysiological studies of the action of GABA on the cortex, there is still no general agreement as to whether GABA specifically blocks cortical excitatory potentials or just generally depresses neuronal membrane. The first hypothesis has been discussed recently by Purpura ( 1959) and Purpura et al. ( 1959). Support for the latter hypothesis, nonspecific depression, is provided by the work of Curtis et d.(1959) and Curtis and Watkins (1960) on spinal cord neurons of the cat. By use of the microelectrode injection technique, these workers were able to show that GABA, appIied iontophoretically in the vicinity of a single spinal cord neuron, depressed the excitability of the neuron to orthodromic and antidromic stimulation as well as to direct electrical stimulation through the intracellular pipette, although the membrane potential was not altered. In addition, GABA also abolished or depressed, to an equal extent, inhibitory as well as excitatory postsynaptic potentials. The precise manner in which GABA depresses excitability in neuronal membrane is not clear. Although an increase in membrane conductance has been suggested by Curtis and Watkins (1960) on the basis of their data, such a change has not yet been related to a change in the membrane permeability of any particular ion or group of ions.

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233

The action of GABA upon K4*outflux from the intact rabbit cortex was investigated by Brinley et al. (1960a) using the perfusion chamber arrangement. This study was undertaken with the thought that if GABA did depress cortical cells either by depolarizing or by “stabilizing” them (in the manner described by Shanes), then one might expect to find significant alterations in potassium flux-an increase in potassium outflux if the cell were depolarized; a decreased outflux if the cell membrane were “stabilized.’’ Actually it was found that surface perfusion of the cortex with 0.1 M GABA solution resulted in a transient flux increase (maximum increase of 20%)which lasted only about 2.5 min even though the drug remained on the cortex indefinitely. Several possible sources of error in the technique that might have lead to an artifactual flux increase were investigated experimentally and found not to be the cause of this transient increase in outflux. It was concluded, therefore, that the observed increase in potassium outfiux was a genuine consequence of topical GABA application. Since the flux increase persisted only for a limited time, even though the GABA remained on the cortex, one cannot argue from these data that GABA had any permanent effect on membrane permeability. The tentative explanation advanced is that GABA interacts in some way with the membrane, possibly by displacing potassium from sites on the membrane. This interpretation of the data is compatible with the conclusion of Curtis and Watkins (1WO) that GABA (as well as other depressant or excitant amino acids) may combine with receptor sites on the membrane. It should be noted that procaine and GABA act upon the spinal motoneuron and cortical cell membrane in quite different ways, although their effects upon evoked potentials can be similar, e.g. reversal of the surface negative wave of the superficial cortical response to surface positive. It appears that the demonstration of similar, drug-elicited effects on evoked electrical activity need not imply similar effects upon cell membranes.

D. METABOLIC INHIBITORS 1. General Consideration of lnhibitor Studies Substances which interfere with metabolism such as dinitrophenoI, azide, iodoacetic acid, etc. have been widely used to study ~

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F. J. BRINLEY, JR.

transport systems which depend upon a supply of metabolic energy for their operation. Despite the usefulness of inhibitor studies, there are certain limitations which should be mentioned. a. Permeability Considerations. The mere application of a metabolic inhibitor to the bathing solution surrounding an excised tissue preparation is no guarantee that it will reach the region of the cell where the energy producing biochemical reactions occur. An adequate concentration of inhibitor at the locus of action may never be reached if the cell membrane is sufficiently impermeable to the agent. Caldwell (1960) has observed an effect of this sort in squid axon. Addition of 0.2 mM dinitrophenol (DNP) to seawater at pH 8 did not affect either the sodium efflux or axoplasm adenosine triphosphate (AT.€') content of axons immersed in this seawater. However, when similar experiments were performed using seawater at pH 6.5-7 there was a marked reduction in both axoplasm AT" content and sodium efflux. Caldwell found that at the lower pH, 7 times more DNP entered the axon than when the preparation was immersed in seawater at pH 8. Presumably, at the higher pH, insuflScient DNP entered the axon to affect ATP synthesis, although at pH 8, the concentration of DNP was 4 x l W moles/kg wet axoplasm, which is high enough to uncouple phosphorylation in most in i)&o biochemical systems. b. Multiple Energy Sources. The results of experiments performed with metabolic inhibitors may be misinterpreted if the energy producing reactions blocked by the inhibitor are not the energy sources for the particular transport process under study. This possibility is also well illustrated by the experiments of Caldwell, just cited. He found that 0.2 m M DNP reduced the axoplasm Content of arginine phosphate at either pH 6-7 or 8. Had the results of the arginine phosphate and sodium flux determinations at the higher pH been available without a knowledge of the ATP level, one could have, erroneously, concluded that sodium outtlux was not dependent upon metabolic energy. c. Changes in Passive Fluxes. Although most of the emphasis in metabolic inhibitor experiments has been upon the changes produced in rates of ion movement against electrochemical gradients, sigdcant alterations in passive ion movements also occur. Some of the relevant data are summarized in Table V.It appears that in some

TABLE V CHANGES IN IONFLUXES Potassium Tissue Toad sciatic nerve Sepia axon Sepia axon

Condition Resting

Agent

Anoxia

Resting or 0.2 m M DNP lightly stimulated Post stimulation 0 . 2 mM DNP

Rat sympathetic Resting ganglion

Glucose lack 0 . 2 mM DNP

Sodium

Influxa OutfluxO Influx0 Outfluxa

Source

0.3

1.5

1.5

0.9

Shanes (1957),Table 22 and Fig.

0.47

-

1.16

-

0.14

1.40

54. Hodgkin and Keynes (1955): Table 5, Exp. 1 and 2; Table 7, Exp. 6 and 7. Hodgkin and Keynes (1955): Table 4; Table 5, Exp. 3 3 ; Table 6; Table 7. Brinley and Larrabee (1961).

1.24 1.22

1.35 1.40

0.57 4 . 1 -

-

-

6

c

b!

8

0

3

236

F. J, BRINLEY, JR.

cases the per cent changes in passive fluxes may be nearly as great as those which occur in the active fluxes and, furthermore, changes in passive fluxes may conceal changes in the active component of the total flux. Shanes (1957) has reported that the sodium outfIux in amphibian nerve is not affected by any combination of anoxia, iodoacetate, or dinitrophenol (see Table V, line 1).One might have supposed, therefore, that sodium outflux in amphibian nerve is essentially passive; however, on the basis of other experiments Shanes has concluded that about 40% of the sodium oudlux does actually depend upon energy sources, It is the author’s opinion that the assumptions underlying experiments designed to distinguish between active and passive transport by use of metabolic inhibitors should be critically re-evaluated. If one accepts the proposition that an active flux depends directly on some energy-producing process and a passive flux does not, then the statement that an inhibitor has altered a passive flux is self contradictory unless one assumes that the inhibitory substance has an effect upon membrane properties quite apart from its effect in disrupting the normal production of metabolic energy. Such an additional effect of inhibitors is possible, but is not generally considered to occur. Attention should be given to the possibility that in nervous tissue a portion of the resting energy production is used for continual resynthesis of the neurolemma. It is possible that t h i s energy requirement, which maintains the passive permeability properties of the membrane, exists independently of any energy requirements for ion “pump priming.” Interference with the normal production of energy by the use of inhibitors would be expected, in this situation, to cause changes in membrane permeability as well as interfering with any particular unidirectional flux. 2. Inhibitor Studies in C d e x and Ganglion a. Cortex. Davies ( 1953), using a technique for local perfusion of surface blood vessels in cats, has reported that 1-5 X M DNP depolarized the perfused region within 4 min. The difference between the effects of DNP in cortex and peripheral nerve where the inhibitor seems to alter fluxes rather than membrane potential is probably due to important differences in the compactness and size of the individual elements in the tissues being studied. Depolarization of nerve membrane by treatment with inhibitors has been pro-

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ION FLUXES IN THE CNS

duced in both frog and squid nerve (Shanes, 1958a, b; Hodgkin and Keynes, 1955) but in each case it was shown to be an experimental artifact due to accumulation of potassium ions in a restricted extracellular space. Rapid depolarization subsequent to DNP exposure does not occur in well washed Sepia or Loligo axons. In these axons there may be a slow depolarization, developing over a period of many minutes, which could be explained on the basis of a slow loss of potassium and gain of sodium because the ion pumps had been poisoned. In toad nerve some residual depolarization remained even in washed, desheathed preparations but whether this was due to the difficulty of completely removing excess potassium from the extracellular space or other factors is not clear. On the basis of these observations on peripheral nerve, the cortical depolarizations observed by Davies after perfusion with DNP could be explained as resulting from potassium accumulation in a restricted extracellular space of the sort that probably exists in the brain. There is, however, another equally valid explanation. Because of the large surface to volume ratio of the small axon terminals and dendrites in the cortex, a reduction of the active sodium outflux and potassium influx by inhibitors will lower the intracellular potassium concentration and raise the sodium concentration relatively rapidly, thus causing membrane depolarization and loss of excitability. Some idea of the rapidity with which the intracellular concentrations might change in the small fibers of the cortex can be gotten from the following simple calculations. Data from the work of Hodgkin and Keynes (1955) shows that in cephalopod axons the net leakage of potassium in fibers poisoned by DNP is about 25 x 10-l2moledcm? membrane/sec. Assuming that the net loss of potassium is roughly the same for mammalian dendrites, and taking the average radius as 0.5 p and the internal potassium concentration as 140 mM, we have for the per cent intracellular potassium lost per second initially: loo

MA

25 X

ci,v = 140 x

10-6

Xk L x rr2L

x

100 =

3.5

x

10-6

r

A similar computation for sodium indicates that the intracellular sodium concentration will increase about 1.5Wsec. Even if the magnitudes of the leaks are overestimated by a factor of 5, about 35%of

238

F. J. BRINLEY, JR.

the intracellular potassium will have been lost, and the intracellular sodium increased by some 70%, at the end of 4 min. These changes appear of the proper order of magnitude to account for the observations of Davies (1953) that depolarization and loss of excitability occur within minutes after cortical perfusion with DNP. (It is interesting to note that McIlwain and Gore (1951) found that the creatine phosphate level of cortical tissue slices was markedly reduced winthin 2 min after the addition of DNP to the bathing media), b. Ganglion. Potassium fluxes in excised rat sympathetic ganglion have also been studied during interference with normal metabolism either by withdraw of glucose or addition of dinitrophenol to the bathing solution ( Brinley and Larrabee, 1961). Either procedure will increase both influx and outflux by about 20 and 40%, respectively. It can be argued from these data that there is a small component of active inward potassium transport occurring in the unpoisoned ganglion. If outflux is all passive, and if the percentage increase in the passive component of influx caused by an inhibitor is equal to the percentage increase in passive outflux, then the following equations hold (see Table V) ,

+ +

resting ganglion : x y = 5.07 (234 poisoned ganglion: 1.4 x 0 = 1.2 X 5.07 (23b) where 2 is the resting passive influx and y, the metabolically dependent influx [the average resting influx in sheathed ganglia is 5.07 mpmoles/min/mg ( dry weight) 1, From these data and assumptions, the metabolically dependent influx can be calculated to be about 10%of the total Mux. This interpretation, however, is by no means unique. It is possible that some membrane depolarization occurred which would have increased the outflux relatively more than influx, without requiring any additional assumptions about active transport. VI. Summary

As might be expected, whole brain exhibits some of the membrane properties of any nervous tissue such as the ability to concentrate potassium and exclude sodium or to alter membrane permeability to these ions in response to a change in membrane potential. However, there are some significant differences. The brain, by virtue

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of having a very small extracellular space, appears to be a far more compact tissue than peripheral nerve. This histological peculiarity may be partly responsible for the phenomenon of cortical spreading depression. The small extracellular space may also allow electrical activity of one neuron to influence the excitability of adjacent neurons by altering the extracellular potassium concentration in the small cleft between them. This nonsynaptic modification of excitability may also be shared by glial cells which might leak potassium into or remove it from the extracellular space under appropriate conditions. The experimental data from tracer experiments relating to the effects of drugs on membrane properties of brain cells is far too sketchy for any definite conclusions to be made at this time. It is this reviewer’s opinion that the separation of ionic transport processes into active or passive fluxes by the use of drugs is less satisfactory in mammalian nerve tissue than it appears to be in other tissues. The practical difficulties involved in attacking these sort of problems with tracer techniques are twofold. First, the histological complexity of the cortex makes the creation of a practical mathematical model for ion kinetics difficult. Second, the small size and stringent metabolic requirements of the various cell types in the brain make it difficult to study the intact brain without producing gross and irreversible alterations in function. The recent papers by Crain (1956) and Hild and Tasaki (1962) dealing with the electrical properties of isolated nerve and glial cells in tissue cultures suggest that one might use these preparations to study the kinetics of transmembrane ion and molecule movements. If the internal concentrations of ions and membrane areas could be measured directly in such preparations, then one could calculate transmembrane fluxes accurately from tracer experiments. Such information would complement the electrophysiological data already available for whole brain, and conclusions concerning membrane transport processes (as well as other membrane phenomena, such as drug effects) in the brain could rest upon independent experimental support rather than upon inferences drawn from other tissues. REFERENCES Allen, J. N. (1955).A.M.A. Arch. Neurol. Psychiat. 73, 241. Ames, A., 111, and Nesbett, F. J. (1958).J . Neurochem. 3, 116. Brink, F. (1954).Pharmacol. Revs. 6,243.

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Brinley, F. J., Jr,, and Larrabee, M. G. (1961). Unpublished results. Brinley, F. J., Jr., Kandel, E.R., and Marshall, W. H. (1960a). J. Neurophysbl. 23,237. Brinley, F.J., Jr., Kandel, E. R., and Marshall, W.H. (1960b). 1. NeurophySiol. 23, 240. BureH, J. (1956). J. Neurochem. 1, 153. BureH, J. ( 1960). Physiol. B o h m o s h e n . 9,202. Burel, J. ( 1962). Personal communication. BureH, J., and BureHovB, 0. (1956). Physlol. Bohemoslouen. 5, 195. BureH, J., BureHovB, O., and Kfivhek, J. (1960). In “Structure and Function of the Cerebral Cortex” (D. P. Tower and J. P. Schade, eds.), p. 257. Proceedings of the 2nd International Meeting of Neurobiologists. Elsevier, Amsterdam. BureHovA, 0. (1956). Physlol. Bohemoslouen. 6, 1. Caldwell, P. C. ( 1960). J . Physiol. (London) 152, 545. Crain, S. M. (1956). J . Comp. Neurol. 104, 285. Crank, J, ( 1956). “The Mathematics of Diffusion.” Clarendon Press, Oxford. Cummins, J. T., and McIlwain, H. (1961). Blochem. J . 79, 330. Curtis, D. R., and Phillis, J, W. (1960). J. Physiol. (London) 153, 17. Curtis, D. R., and Watkins, J. C. (1960). J. Neurochem. 6, 117. Curtis, D. R., Phillis, J. W., and Watkins, J. C. ( 1959). J. Physiol. (London) 148, 185. Darden, E. B., Jr. (1960). Am. J. Physiol. 198,709. Davis, P. W. (1953). Abstr. 19th Intern. Physiol. Congr. held in Montreal, Canada, p. 300. de Lorenzo, A. J. D. (1961).Bull. Johns Hopklns Hosp. 108,258. de Robertis, E., and Gerschenfeld, H. M. (1961). Intern. Reu. Neurobfol. 3, 1. Eccles, J. C. (1957). “The Physiology of Nerve Cells.” Johns Hopkins Press, Baltimore, Maryland. Eccles, J. C., Fatt, P., and Koketsu, K. (1954). J. Physiol. (London) 128, 524. Eccles, R. M. ( 1955). J . Physiol. (London) 130, 572. Eichelberger, L., Kollros, J. J., Walker, A. E., and Roma, M. (1949). Am. I . Phydol. 158, 129. Fleckenstein, A. ( 1951). Arch. exptl. Pathol. Pharmakol. Naunyn Schmledeberg‘s 212, 416. Folch, J., Lees, M., and Sloane-Stanley, G. H. (1957). In “Metabolism of the Nervous System” (D. Richter, ed.), p. 174. Pergamon, New York. F d , K., and Fuortes, M. G. F. (1956). J. Phydol. (London) 131,424. Frank, K., Fuortes, M. (3. F., and Nelson, P. G. (1959). Sclsnce 130 (3366). 38. Frankenhaeuser, B., and Hodgkin, A. L. (1957). 1. Physfol. (London) 137,360. Goldring, S., OLeary, J. L., and Huang, S. H. (1958). Electroeneephalog. and Clin. Neurophyslol. 10, 663. Gossweiler, N., Kipfer, K,, Poretti, G., and Rummel, W. (1954). Arch. ges. Physiol. Pfliiger’s 280, 154. Grafstein, B. (1956). J . Neurophysiol. 19, 154. Guttman, R. (1940). J . Gen. Physiol. 23, 343.

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Harris, E. J., and Burn, G. P. (1949). Trans. Faraday SOC.45,508. Harris, E. J., and McLennan, H. (1953). J. Physiol. (London) 121,629. Hild, W., and Tasaki, 1. (1982). J . Neurophysiol. 25,277. Hodgkin, A. L. (1951). Biol. Revs. 26, 339. Hodgkin, A. L. (1958). Proc. Roy. SOC.B148, 1. Hodgkin, A. L., and Katz, B. (1949). J. Phydol. (London) 108,37. Hodgkin, A. L., and Keynes, R. D. (1953). 3. Physiol. (London) 119, 513. Hodgkin, A. L., and Keynes, R. D. (1955). J . Physiol. (London) 128,28. Horstmann, E., and Meves, H. (1959). 2. ZeUforsch. u. mikroskop. Anat. 49, 589. Jaeger, J. C. ( 1941). Phil. Mag. 32,324, 332333. Kandel, E. R., Spencer, W. A., Brinley, F. J., Jr. (1961). J. Neurophysiol. 24, 225.

Katzman, R., and Leiderman, P. H. (1953). Am. J . Phydol. 175, 283. Kety, S. S., and Schmidt, C. F. (1948). J. Clin. Invest. 27,484. Keynes, R. D. (1951). J. Physiol. (London) 114, 119. Keynes, R. D. (1954). Proc. Roy. SOC.B142,359. Keynes, R. D., and Lewis, P. R. (1951). J. Physiol. (London) 113, 73. Krebs, H. A., Eggleston, L. V., and Terner, C. (1951). Biochem. J. 48,53. KPivAnek, J. (1961). J. Neurochem. 6, 183, 188. KPivhek, J., and Bum”, J. ( 1960). Phydol. BohemosZmen. 9,494. Kmjevic, K. ( 1955). J. Physiol. (London) 128, 473. LeHo, A. A. P. (1944a). 3. Neurophysiol. 7,359. LeHo, A. A. P. (1944b). J. Neurophysiol. 7, 391. L&o, A. A. P, (1951). EZectroencephaZog. and Clin. Neurophysiol. 3, 315. LeFio, A. A. P. (1954). Anais. acad. brad. cienc. 26, XXII-XXIII. L&o, A. A. P., and Morrison, R. S. (1945). J. Neurophysiol. 8, 33. LeHo, A. A. P.,Martins-Ferreira, H., and Marshall, W. H. (1959). Unpublished observations quoted by Marshall, W. H. (1959). Physiol. Reu. 39, 239, 254. Lowry, A. H., Hastings, A. B., McCoy, C. M., and Brown, A. N. (1946). J . Gerontol. 1 ( I ) , 345. McIlwain, H., and Gore, M. B. R. ( 1951). Biochem. J. 50,24. Manery, J. F. (1952). In “Biology of Mental Health and Disease” (Twentyseventh Annual Conference of the Millbank Memorial Fund), p. 1%. Harper ( Hoeber ), New York. Marshall, W. H. (1959). Physiol. Revs. 39,239. Nims, L. F. (1959). Yale J. Biol. and Med. 31, 373. Pappius, H. M., and Elliott, K. A. C. (1954). Can. J. Biochem. and Physiol. 32, 484. Pappius, H. M., and Elliott, K. A. C. (1958a). Can. J. Biochem. and Physbl. 34,1007. Pappius, H. M., and Elliott, K. A. C. (1956b). Can. J. Biochem. and Physbz. 34, 1053. Pappius, H. M., and Elliott, K. A. C. (1962). Can. J. Biochem. and Physbl. 40, 885. Phillips, C. G . (1958). Quart. J. Ezptl. Physbl. 41, 70.

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Purpura, D. P. (1959).Intern. Reu. Neurobiol. 1,48. Purpura, D.P.,Girado, M., Callan, D. A., and Grundfest, H. (1959).1. Neurochem. 3, 238. Rall, D. P., and Patlak, C.S. (1962).Federation Proc, 21, 324. Robertson, J. S. ( 1957).Physiol. Reus. 37, 133. Rothenberg, M.A. (1950).Biochem. et Biophys. Acta 4, 96. Scholl, D. A. ( 1956).“The Organization of the Cerebral Cortex.” Wiley, New York. Shanes, A. M. (1957).In “Metabolic Aspects of Transport Across Cell Membranes” (Q. R. Murphy, ed.), p. 127. University of Wisconsin Press, Madison, Wisconsin. Shanes, A. M. (1958a). Pharmacol. Reus. 10, 59. Shanes, A. M. (1958b).Pharmncol. Revs. 10, 165. Shanes, A. M.,and Berman, M. D. (1955a).1. Cellular Comp. Physiol. 45, 178. Shanes, A. M.,and Berman, M. D. (1955b).J . Celluh Comp. Physbl. 45,199. Shanes, A. M., Frekgang, W. H., Grundfest, H., and Amatniek, E. (1959).J . Gen. Physiol. 42, 793. Solomon, A. K. (1960).In “Mineral Metabolism” (C. L. Comar and F. Bronner, eds.), Chapter 5. Academic Press, New York. Sonnenschein, R. R., and Walker, R. M. (1956).Federatson Proc. 15, 175. Steinbach, H. B., Spiegelman, S., and Kawata, N. (1944).J. Cellular Comp. Physbl. 24, 147. Streicher, E., and Press, G. D. (1960).Federation Proc. 19, 285. Tasaki, I. (1960).Science 132, 1661. Taylor, R. E. (1959).Am. J . Physiol. 196,1071. Tschinigi, R. D.,Inanaga, K., Taylor, J. L., Walker, R. M., and Sonnenschein, R. R. (1957).Am. I. Physiol. 190,557. Van Haareveld, A. ( 1960).J . Neurochem. 3,300. Van Haareveld, A,, and Ochs, S. (1957).Am. J. Physiol. 189,159. Van HaareveId, A.,and SchadB, F. P. ( 1959).J . Cellular Comp. Physiol. 54,65. Van Haareveld, A,, and Stamm, J, S. (1952).J. Neurophyslol. 15, 487. Van Haareveld, A., and Stamm, J. S. (1955).Electromcephalog. and CNn. Neurophysiol. 7, 363. Wang, J. H., Robinson, C. V., and Edelman, I. S . (1953). J . Am. Chem. SOC.

75, 466. Weidmann, S . ( 1955).J. Physiol. (London) 129,568. Woodbury, D. M. (1958).In “Biology of Neuroglia” (W. F. Windle, ed.), p. 120. Charles C Thomas, Springfield, Illinois. Woodbury, D. M., Timiras, P. S., Koch, A,, and Ballard, A. (1956). Federation Proc. 15, 501. Yannet, H.,and Darrow, D. C. (1938).J . Clin. Inuest. 17, 87. Yeandle, S. (1961).Abstracts, 5th Annual Meeting of the Biophysical Society. Code No. FE 11. Zierler, K. L. (1980).Am. J . Physiol. 198, 1086.

INTERRELATIONSHIPS BETWEEN THE ENDCXRINE SYSTEM AND NEURlOPSYCHlATRYl By Richard P. Michael and James

L. Gibbons

Department of Psychiatry, Institute of Psychiatry, Maudsley Hospital, London, England

. . . . . . . . . . . . . . . . . . . . . Experimental and Physiological Foundations . . . . . .

Introduction

I. Adenohypophysis, the Adrenal Cortex, and Emotion A. B. Human Clinical Studies . . . . 11. The Thyroid and Psychiatry . . . . A. Thyroidal Function in Mental Disorder B. Psychiatric Changes in Thyroid Disease References . . . . . . . . .

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. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

. . . . .

243 245 245 258 282 282 288 292

Introduction

The association between certain critical periods in life, such as puberty, pregnancy, and menopause, and changes in mental state has directed speculation from the earliest times to the possible effects of “humours” on the mind. Although it is only very recently that endocrinologists have interested themselves in the effects of hormones on the brain, it is true to say that for the past 70 yr psychiatrists have treasured the illusion that the solution of several etiological problems in psychiatry only awaited advances in the endocrinological field. At one time, Kraepelin (1896) regarded dementia praecox as basically an endocrine disorder, and Freud ( 1905) observed that certain disorders, which he was seeking to understand by psychological means, might eventually be treated successfully with hormones. A whole series of speculative treatments has at one time or another been attempted with every variety of endocrine 243

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RICHARD P. MICHAEL AND JAMES L. GIBBONS

preparation. The inevitable failure of such methods caused endocrinological psychiatry ( a term introduced by Laignel-Lavastine in 1908) to fall into disrepute, During the twenties, an endocrine psychology was evolved which attempted to explain the variations of normal personality on a hormonal basis, hence, the introduction of such meaningless terms as a “thyroid type” and a “pituitary type.” Despite the period of disillusionment, a feeling has persisted that endocrinology would contribute importantly to psychiatry when appropriate advances in methodology had been made. Within the last decade, such methods have indeed become available and, after the first flush of enthusiasm, in which it became apparent that the problem of schizophrenia would not be solved overnight, attention has been directed with increasing profit to specific aspects of the relationships between endocrine function and mental disorder. The introduction of ACTH and cortisone into therapeutics provided a very great impetus to basic research both in medicine and in endocrinology. This activity soon spread, particularly as the concept of stress developed, to psychiatry and psychosomatics. The interrelationships between endocrinology and psychiatry are obviously too vast a field to be covered in a short review, but certain areas are developing rapidly and it is to these growing points we have addressed ourselves. The main aim has been to consider the experimental basis for the control of changes in pituitary-adrenocortical activity as it impinges upon psychiatry; the role of other endocrines has been merely touched upon. The subject can be considered from 2 main points of view: (i) the psychiatric symptomatology occurring in endocrine disorder; and (ii) the changes in endocrine activity occurring in psychiatric conditions. The former viewpoint was that taken by Bleuler (1954) in his extensive monograph and has since been the subject of several studies by the Zurich school. The fascinating topic of the possible psychogenesis of endocrine disorder, though most important, could not be dealt with in the present article. When considering the literature of a field in which so much has been written, the authors have been at some pains to exclude those reports which either fail to advance the issue or whose methodology appears open to question. * The authors thank Prof. Sir Aubrey Lewis for the opportunity of referring to case material from the Department of Psychiatry.

ENDOCRINES AND NEUROPSYCHIATRY

I. Adenohypophysis, the Adrenal Cortex, and Emotion

A. EXPERIMENTAL AND

PHYSIOLOGICAL FOUNDATIONS

1. Endocrine Aspects a. Characterization of ACTH The dependence of the adrenal cortex on the pituitary gland was first clearly demonstrated by Smith (1927, 1930), who showed that in the rat hypophysectomy caused an adrenocortical atrophy which could be prevented to some extent by transplanting anterior pituitary tissue. Evans (1933) and Collip d al. (1933) showed that this atrophy could also be prevented by daily injections of anterior pituitary extract. Attempts to isolate the hormone concerned were made by Li et al. (1943), and Sayers et al. ( 1943), and later, Astwood et al. (1951) prepared more active material using an adsorption technique on oxycellulose. Li et aZ. (1954) and Bell (1954) have since isolated pure peptides from the sheep and pig gland with high corticotropic activity. Subsequently, these workers (Bell, 1954; Howard et d.,1955; Li et al., 195.5; Li, 1956) established pig and sheep corticotropin as straight-chain polypeptides, the latter containing 39 amino acids with a molecular weight of approximately 4,500.

b. Determination of ACTH Activity In assessing pituitary ACTH activity, use has been made of changes occurring in the adrenal cortex, for example, hypertrophy, depletion of sudanophilic lipids, depletion of adrenal cholesterol, and ascorbic acid (Sayers et al., 1948; cf. also Tepperman et al., 1943; Sayers, 1950). Decreases in the number of circulating lymphocytes and eosinophils also depend upon the activity of the pituitaryadrenocortical system (Dougherty and White, 1947; Forsham et d., 1948; Dougherty, 1952), a measure which has been widely used in the clinical field. In the laboratory, McDennott et al. (1950) established close agreement between the eosinopenic responses and adrenal ascorbic-acid depletion. Attempts to estimate ACTH chemically in normal human blood have been largely unsuccessful (Harris and Li, 1954). Bioassay methods, using adrenal ascorbic-acid depletion following the intravenous administration of test-blood into

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hypophysectomized rats, ( Sydnor and Sayers, 1952, 1953) have proved more satisfactory (see Sayers, 1955). Short-term changes in blood ACTH levels have been studied by means of a cross-circulation technique between intact and hypophysectomized rats which is sufficiently sensitive to detect the ACTH activity in as little as 10 ml of blood (Brodish and Long, 1956). With this technique it has been demonstrated that blood ACTH activity is no longer detectable 6 hr after the severe stress of either unilateral or bilateral adrenalectomy. In the latter instance, the disappearance of ACTH cannot be due to increased blood levels of adrenocortical hormones-a finding which is not in line with the simple hypothesis which supposes that the level of circulating corticoids controls the rate of ACTH release. The measurement of ACTH by bioassay has been further developed by Hodges and co-workers who avoid the need for hypophysectomy by using rats whose endogenous ACTH secretion is blocked with either deoxycorticosterone acetate, hydrocortisone, or prednisone ( Hodges, 1954, 1955; Barrett et al., 1957; Hodges and Vernikos, 1959). The intravenous administration of ACTH to hypophysectomized rats and measurement of the increase in the concentration of ascorbic acid in adrenal venous blood, rather than the content in the gland, has been developed into a sensitive bioassay by Munson and Toepel (1958). The increase in the formation of corticoids in vitro by incubated adrenal cortex to which ACTH is added has also been satisfactorily used for bioassay purposes (Saffran et al., 1952; Saffran and Schally, 1955; Safian and Saffran, 1959). Bush (1951) demonstrated that ACTH caused a marked rise in the output of corticosterone and 17hydroxycorticosterone in adrenal venous blood and showed that cortisol is the main hormone secreted by the human adrenal (Bush and Sandberg, 1953). These substances were also found in normal human urine after ACTH administration ( Mason, 1950; Schneider, 1950; Zaf€aroni d al., 1950). Vogt has estimated the corticoids in adrenal venous blood by both biological (Vogt, 1943) and chemical methods (Vogt, 1957). Such direct determinations in the adrenal effluent must clearly be a more reliable index of adrenocortical and pituitary ACTH activity than determinations of corticoids in peripheral blood (Samuels et al., 1957). Following the work of Vogt, it was shown by Rauschkolb et al. ( 1954) that ACTH increased the output of cortisol in the adrenal effluent blood of hypophysectomized dogs -an effect used by Nelson and Hume (1954, 1955) for bioassay

ENDOCRINES AND NEUROPSYCHIATRY

247

purposes, A linear relationship was found by Cohen and Kleinberg (1959) between the dose of ACTH administered and plasma corticoid concentrations; this has been used to standardize ACTH preparations. c. Determination of Adrenal Corticosteroids

(1) In PEarma. A major advance in the development of this field has been due to the introduction of chemical methods of sufficient specificity and sensitivity for the estimation of adrenal steroids in blood. Most frequently used, particularly in psychiatric studies, is the Porter-Silber reaction (Porter and Silber, 1950), first elaborated into a usable method by Nelson and Samuels (1952) and Eik-Nes et al. (1953). This has been subjected to a critical evaluation by Harwood and Mason (1956), who determined the precision of the 3 steps which comprise the method. Another and simpler version was introduced by Silber and Porter (1954) and subsequently modified by Peterson et al. (1957); the Peterson technique has been found satisfactory in our laboratory. Under normal conditions cortisol accounts for 95% of the unconjugated Porter-Silber chromogens in human blood. Methods have now been developed which separately measure cortisol and corticosterone [see reviews by Braunsberg and James (1961) and Bush (196l)l. Sweat devised a technique for the measurement of cortisol and corticosterone in which the steroids are isolated from plasma by silica gel chromatography (Sweat, 1954a) and estimated by measurement of sulfuric-acid-induced fluorescence (Sweat, 195413, 1955). Bondy et a2. (1957) using paper chromatography isolated cortisol from plasma and estimated it by fluorimetry. 0 la marker. A Losses were corrected for by the use of C i 4 - ~ r t i ~as more specific, but more elaborate technique, also developed by Peterson, involves a dual isotope derivative method in which cortisol, isolated by paper chromatography, is acetylated with tritiated acetic anhydride of known specific activity. The cortisol acetate is purified further and estimated by tritium counting and losses are compensated for by the use of C14-cortisolas a marker throughout the procedure ( Schedl et d.,1959). ( 2 ) Plasma Corticoids; Protein Binding and Coniugutes. The plasma level of cortisol, which reflects the balance between production by the gland and disposal by the tissues, is a useful index of adrenocortical activity and is particularly applicable to the study of

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RICHARD P. MICHAEL AND JAMES L. GIBBONS

short-term changes (Perksy et at., 1958). In certain circumstances, however, plasma levels can be misleading since the methods referred to measure total plasma cortisol, the bulk of which is bound to protein, while it is probable that only the free steroid is biologically active ( Mills, 1961) , An increased plasma level of cortisol may represent increased protein binding rather than an increased output of steroid by the adrenal cortex, Such an effect, an increase in bound steroid, can be produced by the administration of estrogens and appears to be responsible for the elevated blood levels found in pregnancy. Moreover, a rise in blood cortisol has been produced by the administration of estrogens to an Addisonian patient on fixed maintenance doses of steroid (Taliaferro et al., 1956; Mills et al., 1960; Peterson et al., 1960; Wallace and Carter, 1980; Daughaday et al., 1961). Approximately 45% of the total 17-hydroxycorticosteroids in plasma are present in a conjugated form, mainly as glucuronides (Brown et al., 1957). This fraction is not measured by the NelsonSamuels method. Bongiovanni (1954) introduced a technique for its estimation after hydrolysis with p-glucuronidase. It has been suggested by Reddy et al. (1956) that the total plasma level of 17hydroxycorticosteroids ( conjugated and unconjugated ) might afford a measure of adrenal steroid production which would be independent of the rate of utilization, With rapid rates of utilization, the proportion of conjugated steroids would be high; with a slow rate of utilization it would be low. These expectations were confirmed in a study of hyper- and hypothyroidism (Brown et al., 1958). Determination of total 17-hydroxycorticosteroids has not been widely used in psychological investigations and recently Steenburg et al. (1961) have shown that the blood level of conjugated 17-hydroxycorticosteroids depends on factors other than the rate of metabolism of cortisol. (3) In Urine. Adrenal steroids have been measured in urine as well as in blood, and the amount of free cortisol excreted is a good index of adrenocortical activity (Greaves and West, 1960), although it is not routinely measured. The usual methods estimate various metabolites of cortisol, which are present in urine in the conjugated form and which are measured after hydrolysis. The methods of Reddy d al. (1952) and of Glenn and Nelson (1953) depend on the Porter-Silber reaction and both have been widely used in the United

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249

States. The results are expressed BS 17-hydroxycorticosteroids. In Britain and Europe, the method devised by Norymberski et al. (1953) is more often used; it measures a rather wider range of metabolites, expressed as 17-ketogenic steroids. These techniques have been critically reviewed by Loraine (1958) and compared by Golub et al. ( 1958) . In general, the Glenn-Nelson and Norymberski techniques correlate well. It has also been shown that, when outputs are high, as in Cushing’s disease, the urinary excretion of 17-ketogenic steroids correlates with the adrenal output of cortisol as measured by isotope dilution (Cope and Black, 1959). That this latter, empirical technique provides a true measure of adrenal output has now been demonstrated mathematically (Laumas et d.,1981a, b; Gurpide et al., 1962). 2.

The Influence of the Central Nervous System

a. The Problem of Nerve Supply

Despite the intensity of experimental activity in this field, the extent of the nervous system’s control of anterior pituitary function and the physiological pathways by which this control is exerted continues to present a problem. In contrast to the posterior lobe, the pars distalis, although containing abundant secretory cells, possesses but a scanty nerve supply. Several workers have described either fiber bundles or actual nerve fibers in relationship to secretory cells (Brooks, 1938; Hair, 1938; Vasquez-Lopez, 1949; Metuzals, 1956). Brooks and Gersh (1938), in young rats, noted nerve fibers terminating upon both acidophil and basophil cells-a relationship they thought was lost after stalk section. In his extensive study of the innervation of the hypophysis, Rasmussen (1938) located sympathetic fibers passing to the pars distalis from the pericavernous plexus, although he concluded they subserved only a vasomotor function. Following hypophysectomy in rat, dog, and man, Rasmussen (1940) was unable to demonstrate retrograde degeneration in any region of the hypothalamus other than the supraoptic nuclei; this degeneration only involved the paraventricular nuclei when the median eminence of the tuber cinereum was also damaged. Green (1951) in his comparative study of the vertebrate gland was unable to identify either secretomotor or perivascular nerve fibers, findings confirmed by Wingstrand (1951) who

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found reticular fibers only. Recent studies with the electron microscope confirm that the majority of fibers revealed by silver staining techniques are not axons (Palay, 1953).Despite the divergent views concerning the presence or absence of nerve fibers in the pars distalis, it is clear that, if present, they are both difficult to visualize and very few in number when compared with those of the neural lobe. b. The Role of the Portal Vessels; Stalk Section and Transplantation Studies The marked differences in the pattern of innervation of the anterior and posterior lobes of the pituitary raised early on the question of the functional significance of alternative vascular pathways. Descriptions of the pituitary portal system (Popa and Fielding 1930; Wislocki, 1938) led several workers to investigate the direction of blood flow in these vessels; it was established to be from tuber cinereum to gland (Houssay et at., 1935; Green and Harris, 1947; Worthington, 1955). Green and Harris have directed attention to the possible functional significance of this structure as a specialized vascular pathway, and the nature of the link between hypothalamus and pars distalis has been investigated in a series of transplantation and stalk-section experiments. It has been established for several years that well vascularized homografts and transplants of anterior pituitary tissue are largely nonfunctional in hypophysectomized animals when placed at a site remote from the median eminence, e.g., beneath the temporal lobe or in the anterior chamber of the eye (Westman and Jacobsohn, 1940; Cheng et al., 1949a,b; McDermott et al., 1950; Harris and Jacobsohn, 1952). On the other hand, in animals with grafts placed in the sella turcica and revascularized from the portal system, adrenal weights are maintained and the gland responds normally to stress. Fortier (1951) noted that the responsiveness of certain intraocular transplants, not possessing an innervation, depended upon the type of stress employed. So-called neurotropic stressors, needing the mediation of the central nervous system, no longer evoked an adrenal response, whereas so-called systemic stresses, apparently mediated by the general circulation, continued to evoke an eosinopenia. In many of the older studies, it was found that simple stalk-section failed to prevent the adrenocortical response to stress. It is now known that the portal vessels possess a marked regenerative capacity and it has been suggested

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that they are under the influence of trophic stimuli not common to capillaries elsewhere in the body. De Groot (1952), by inserting a plate beneath the median eminence in the mouse, was able to prevent portal vessel regeneration following stalk section, and prevent also the return of a stress response, In successful preparations, without revascularization, adrenal atrophy also resulted. These findings have been confirmed in the ferret (Donovan and Harris, 1956) and in the rabbit (Fortier et al., 1957). However, using a sheet of polythene to prevent portal vessel regeneration in the stalk-sectioned monkey, Hume (1958) was unable to abolish completely the rise in plasma corticoids produced by surgical stress. The possibility that the loss of pituitary-adrenocorticalactivity produced by stalk-section with plate insertion or resulting from portal vessel cauterization is due merely to an ischemic atrophy of the gland has received considerable attention (Westman et al., 1943; Daniel and Pritchard, 1956). Campbell and Harris (1957) estimated pituitary volumes in the rabbit and found the decrease in volume to be of a similar order (2647%) in groups which had been stalk-sectioned only and those stalk-sectioned with plate insertion. However, only those with successful plate insertions showed marked diminution of adrenocortical function; these findings make it improbable that interference with the glands' general blood supply and nutrition would account for the difference.

c. Hypothalamic Lesions and Ablation Studies It is well known that hypophysectomized animals maintain life better than those subjected to bilateral adrenalectomy, an observation which suggests that the adrenal cortex has some capacity for autonomous activity and perhaps a base-line level of secretion in the absence of the anterior lobe. Rauschkolb et al. (1956) have shown in the hypophysectomized dog that the rate of secretion of 17-hydroxycorticosterone, corticosterone, and ll-deoxy-l7-hydroxycorticosterone is 10% of normal. Lesions in the hypothalamus, although clearly affecting the response to stress, produce somewhat variable changes in adrenal size. Greer (1952), Bogdanove et a2. ( 1955), Bogdanove ( 1957),and Slusher ( 1958) all found that lesions in the anterior portion of the median eminence in rats cause atrophy of the adrenal cortex, while the findings of McCann (1953) in the rat, Ganong and Hume (1954) in the dog, and Laqueur et a2. ( 1955) in the cat, indi-

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cated that the effect of such lesions on adrenal size is inconstant. However, hypothalamic lesions are clearly effective in blocking the compensatory hypertrophy of the remaining gland which usually follows unilateral adrenalectomy ( Ganong and Hume, 1954; Fulford and McCann, 1955). More clear-cut are the results of hypothalamic lesions in animals subjected to stress. Using adrenal ascorbic-acid depletion, McCann (1953) showed that such lesions effectively block stress responses. Similar results are reported by Porter (1954) and Schapiro et al. (1956) using eosinopenia. Ganong et al. (1955) found that hypothalamic lesions prevented the rise in 17-hydroxycorticoids normally occurring in the venous effluent of dogs following surgical stress, It is perhaps difficult to reconcile the ability of transplanted pituitaries to respond to stress with the effectiveness of hypothalamic lesions in blocking stress responses. Using the method of Nelson and Hume (1955) for the measurement of blood ACTH together with the measurement of 17-hydroxycorticosteroids in adrenal venous blood, Hume (1958) has reported a study of the effectiveness of hypothalamic lesions in blocking the response to operative trauma in dogs. In normals, the adrenal venous-blood corticosteroid output, 2 hr after operation, expressed in micrograms per min, averaged 12.2, while in those dogs carrying anterior median eminence lesions, the rate was 2.5; this should be compared with a mean of 10.9 pg in dogs with lesions at other hypothalamic sites, including the mammillary body area, In contrast to other groups, dogs with anterior median eminence lesions also failed to show a rise in ACTH levels. Earlier studies [de Groot and Harris (1950) in the rabbit, Porter (1953) in the cat, and Slusher and Roberts (1956) in the rat] had implicated the mammillary body region in the control of ACTH release, while the investigations of McCann and Brobeck (1954) implicated the median eminence. These discrepancies may well be due to species differences. Further studies by Slusher (1958) have indicated a dissociation between adrenal ascorbic-acid depletion and plasma corticosterone changes as the result of hypothalamic lesions in the rat. Lesions situated posteriorly and in the midportions of the hypothalamus prevented corticosterone release without altering adrenal ascorbic acid depletion or adrenal weights, while lesions in the tuberal region blocked adrenal ascorbic acid depletion without preventing stress-induced rises in adrenal vein corticoids. Thus, the stress-induced liberation of corticosterone into the adrenal vein

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does not appear to depend upon a parallel depletion of ascorbic acid in the gland. These findings raise the possibility of the existence of 2

pituitary ACTH factors controlled by different hypothalamic zones. A further complexity is revealed by studying dogs with isolated pituitaries in which all the brain above the level of the inferior colliculus is removed. Animals so prepared show high resting-levels of blood corticoids. Two-thirds of these animals, with isolated pituitaries in situ, still respond to burn trauma with an increased corticosteroid output (Egdahl, 1960). It has been suggested (Egdahl et al., 1959) that these data indicate the presence of an ACTH-releasing center located in the hindbrain and that their results could be due to the removal of a tonic CNS inhibition of ACTH release (see review by Fortier, 1962). The view, that the activity of the anterior pituitary is controlled by hypothalamic neurohumoral agents which, in their turn, regulate the release of the corresponding trophic hormone, is now receiving increasing experimental support ( see Schindler, 1962). A lipoprotein extracted from bovine posterior hypothalamus with corticotropinreleasing activity (CRF) was reported by Slusher and Roberts (1954). Working with dogs, Porter and Jones (1956) and Porter and Rumsfeld (1956) showed that blood collecting in the sella turcica from the cut pituitary stalk of hypophysectomized animals possessed corticotropin-releasing activity in hydrocortisone-blocked rats. Guillemin and Rosenberg (1955), Saffran et d. (1955), Guillemin et d. ( 1957) have succeeded in isolating a polypeptide from hypothalamic tissue with the ability to release ACTH from pituitary tissue in vitro. Guillemin’s CRF (fraction D ) has now been shown to release ACTH in human subjects as measured by an increase in plasma 17hydroxycorticosteroids (Clayton et aE., 1957). Schally et al. ( 1980) have demonstrated 2 CRF’s and shown that P-CRF is very potent as an ACTH releasing agent; it is a polypeptide and contains all the amino acids of lysine vasopressin, with alanine, arginine, histamine, and serine in addition, and has 10 times the ACTH-releasing power of vasopressin.

d. Electrical Stimulation: Hypothalamus and Limbic System Hume and Wittenstein (1950) using the eosinopenic response in the dog and de Groot and Harris (1950) using lymphopenia in the rabbit first showed that ACTH-release resulted from electrical stimu-

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lation of the hypothalamus in the conscious animal. These findings have been confirmed in the cat (Porter, 1953; Anand and Dua, 1955) and in the monkey (Porter, 1954). In rabbit, cat, and monkey the effective site for electrode placement was found to be the region of posterior tuber and mammillary body. When the electrode tip was situated in the anterior pituitary itself, stimulation was found to be ineffective. Using chronically implanted electrodes in the unanesthetized monkey, confined in a chair, with plasma 17-hydroxycorticoid determinations, Mason (1958a) has shown that a marked rise in plasma corticoids results from hypothalamic stimulation. The rate of steroid rise during hypothalamic stimulation (20-25 pg/loO ml/hr ) was similar to that produced by the administration of large doses of ACTH. Maximal corticoid rises resulted from stimulation in both anterior and posterior hypothalamus but not in control regions such as the putamen. Porter (1954) directed attention to areas other than the hypothalamus which appear to be capable of influencing ACTH release during electrical stimulation. Stimulation of both hippocampus and uncus inhibited the eosinopenia induced by surgical stress in the monkey, while stimulation of the orbital surface of the frontal lobe was found to produce an eosinopenia. Anderson et al. ( 1957) have implicated a mesencephalic-hypothalamic mechanism in the activation of stress-induced rises in urinary corticosterone output. Recent studies have drawn attention to the limbic system. Mason ( 1959a) has shown marked rises in plasma 17-hydroxycorticosteroids during electrical stimulation of the amygdaloid complex in unanesthetized monkeys with chronically implanted electrodes, It has also been shown in the lightly anesthetized cat (Setekleiv et al., 1960) that acute rises of the plasma level of 17-hydroxycorticoids can be produced by stimulation of the anterior cingulate cortex, of the lower portion of the posterior ecto- and suprasylvian gyri of the temporo-occipital cortex, and of the amygdala. In line with the findings of Porter are those of Ganong and Goldfien (1959) who have reported a rise in plasma corticoids resulting from stimulation of the orbital surface of the frontal lobe in the dog. Both hippocampus and amygdala have projections to the hypothalamus via fornix and stria terminalis, and Nauta and Kuypers (1958) have demonstrated that there are midbrain projections from the dorsal tegmental nucleus of Gudden via the bundle of Schutz to the periaqueductal gray matter and, together with the system of the mammillary peduncle, to the

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hypothalamus, preoptic area, and septum. Elevations in plasma corticoids by stimulation of the amygdala have already been referred to, but Mason (1958b) has found a marked suppression of levels in the range 0-6 pg/lOO ml plasma, 24 hours after a period of hippocampal stimulation; bilateral hippocampal ablation or bilateral fornix section appears to abolish the normal steroid diurnal variation in the monkey.

e. Behavior Studies: Environmental Stress and Conditioning The brilliant interdisciplinary studies of the group at the Walter Reed Army Institute of Research have opened up the area of interaction between psychological influences and the neural mechanisms which underly the control of ACTH release. They have shown that marked rises in plasma corticosteroids in monkeys result from changes in the environmental situation-for example, when first moving animals from home to experimental cages or when first confining animals in restraining chairs. In the latter case, the steroid rise returned to normal after 5 days and a normal response to ACTH administration was demonstrated after this initial period of adaptation (Mason et al., 1957a). The stress to a monkey of being caught and held and then being subjected to venipuncture resulted in plasma steroid rises which persisted for several hours, These workers noted, however, that adaptation occurred rapidly and that similar steroid rises failed to occur on subsequent occasions. A further example of the sensitivity of the pituitary-adrenocortical system to environmental changes is provided by the steroid rises commonly found in the Walter Reed monkeys on Mondays, presumably due to the increased activity in the laboratory following the quiet of the weekend. Applying precise modifications of the well-established techniques of operant conditioning (Skinner, 1938; Estes and Skinner, 1941; Brady and Hunt, 1955), the relationships between conditioned emotional responses and the activity of the pituitary-adrenal axis has been investigated in the monkey using plasma 17-hydroxycorticoid determinations (Mason et al., 19571,). Three main types of behavioral situation have been studied: lever pressing for food reward under conditions of anticipated distress, i.e., anxiety; lever pressing in order to avoid receiving painful electric shocks; and a combination of both. Whereas animals that were simply lever pressing to

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obtain food reward showed no elevation in plasma steroids, those in the situations involving conditioned anxiety and conditioned avoidance behavior showed marked elevations of plasma steroids (20 pg/ 100 ml/hr ) . Elevation in the adrenal-vein 17-hydroxycorticoidlevel following reserpine administration to dogs was reported by Egdahl et d. (1956), and similar elevations in the blood levels of schizophrenic patients treated with reserpine were reported by Tui et al. (1956). Harwood and Mason (1957) studied the effects of tranquilizing drugs on ACTH release in more detail in the monkey. They demonstrated that doses of reserpine (1.0 mg/kg) produced a rate of rise of 17-hydroxycorticoidscomparable with that evoked by large doses of ACTH. Very similar but less marked elevations were produced by chlorpromazine. It is, of course, interesting to find that tranquilizing agents can behave as “stressors” in terms of ACTH release when short-term changes are being studied, although they abolish the pattern of conditioned avoidance and conditioned emotional behavior. However, chronic administration of tranquilizing agents does appear to block the steroid rise normally associated with a conditioned anxiety session ( Harwood and Mason, 1958). Mirsky et al. (1953) have treated monkeys with ACTH during the period of acquisition of a conditioned fear response and demonstrated that these animals show a diminished reaction and unusually rapid extinction of the conditioned avoidance behavior so produced. B. HUMANCLINICALSTUDIES

1. Psychological States and Adrenocolticd Function Any stimulus which threatens the integrity of the organism has been shown by Selye and many others to result in a change in the activity of the adrenal cortex. The effects of injury and physical stresses on the organism have been reviewed elsewhere (Selye, 1950; Moore, 1957). More interesting in the present context are the striking changes in the activity of the pituitary-adrenocortical system brought about by stresses of a psychological nature; here the main effects are on the mood and emotional responses. The older clinical studies, prior to the introduction of chemical methods for determining 17hydroxycorticoids, will not be reconsidered here, since the findings are very difficult to interpret when based upon indirect measures of

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adrenocortical activity-eosinophil counts, urinary uric acid-creatinine ratios, and the urinary excretion of 17-ketosteroids, The influence of psychological and emotional factors on adrenocortical activity may be conveniently grouped into 3 types of study: ( 1) those involving healthy subjects in naturally occurring stressful life situations; (2) those in which healthy and anxious subjects are exposed experimentally to stressful procedures; and ( 3 ) clinical studies of psychiatric patients either during the natural course of their illness or when subjected to therapeutic interventions. a. Normal Subjects Experiencing Environmental Stress

Data have been collected upon the response of the adrenal cortex of normal subjects in the following types of presumably stressful situation: patients in hospital on the day prior to an anticipated major surgical procedure (Franksson and Gemzell, 1955; Price et id., 1957); relatives accompanying injured or severely ill patients to hospital; students about to take final examinations (Bliss et al., 1956); oarsmen taking part in a university boat race (Hill et al., 1956; Marchbanks, 1958). In the above studies, the common response is an increase in the plasma level of cortisol or the urinary excretion of 17-hydroxycorticoids. In 33 randomly selected surgical patients at the Karolinska Hospital it was found that the plasma 17-hydroxycorticoids approximately doubled on the morning of the day of operation, the authors attributing the increase to the emotional state of the patients (Franksson and GemzeU, 1955). In 24 patients admitted to the Walter Reed Hospital (Price et al., 1957) for thoracic surgery, a mean A.M. 17-hydroxycorticoidlevel of 21 pg/loO ml was found on admission. This had dropped to 15 pg/loO ml a few days before operation and rose again to 18 pg/100 ml on the preoperative day (normal mean: 12 p g ) . Each patient’s emotional state was rated by a psychiatrist and psychologist as well as by Rorschach testing. The authors concluded that the 17-hydroxycorticoidelevations were associated with a variety of emotional states but more especially with the amount of general emotional involvement of the patients in a given situation rather than the degree of clinical anxiety exhibited. Bliss et al. (1956) found a mean plasma value of 23 pg (corrected to &A.M. values) in the anxious relatives of patients seen in the emer-

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gency room of a general hospital and mean levels of 18 pg and 20 pg in two groups of students on the morning prior to important examinations (normal mean, 13 pg/lOO ml), Hale et al. (1958) measured plasma corticosteroids by fluorimetry in 44 flight crew members, 1% hr before, and % hr after, long-range training flights lasting 9-12 hr. The mean preflight cortisol level was 11.3 pg; this had risen to 16.8 pg at the end of the flight. In a preliminary communication, Hodges et al. (1962), although unable to detect ACTH in the plasma of control subjects, found a marked elevation (6-9 milliunits ACTH/100 ml) in the plasma of students immediately following a viva voce examination. Levels of corticosteroids were approximately double in the stressed subjects. Urinary corticoid excretion has also been measured in stressful situations. Hill et al. (1956) collected timed urine samples from Harvard boat-race crews on a control day, a timed-trial day, and the actual day of the race in two consecutive years. 17-Hydroxycorticoid excretion in the evening sample, which included the race period, was increased as compared with the control day (in 1953,1.5mg in 4 hr specimens as compared with 0.2 mg; in 1954, 1.4 mg as compared with 0.6 mg) . When 24-hr specimens were collected and compared, there was seen to be a significant increase in excretion on both race and timed-trial days (8.6 mg as compared with 4.0 mg). Strenuous exercise without the stimulation of a competition was not associated with a significant increase in the urinary corticoid excretion. The relative insensitivity of urinary methods when studying short-term changes is suggested by the observation that no consistent increases were observed in students taking examination [cf. plasma levels in the study of Bliss & al. ( 1956)l. However, 4 members of a bomber crew on a 22%-hrflight showed a marked rise from a control mean of 9 mg to 15mg per 24-hr specimen ( Marchbanks, 1958).J. W. Mason (195913) noted the fact that bomber crews and their instructors may show high control values and draws attention to the possibility of social as well as group influences determining patterns of hormone excretion. An attempt to study the influence of personality on the pattern of urinary corticoid excretion has been made by Fox et al. (1961), who measured daily urinary excretion for 30 days in 18 university students and compared subjects with consistently high and low excretion rates, A tentative correlation was found between high urinary excretion and a high degree of emotional involvement and

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reactivity, while emotional coldness and detachment appeared to be associated with low outputs.

b. Healthy and Anxious Subjects Exposed Experimentally to Stressful Procedures An effort has been made to obtain more information about the psychiatric aspects of stress by exposing healthy subjects to emotionally disturbing procedures under controlled conditions. Bliss d al. (1956) subjected university students to the following stressful procedures: ( i ) the administration of lysergic acid; (ii) forced selfrevelation before a one-way screen; and (iii) exposure to a situation containing a threat to future careers. In general, subjects reacting with emotion showed a modest rise in 17-hydroxycorticosteroids of 4-5 pg/lOO ml of plasma. One subject became severely disturbed by the one-way screen procedure and his steroids rose by 13 p g . Lysergic acid produced a less marked emotional reaction with a steroid rise of 6 pg. When a third attempt to produce an emotional response failed, and he remained calm, his steroids followed a normal, downward diurnal course. The introduction of healthy subjects to an experimental situation, itself constitutes a stress. A group of soldiers admitted to hospital for a series of sleep-deprivation experiments were found to have raised blood levels of 17-hydroxycorticosteroids (21 pg/lOO ml) prior to the start of the actual experiment (Mason, 195913). Twelve normal subjects, assembled for an investigation (Persky et al., 1959a) also showed steroid levels of 21 pg. However, in 5 normal subjects who submitted themselves to a perceptual-distortion situation, the rise in 17-hydroxycorticosterone was less (Persky et al., 1 9 5 9 ~ ) Sponta. neously occurring stressful situations appear to be more effective in producing steroid rises than those contrived in the laboratory. The detection of small steroid rises in this situation is not easy because rises are superimposed on the background of a declining plasma level due to the normal diurnal variation, (e.g., 9 ~ . ~ . - 1 3 . 5p g ; 3 P.M.-9 pg; 6 P.M.-~ pg) (Doe et al., 1960b). Because of this, the net steroid change may actually be a decrease, although a lesser one than on a control day. A statistical technique for handling such data has been presented by Persky et al. (1959a). The highest reported increase in steroid levels resulting from a psychological technique is caused by hypnotically induced anxiety (12.5 p g to 20.2 pg). On

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termination of the anxiety, the steroids returned to the prestress levels. Similar experiments with similar results have been carried out on psychiatric patients, almost all of which were anxious or anxietyprone. Bliss et al. (1956) induced modest steroid rises in patients subjected to stressful interview and to the distress produced by the delayed speech-feedback apparatus. The effect of a stressful interview on 19 anxious patients was studied in more detail by Persky et a2. (1958). The emotional reaction of these patients was assessed in terms of anxiety, anger, and depression. The intensity of each reaction correlated with the level of plasma 17-hydroxy~orticosteroids. The authors concluded that emotional arousal in general, rather than the particular type of emotional reaction, or the nature of the stressinducing stimulus, was responsible for the increased adrenocortical activity. The same workers investigated a similar group of anxious patients who were stressed by means of a perception-distortion situation (Persky et al., 1 9 5 9 ~ )Similar . correlations were found between affect ratings and 17-hydroxycorticosteroid levels. In all these patients the experimentally induced increases were modest, and less than those occurring as a result of simple exposure to the laboratory milieu ( Sabshin et al., 1957). Investigations employing measures of urinary steroid excretion are generally in line with the studies referred to above. Sloane et al. (1958) found a high rate of 17-hydroxycorticosteroid excretion in normal subjects attending a laboratory for the first time as controls, and Hetzel et al. (1955) noted a small but significant increase in the excretion rate in patients subjected to stressful interview. The effect of a novel experience was clearly shown by Fishman et al. (1962) in a study of several groups of college students admitted to a hospital research unit as volunteer control subjects. Generally, urinary steroid levels fell during the first week as the subjects accustomed themselves to the ward. The highest initial levels were found in groups of students who were strangers to each other and who had little experience of living away from home. In one group, steroid levels were low on the first day and thereafter became higher. In this group were students experiencing financial hardship and the threat of academic failure. Admission to the ward afforded transient respite from their difficulties. The same workers (Handlon et at., 1962) employed films as a

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means of inducing various emotional states in students. Whereas “arousing films (e.g., “High Noon”) produced only a slight rise in plasma 17-hydroxycorticosteroids, “bland” films ( e.g., Walt Disney’s nature films) induced a considerable fall of 4 3 pg/lOO ml. It is suggested that bland films hold the subject’s attention without causing emotional arousal and prevent his attention being engaged by other, potentially stressful stimuli.

c. Clinical Studies in Psychiatric Patients Interest in the possibility of an endocrine role in the etiology of mental disorder revived in the early 1950’s as the concept of stress impinged upon psychiatry. Although the earlier studies may now strike us as somewhat naive, nevertheless the whole field of interaction between emotion and the adrenocortical system was opened up for the first time (see review by Altschule, 195313). Applying newer techniques, Bliss et al. (1956) have concluded that any psychiatric disorder accompanied by emotional turmoil is likely to be associated with increased plasma 17-hydroxycorticoid levels. They found a mean of 22 pg/lOO ml in 19 recently admitted, disturbed psychiatric patients. There were, however, individual exceptions: One patient, judged to be experiencing extreme tension and anxiety, had the low blood level of 6 pg. Similarly, Board et al. (1956) reported a mean level of 19.8 pg in 30 patients newly admitted to a psychiatric unit. The mean level of the 5 patients assessed as most distressed was 22.1 pg, compared with 16.0 pg in the five least distressed. (1) Anxiety. Patients with anxiety as the presenting sympton have been studied extensively by Persky and his colleagues. It has been shown that anxious patients have a raised plasma 17-hydroxycorticosteroid level of 20 pg or more per 100 ml ( Persky et uZ., 1956), and a urinary excretion of 17-hydroxycorticosteroids about twice . plasma 17-hythat of normal controls (Persky et al., 1 9 5 9 ~ )The droxycorticosteroid level in anxious patients is similar to that found in otherwise healthy subjects involved in an emotionally stressful situation, as already discussed. The administration of ACTH causes a greater urinary 17-hydroxycorticosteroid output than in normal subjects, although the increase of plasma steroids produced by ACTH is not significantly greater than normal ( Persky, 1957a). The rate of disappearance of exogenous cortisol from the plasma is in-

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creased (Persky, 1957b), as is the turnover rate of radioactive cortisol (Persky et al., 1959b). In other words, cortisol is produced in greater amounts and is metabolized more quickly in anxious patients. The natural assumption is that this adrenocortical overactivity is a result of increased secretion of ACTH, and recently Persky d al. (1959b) have shown that a group of anxious patients, with the expected high plasma levels and urinary output of steroids, had a mean plasma level of adrenal weight maintenance factor more than twice that of normal controls. It was also shown that the plasma levels of this factor and of “classical” ACTH were significantly correlated with the clinical anxiety rating. (2) Depression. A group of 33 depressive patients, investigated within a few days of admission to hospital by Board et al. (1957) had a high mean plasma 17-hydroxycorticosteroid level of 19.5 pg, 604: higher than normal controls; the mean level of the 9 more severely ill patients was 23.7 pg. Those with retarded depression (who showed psychomotor retardation and an inability to cry) had higher mean levels than those who were agitated and tearful. Clinical improvement was accompanied by a fall in the plasma steroid level over the next fortnight. A small group of 6 more chronically ill depressives, whose initial plasma 17-hydroxycorticosteroid levels were somewhat lower ( 17.1 pg) received electroconvulsive therapy (ECT). Clinical improvement in these cases was, however, associated with a rise in plasma steroids to 28.1 pg. Gibbons and McHugh ( 1962), using the technique of Peterson et al. ( 1957),studied 17 depressives at weekly intervals over periods of 8 to 12 weeks. The mean level before treatment was 22 pg, falling to 10 pg with clinical recovery, due to ECT or antidepressive drugs (imipramine), or without specific treatment. It may be noted that the later authors did not see the sharp rise following ECT referred to above. Another hospitalized patient, with untreated, recurrent depression, showed abrupt rises in plasma steroids coincident with each depressive episode. ( 3 ) Mania. The activity of the adrenal cortex during pure mania, as distinct from emotional excitement in other psychiatric conditions, has not been extensively studied and the findings which have been reported are somewhat conflicting. There is also, nowadays, the dirsculty of obtaining manic patients who have not been treated with large doses of phenothiazines, which may influence

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adrenocortical activity (see Section A. 2. e). In a study of a woman with manic-depressive psychosis extending over a year, Rizzo et al. ( 1954) found a consistently low urinary 17-hydroxycorticosteroid output (1mg or less per 24 hr) during a manic period lasting several months. The steroid output returned to normal as the symptoms of overactivity disappeared. Despite the low urinary output during the manic phase the response to ACTH was normal. Although it is difficult to draw conclusions from isolated case reports, experience in our own laboratory has been in line with the findings described above. A 15-yr-old cyclothymic girl studied for 6 months has shown low plasma levels of 17-hydroxycorticosteroids ( 7 pg/lOO ml) by the method of Peterson et a2. (1957) during periods of mild elation, while the onset of periods of depression has been accompanied by a marked rise in these levels (27 pg/lOO ml) , Bliss et al. ( 1956),however, reported an overactive, manic patient whose plasma steroid level was 28 pg, which fell to 14 pg when he became calm. Clearly, the subject of adrenocortical activity during mania requires further study. (4) Schizophrenia. A series of investigations by the Worcester Foundation group let to the conclusion that, in two-thirds of their schizophrenic patients, the adrenocortical responsiveness to ACTH and to the following stresses was diminished-heat; cold; ingestion of glucose; a pursuit meter task; a target-ball frustration test. The measurements used were indirect, consisting of blood eosinophil and lymphocyte counts and estimations of urinary uric acid, phosphates, electrolytes, and 17-ketosteroids. The differences from normal lay in urinary excretion patterns and not in lymphocyte and eosinophil responses (Pincus and Hoagland, 1950). It has also been suggested that the pattern of individual 17-ketosteroids in the urine was abnormal in schizophrenic patients (Mittelman et al., 1952; Reiss and Stitch, 1954). On the other hand, Altschule (1953a), using similar indirect measures, concluded that the adrenal cortex of schizophrenic patients is hyperactive. Intermediate results have been obtained by other groups (see Altschule, 1953b; Freeman, 1958). Despite the immense amount of effort devoted to the investigations referred to above, their value must inevitably be restricted by the indirectness of the indices employed for the assessment of adrenocortical activity. Even the measurement of urinary 17-ketosteroids has little relevance, as Sayers (1950) has pointed out, be-

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cause of the lack of correlation between the rate of 17-ketosteroid excretion and other indices of adrenocortical activity. For this reason we have excluded from review papers dealing exclusively with the urinary 17-ketosteroid output in schizophrenics. To these difficulties must be added those inherent in any study of schizophrenia, and, in particular, the lack of homogeneity of the clinical material. Bliss et al. (1955) were able to overcome some of these problems by measuring plasma 17-hydroxycorticosteroids and by considering separately acute and chronic schizophrenic illnesses. Of 27 newly admitted patients, diagnosed as schizophrenia of recent onset, 19 were emotionally disturbed and had a mean plasma 17-hydroxycorticosteroid level of 22 pg/lOO ml, compared with a level of 13 pg in the 8 patients who were emotionally calm. When acute schizophrenics were treated with ECT, a transient mean rise of 7 pg/lOO ml occurred, reaching a maximum within an hour after the convulsion and returning to the previous level within 4 hr (Bliss et al., 1954). More detailed study of individual cases revealed considerable variation in steroid response in the same subject on different occasions. There was no correlation between the degree of the steroid rise and the therapeutic effect of ECT, and exactly similar rises were seen in a group of chronic, deteriorated hebephrenic patients in whom treatment produced no psychological improvement, There are no data on the long-term changes in adrenocortical activity which might result from ECT. Insulin coma treatment of similar cases was also associated with transient steroid rises of similar magnitude. A study of 26 chronic, affectless schizophrenics, all of whom had been hospitalized for more than 5 yr, and none of whom were malnourished, revealed marked differences from the acute schizophrenic group (Bliss et al., 1955). These patients, when compared with a group of normals of the same age range, showed similar plasma levels (14 pg/lOO ml) and a normal response to several dose-levels of ACTH. The scatter of results in normal and chronic schizophrenic groups was similar. Pyromen and insulin stresses also produced normal steroid rises. Paper chromatography of samples of schizophrenic plasma, taken before and after injection of ACTH, failed to reveal the presence of any abnormal steroids. It is interesting to note that the affectless schizophrenics appear to show relatively normal adrenocortical activity, whereas those with

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emotional turmoil resemble the depressive and anxious groups in this respect. ( 5 ) Anorexia Neruosa. Separate mention is made of this difficult psychiatric condition because of its confusion with hypopituitarism (see Section B.2.d) and the wide-spread belief among otherwise well-informed psychiatrists that it is associated with marked pituitary dysfunction. We only intend to deal with its relationship to adrenal cortical function, as the whole topic of anorexia nervosa has been the subject of a recent monograph by Bliss and Branch ( 1960). While the psychogenic amenorrhea is perhaps associated with a decreased or disordered output of pituitary gonadotropins (although the evidence for this is tenuous) , thyroid function appears in general to be normal. It has been shown by Bliss and Migeon (1957) that the plasma level of 17-hydroxycorticosteroids is within the normal range in this disorder and that the adrenal cortex responds normally to the administration of ACTH. The low urinary output of steroids reported in this condition is seen to be spurious when the body weight of patients is taken into consideration. Further, in many chronic wasting diseases a normal plasma steroid level is found in association with a low urinary output of total 17-hydroxycorticosteroids because of a diminished rate of conjugation ( Shuster, 1960). In the present state of knowledge, anorexia nervosa cannot be regarded specifically as an endocrine disorder. 2. Psychiatric Aspects of Pituitary and Adrenocortical Disorders a. Cushing’s Syndrome

This syndrome covers that group of disorders which have in common the sustained, excessive production of cortisol and, to a variable extent, of adrenal androgens. These patients are obese, plethoric, show a characteristic “buffalo hump” distribution of fat, and have purple striae on abdomen and thighs. Women are frequently hirsute and show signs of virilism. Moderate hypertension is the rule and mild diabetes is often present. The increased output of adrenal steroids is shown by the elevated urinary excretion of 17-hydrocorticosteroids or of 17-ketogenic steroids and by raised plasma levels of cortisol (20-50 pg/lOO ml) , It has recently been shown that there is an enormous increase in the unbound fraction of plasma cortisol in

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such patients (0.9 pg raised to 16.6 pg/lOO ml: Doe et al., 1960a). Urinary excretion rates of free cortisol also increase many times; up to 710 pg a day compared with a normal mean output of 15pg (Ross, 1960). These patients fail to show the normal diurnal variation, the plasma 17-hydroxycorticosteroids remaining high throughout the 24 hr, (Lindsay et al., 1956; Doe et al., 1960b; Ekman et al,, 1961). Some patients additionally show a marked elevation in the urinary output of 17-ketosteroids. The most frequent cause-in 70-80% of cases-is bilateral adrenal hyperplasia, while the remaining 2&30% are due to either adenoma or carcinoma of the adrenal cortex (with about equal frequency) (Soffer et al., 1955; Sprague et al., 1956; Liddle, 1960). Basophil adenoma of the adenohypophysis is found in association with a variable proportion of cases with bilateral hyperplasia. While the neoplasms secrete autonomously, the hyperplastic glands are thought to be responding to an increased secretion of ACTH because of failure of a normal regulatory mechanism (Nugent et al., 1960). This has led to the introduction of tests aimed both at diagnosis and at distinguishing between the two main causes of the syndrome. Most cases show overresponsiveness to ACTH in terms of urinary steroid output, and this is true of the small number of cases who have urinary and plasma steroid levels within the normal range (Nabarro et al., 1958). In using potent ACTH-suppressing agents such as dexamethasone or triamcinolone, it has been found that small doses, sufficient to suppress ACTH secretion in healthy subjects, fail to influence the output of steroids in Cushing’s syndrome. The administration of high doses of these substances results in a decrease in urinary 17-hydroxycorticosteroids in cases of adrenocortical hyperplasia, but not in those due to neoplasm (Liddle, 1960). Although Davies et al. (1960) claimed to find raised ACTH plasma levels in two patients with Cushing‘s syndrome, this is not the general finding. Vance et al. (1962), measuring the minute output of corticosterone in the adrenal venous effluent of hypophysectomized rats, were able to detect ACTH in the plasma of normal human subjects (O.Pl.0 milliunits/100 ml) (see Hodges et al., 1962). Six cases of Cushing’s syndrome due to hyperplasia were in the range 0.6-0.8 m a , whereas in 4 patients who developed pituitary tumors after bilateral adrenalectomy the levels rose to 12-30 m.u. From the earliest delineation of the clinical features of the syn-

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drome (Cushing, 1932; Albright, 1942) reference has been made to the occurrence of psychiatric abnormalities. However, the importance of the psychological symptomatology was, perhaps, not at first appreciated, as it was looked upon as being merely reactive to the sometimes gross disfigurement the disease produces. In fact, psychiatric symptoms constitute a major part of the syndrome. Symptoms which could be regarded as being primarily somatic, but which certainly have psychological importance, include impotence in men, amenorrhoea and loss of libido in woman (66% of cases), and also excessive fatigue and asthenia (60% of cases). The reported incidence of psychiatric disturbance generally exceeds 50%,for example, 42% (17) of 40 cases (Soffer et al., 1955), 58% (19) of 33 cases (Glaser, 1953), 64% (16) of 25 cases (Trethowan and Cobb, 1952), 75% (40) of 53 cases (Starr, 1952), 100%of 20 cases ( Bleuler, 1954), 88% (30) of 34 cases (Hurxthal and OSullivan, 1959). The series referred to above were collected from general hospitals and were not selected primarily for their psychiatric interest. There is fair agreement that severe mental disturbance, warranting the description “psychotic,” is found in 1EL20%of cases (Jonas, 1935; Starr, 1952; Trethowan and Cobb, 1952; Glaser, 1953; Hurxthal and O’Sullivan, 1959). Depression, associated with anxiety or retardation, is extremely frequent; elevation of mood is rare and frank euphoria scarcely ever encountered (in Starr’s series, 1 case in 53). In addition to the marked depressive features, a wide range of psychiatric symptoms has been reported, including the appearance of paranoid delusions and auditory hallucinations. These occurred in 4 of the 7 cases reported by Glaser. A fairly common feature of the mental changes in Cushing’s syndrome is the occurrence of acute, brief episodes of grossly disturbed behavior; excitement, acute anxiety, and apathy verging on stupor have all been reported. Bleuler remarks that the acute psychotic episodes do not resemble typical schizophrenia or manic-depressive psychosis. In general, a typical schizophrenic illness is not seen in patients with Cushing’s syndrome nor is Cushing’s syndrome found with increased frequency in patients with schizophrenia. With the exception of the acute psychotic episodes referred to, diagnostic confusion with a major psychosis is unlikely and although organic features are mentioned by some authors, a typical acute organic reaction is rare. A series of 13 patients, 12 of them women, has been reported by

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Furger ( 1961). An interesting finding was that 7 patients developed Cushing’s syndrome during a long period of severe psychological stress. In each case this was imposed on the patient by the behavior and attitudes of close relatives. In every case, psychological symptoms appeared during the illness and, in 8 of them, at an early stage. Furger was impressed by the unexpected uniformity of the clinical picture; almost all patients were consistently or intermittently depressed (findings supported by those of Koster and Thiel, 1961) and many of them complained of considerable memory difficulty, although objective evidence of memory defect was slight. Emotional lability was a universal feature and took the form of gross overreaction to emotional stimuli of any sort. For example, one patient stopped going to the cinema and another no longer listened to the radio because they were too deeply moved by the experience. In several cases, the affective overresponsiveness led to difficulties in relationships with other people. In addition to these typical symptoms, one patient had a paranoid illness and another required hospitalization for an episode of acute excitement. An attempt was made to correlate psychological features with certain physical aspects of the syndrome but without success. In particular, there was no relationship between strength of libido and 17-ketosteroid output, nor between appetite and 17-hydroxycorticosteroidoutput, nor was the severity of psychological and physical symptoms closely related. However, the tempo of the illness was important and psychological symptoms were more severe when the syndrome developed rapidly. Nevertheless, in the majority of case studies, obvious psychological symptoms tend to occur when the disease is well advanced. The fact that there is no specific psychiatric syndrome associated with this glandular disorder and that the range of symptoms encountered is very wide, suggests the combination of a “toxic” effect with a psychological response depending upon previous personality and constitution. This view is supported by the observations of Furger ( 1961), who concluded that severe psychological symptoms were encountered more frequently in patients whose previous personality had been more disturbed. Three patients who reported increased libido after the development of Cushing’s syndrome had all previously possessed a strong sexual drive, while those who reported loss of libido, previously had weak sexual interests. Again, the previously more stable patients reported more persistent mood changes, while the more

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temperamental showed fluctuations in mood. It is clear that the sustained high level of circulating cortisol affords no protection against the development of mental symptoms. Despite what has been said, interesting and well-authenticated exceptions occur. The two reports quoted below illustrate cases in which psychiatric symptoms long preceded the diagnosis of endocrine disorder, and where diagnostic confusion with a major psychosis occurred. The first case (Spillane, 1951), a man of 24, was treated for vague rheumatic pains while in the Army and retired as an invalid to the U.K. complaining of anxiety, sleeplessness, and bad dreams. He later became unruly and truculent and developed frank paranoid delusions. After treatment with continuous narcosis and ECT he was discharged home, when his mother, who had not seen him for many months, observed a profound change in his physical appearance. He had become obese, red-faced, and bull-necked. Previously energetic and well-mannered, he had become idle, coarse, and surly. He deteriorated and was readmitted to a mental hospital with auditory hallucinations and again treated with ECT. It was not until 2 yr after his first admission that Cushing’s syndrome was fiimly diagnosed. Deep irradiation of the pituitary was commenced but the patient discharged himself before treatment could be completed and continued to lead a solitary, antisocial life. The second case (Trethowan and Cobb, 1952), a married woman of 31,developed signs of Cushing‘s syndrome (obesity, asthenia, and amenorrhoea), at that time unrecognized, a year prior to her admission to hospital. She had suddenly become excited, overactive, disorientated, and rambling in speech, and refused all nourishment. Treatment with ECT was commenced with the result that she became quiet, withdrawn, and apathetic. Three months later, a diagnosis of Cushing’s syndrome was suggested and irradiation of the pituitary was started. She again became excited and uncooperative and was given a second course of ECT. At this time the diagnosis was “schizophrenic reaction of paranoid type with a depressive component but with lucid intervals.” On her third admission, this time to a general hospital, the diagnosis of Cushing’s syndrome was confirmed and her mental state was suggestive of schizophrenia in partial remission. After operation, at which an adrenal adenoma was removed, her mental state improved rapidly and within a month she

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was apparently normal. At followup, the mental and physical improvement was maintained. Similar cases have been reported by Hamm ( 1955) and Hertz et al. ( 1955). There is general agreement that successful treatment of Cushing’s syndrome also leads to psychological improvement. This is strikingly illustrated by a case reported by Hickman et al. (1961) whose Cushing‘s syndrome was associated with severe paranoid and catatonic schizophrenic features which necessitated heavy sedation and restraint. The symptoms were relieved by adrenalectomy and the patient was regarded as mentally normal ll days after operation. In Furger’s series, there was almost complete parallelism between the physical and mental improvement; eight patients successfully treated by total adrenalectomy recovered fully from their psychiatric s p p toms, Most of them also reported a change in personality, which they regarded as a gain, and which consisted of a more balanced serenity and detachment.

b. Psychological Effects of ACTH and Cortisone Soon after the introduction of cortisone and ACTH into medical treatment it became obvious that the administration of these substances was commonly associated with alterations in mood (Hench et al., 1950). The overall incidence of mood change is high, particularly when large doses are used ( McLaughlin et al., 1953)--84% in the series reported by Browne (1952), 80%in the series of Goolker and Schein (1953). When stricter criteria were used by the latter authors, and mild mood changes-which in themselves would be consistent with relief of physical symptoms-were excluded, significant alteration in mood was still found in 46% of cases. The most commonly observed mood change, and the first to be reported, was euphoria (Taylor et al., 1950; Deb& et al., 1952; Ebaugh, 1951; Brody, 1952; Cleghorn, 1952; Lidz et al., 1952; Rome and Braceland, 1952). Browne reported euphoria in 80 of his 125 cases, and Taylor in 22 of his 26 cases. Of the 45 instances of unequivocal alteration in mood reported by Goolker and Schein, this was in the direction of increased cheerfulness and euphoria in 70%. Depression occurs in a small proportion of cases (Pearson and Eliel, 1950; Soffer et al., 1950; Rome and Braceland, 1952), and alternating moods have also been noted. Other changes occur beside simple euphoria or depression, as shown by the use of such terms as “ten-

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sion” (Ward et al., 1953), “ambivalence” (Brody, 1952),“irritability and excitability” (Kountz et d.,1953), “restlessness” (Taylor et d., 1950), “enhanced aggressiveness” ( McLaughlin et al., 1953). Such changes are frequently short-lived and are not always immediately related to the period of hormone administration. They may occur at the beginning of treatment, after several days, or upon withdrawal of the hormones. Mood may also alter in one direction at the start and in the reverse direction at the cessation of treatment. However, in their detailed and careful report, Goolker and Schein (1953) were able to detect a change in the mental state, which they found rather difficult to describe, within 6 hr of the commencement of treatment. A clinically recognizable and formed reaction emerged in 3 4 days. More severe mental disturbances are encountered in approximately 5% of cases (Gildea et al., 1952; Ritchie, 1956; Cobb, 1960). The symptomatology of these frank psychoses is extremely varied, There may be mania or depression (Soffer et al., 1950; Borman and Schmallenberg, 1951; Browne, 1952), severe disorientation, delusions, hallucinations, and catatonic features. Great excitement or stupor may occur-in other words, all the symptoms of those acute psychoses that are so hard to place in classical nosology. Thus, the clinical picture may be indistinguishable from acute paranoid schizophrenia (Glaser, 1953), from acute catatonic schizophrenia ( Mach, 1951), or from acute organic psychoses (Ritchie, 1956). The condition is reversible, recovery usually following closely upon the withdrawal of the hormones. Most patients that develop psychoses have had either high dosage or prolonged treatment. Cases are seen that show no mental disturbance on moderate dosage, but become disturbed when the dose in increased ( McLaughlin et aZ., 1953). However, others find no correlation between dosage and the severity of psychological changes. When the pretreatment psychological condition was also taken into consideration, it appeared that those patients who were grossly disturbed initially were liable to deteriorate further when high doses of ACTH or cortisone were administered (Goolker and Schein, 1953).The latter writers gained the impression that psychological changes were more likely to be associated with ACTH than with cortisone administration, a finding that is in line with that of Irons et al. (1951), and confirmed by more recent series (Truelove and Witts, 1959). The question of a possible differential effect of

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ACTH and cortisone on mood has been considered by several authors (Hench et al., 1950; Brown, 1951; Lidz et al., 1952). There is no general agreement but Fleminger (1955) reported details of a case under constant observation for 38 weeks in whom cortisone led to an elevation of mood and in which ACTH administration appeared to provoke depressive symptoms. Although, as has been stated, patients already mentally disturbed may become worse with hormone treatment, the history of previous psychotic illness or of unstable personality does not imply a greater liability to develop a cortisone or corticotropin psychosis ( Lewis and Fleminger, 1954). No less than 35 hypotheses have been put forward with the aim of suggesting mechanisms which underlie the mentaI disturbances associated with ACTH and cortisone administration (see review by Quarton et al., 1955). The present writers would merely like to contrast the high incidence of depressive features in Cushing's syndrome with the high incidence of euphoria induced by hormone administration. Though it is difficult to generalize, the administration of ACTH does not appear to produce a mental state more closely resembling Cushing's syndrome than that produced by the administration of cortisone. Further, the instance of severe mental disorder is 3-4 times higher in Cushing's syndrome than in the iatrogenic condition. These contrasting findings must be due either to some totally unexplained factor or to differences in plasma levels of biologically active steroids or the chronicity of Cushing's syndrome. It is unlikely that ACTH itself, as distinct from its effect via the adrenal, plays a causative role in the development of mental symptoms since in the adrenogenital syndrome, in which extremely high levels of blood ACTH occur, the incidence of gross mental disturbance is low (Bleuler, 1954; Woodbury, 1958). c. Addison's Disease

This condition, described by Addison in 1855, is due to chronic adrenocortical insufficiency, the gland failing to secrete adequate amounts of cortisol and other hormones such as aldosterone, corticosterone, and androgens. Until recently, the commonest cause was bilateral destruction of the gland by tuberculosis, the remaining cases being due either to primary atrophy or to rare causes such as secondary neoplasm. With the decline of tuberculosis, primary atrophy now accounts for up to half the cases. This disease of middle

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life affects men 2-3 times as often as women and is characterized by: asthenia; loss of appetite, weight, and libido; progressive pigmentation; hypotension; hypoglycaemia; and a markedly diminished resistance to stress. The most striking biochemical abnormalities are loss of sodium, retention of potassium, and extracellular dehydration with or without intracellular hydration. The evidence that these general electrolyte disturbances also affect the brain is mainly derived from studies on adrenalectomized animals. Although the total brain sodium and potassium remain unchanged (Flanagan et al., 1950; Bergen and Hoagland, 1951; Stern et al., 1951), there is evidence, derived from calculations based upon the assumption that chloride space is a measure of extracellular fluid volume, that the concentration of intracellular sodium and extracellular potassium increases, leading to a decrease in the ratios of extracellular to intracellular sodium and of intracellular to extracellular potassium (Timiras et al., 1954). These electrolyte changes are associated with an increase in brain excitability. The “electroconvulsive seizure threshold” is decreased by 25%in both adrenalectomized rats and mice, and is restored to normal by sodium chloride or deoxycorticosterone acetate ( DOCA ) ( Davenport, 1949; Woodbury, 1954). This suggests a possible basis both for the proneness to seizures of patients in Addisonian crisis and for the effectiveness of DOCA in the treatment of certain refractory epileptics ( McQuarrie et al., 1942; Aird and Gordan, 1951). Attention was first drawn to the EEG abnormalities found in untreated patients with Addison’s disease by Engel and Margolin ( 1941,1942). Although many workers (Hoffman et d.,1942; Thorn et al., 1949; Bricaire et aE., 1953; Condon et al., 1954) have confirmed the presence of diffuse, high amplitude, slow activity associated with an exaggerated response to hyperventilation, it is doubtful whether these changes are of diagnostic value. While the administration of cortisone normalizes the EEG in Addison’s disease (Forsham et d.,1949), DOCA alone does not. Its administration may in fact lead to abnormal delta activity with prominent high voltage, slow activity in response to overbreathing (Bricaire et al., 1953; Thiebaut et al., 1958). These findings are, in general, confirmed experimentally in adrenalectomized rats, whose EEG‘s are restored to normal by adrenocortical extract and pregnenolone, but not by deoxycorticosterone ( Bergen, 1951; Bergen et al., 1953).

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In his original description, Addison mentioned the occurrence of such psychological symptoms as anxiety, insomnia, and confusion. Klippel (1899), noting that mental symptoms were rarely absent, referred to “endphalopathie Addisonienne.” Bonhoeffer ( 1912) and Kraepelin (1913) also concerned themselves with the psychiatry of Addison’s disease and a considerable literature developed, consisting mostly of individual case reports. Psychological symptoms are found in almost all patients with severe disease, being absent in only 2 of Cleghorn’s 25 cases, and in 3 of the 40 cases mentioned by Bleuler ( 1954). Most observers have stressed the appearance of an apathetic or depressive mood, poverty of thought, and lack of initiative (Sainton, 1906; Tucker, 1922; Ewald, 1928; Goldzieher, 1945). Cleghorn (1952) noted apathy and negativism in 80%of his patients, while seclusiveness, depression, and irritability each occurred in about 50%of cases. In the comprehensive survey of Stoll (1953) who collected, mainly from general medical clinics, 29 cases of chronic Addison’s disease (4-5 yr duration), 8 gave a prior history of some personality disorder. Many were leptosomes suffering from tuberculosis, itself often associated with personality changes. At the time of examination, Stoll noted frank psychiatric abnormalities in 27 of the cases. These were of two main types: mood changes and memory defects. The predominant mood was: in 25%of cases, apathy; in 25% of cases, depression; and, somewhat surprisingly, in 50%of cases, euphoria. Indifference, diminished initiative and fluctuations in mood were very common. With the single exception of his reference to euphoria (by which Stoll implies a state of mild, shallow cheerfulness) there is general agreement upon the changes commonly observed. Most cases showed a mild to moderate chronic organic reaction with memory defect as the main symptom. When patients over 55 yr of age were excluded, this was observed in 75%;it resembled mild senile dementia and its severity generally varied directly with the severity of the Addison’s disease. The organic syndrome improved with substitution therapy, Hallucinations and frank psychoses are rare (Stoll found 30 cases in the literature) and only a few cases are reported with paranoid delusions. Addisonian crises are accompanied by acute psychotic episodes of the organic type; clouding of consciousness, delirium, and stupor. A case of this type has recently been reported from this hospital (Cohen and Marks, 1961), in which a psychosis of organic type associated with hypogly-

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cemia persisted for 2 months after an Addisonian crisis. The very variable acute psychiatric episodes which are a feature of Cushing’s syndrome are never seen in Addison’s disease. Treatment of the physical condition in most instances improves the mental state, and this restoration is best achieved by the administration of cortisone (Thorn, 1951). The improvement produced by DOCA and sodium chloride is less marked and less well-maintained than that brought about by cortisone; Stoll noted, in cases receiving treatment of the former type, that their social efficiency remained below the premorbid level. In a few cases, treatment of Addison’s disease with large doses of cortisone has resulted in the development of a cortisone psychosis (Cleghorn and Pattee, 1954). The present authors have seen such a case recently in which severe depression occurred in a woman receiving 200 mg of cortisone a day, but disappeared as the dose rate was gradually decreased to 25 mg daily.

d. Hypopituitarism This term is applied here to the syndrome of chronic anterior pituitary failure. In his original description Simmonds (1914) placed undue emphasis upon cachexia as a clinical feature of the syndrome; this has been a source of diagnostic confusion ever since. It remained for Sheehan (Sheehan and Summers, 1949) to delineate the essential clinical characteristics associated with loss of anterior pituitary function. This authority has stressed that, except occasionally as a terminal event, severe weight loss is not a feature of the disease. Many of the early cases reported as Simmonds’ disease were, in fact, cases of anorexia nervosa, and as a result much of the psychiatric symptomatology attributed to hypopituitarism is spurious. The continued use of the misleading and archaic term “pituitary cachexia” still gives rise to diagnostic confusion. The commonest cause of chronic pituitary failure, to which Sheehan first drew attention, is ischemic necrosis of the gland associated with postpartum hemorrhage and a hypotensive episode ( Sheehan, 1939). Rarer causes include fibrous or cystic degeneration of uncertain etiology and involvement of the pituitary in inflammatory or neoplastic processes ( syphilis, tuberculosis, intrasellar tumors, etc.) and head injury (Sheehan and Summers, 1949). In some of these conditions, there is a concomitant involvement of adjacent basal regions of the brain. Surgical hypophysectomy has recently

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become a more common cause of loss of pituitary function. Examination of such cases before and after operation is less informative than might be anticipated, because they are already suffering from severe physical illness and, in any case, receive prompt substitution treatment. For moderate signs of hypopituitarism to appear, at least 751 of the gland must be destroyed, and 90%for the condition to become severe. The disorder is 4 3 times more m m o n in women than in men. There is a gradual onset of chronic ill-health with evidence of gonadal, thyroid, and adrenocortical failure. Gonadal function appears to be first affected in that amenorrhea and impotence are early symptoms. Other features include the complete loss of pubic and axillary hair, increased sensitivity to cold, dryness of the skin, expressionless facies, physical weakness, and mental torpidity. These patients are especially liable to go into coma (Caughey, 1958). Diagnosis is confirmed by demonstrating a low urinary output of gonadotropin, a diminished thyroid uptake of radioiodine, and a low urinary excretion of adrenal corticoids. Both the iodine uptake and blood PBI are not usually as low as in myxedema and can generally be increased by the administration of TSH (Van Arsdel and Williams, 1956; Bowers et aE., 1961). The urinary steroid excretion, though as low as in severe Addison’s disease, can be increased by administration of ACTH (Diczfalusy et d.,1956). These findings indicate that a basal secretion of both thyroid and adrenocortical hormones persists. The earliest reports of hypopituitarism stress the frequency of psychiatric symptoms. Thus, Jakob (1923) spoke of apathy, somnolence, and states of confusion, as have many others (Biichler, 1923; Hirsch and Berberich, 1924; Redlich, 1927; Silver, 1933; Wadsworth and McKeon, 1941) . Although many earlier reports are of doubtful value, because of the diagnostic confusion already referred to, more recent authors have described similar symptoms (Sheehan and Summers, 1949; Farquharson, 1950; Cleghorn, 1952; Staehelin, 1953; Bleuler, 1954). In a detailed review of the literature, Kind (1958) collected 78 case reports of proven hypopituitarism and in only 6 was psychological disorder noted as being absent. In one-third of the cases, the psychiatric symptoms resulted in considerable social disability, the patients neglecting themselves and becoming dependent upon relatives. Patients were usually described as apathetic, depres-

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sive, dull and drowsy, and lacking initiative and drive. Severe psychological symptoms tended to appear several years after the first appearance of physical symptoms. Although acute organic reactions with confusion and clouding of consciousness were quite frequent, chronic psychoses resembling schizophrenia were extremely rare, Using Sheehan’s strict diagnostic criteria, Kind ( 1958 ) personally made a detailed study of 22 adult cases, 7 of which were untreated, Evidence of previous personality disorder was infrequent. An almost universal early complaint was loss of libido in the female and impotence in the male. General loss of initiative was also reported as an early symptom in 90% of cases, often experienced as loss of interest and drive. Patients spent longer in bed, sleeping time was probably increased and there was intolerance of cold. Appetite was relatively well-preserved, being markedly diminished in only 4 of the 22 cases and actually increased in 2. A quarter of the patients noticed increased thirst and in 3 there was evidence of diabetes insipidus which subsequently decreased as the anterior pituitary failure progressed (Herrmann, 1955).All patients showed mood disorder: indifference, apathy, and mild depression, occasionally interrupted by brief episodes of irritability and quarrelsomeness. This latter feature was more noticeable when loss of initiative was less marked. Only very few patients showed a transient state of superficially serene unconcern-the so-called “hypophysial mood” ( Frankl-Hochwart, 1912). In untreated cases of long-standing, the symptoms appear in an extreme form. Apathy, indifference, and inactivity may become so profound that patients rarely leave their living quarters, they lie in bed for much of the day and neglect even their personal hygiene. Although such severe psychological disability is seen only in cases of advanced and severe hypopituitarism, the physical condition itself seems insufficient to account for these psychiatric symptoms which are not seen in other chronic debilitating diseases. Further, some patients with very severe hypopituitarism fail to develop mental symptoms of equal severity, Eight such cases occurred in Kind‘s series, all of them women, and most of them with signs of thyroid deficiency. A typical example is afforded by a woman with a 10 yr history of postpartum hypopituitarism who lay abed more than half the day and spent most of the rest of the time huddled over the fire; she

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had been abandoned by her husband and needed to be fed by her daughter. Before her illness she had been an active and efficient housewife. In addition to the changes in mood and initiative already referred to, there is evidence in many patients of a chronic brain syndrome. This may occur quite early in the illness, taking the form of a demonstrable memory defect ( 5 of 7 untreated cases seen by Kind). Patients themselves complain of forgetfulness and a case is reported of a nursing sister being obliged to give up work because of inability to remember her instructions. Symptoms of the acute organic type are seen in association with physical crises and take the form of stuporose or comatose states, often preceded or followed by phases of confusion and delirium ( Blau and Hinton, 1960). Although a high proportion of cases die in coma, this may be the occasion when the diagnosis is first made. Chronic psychoses with florid symptomatology, whether of a depressive or schizophrenic type, were not seen by Kind and are very rare in this disorder. Only two cases of typical schizophrenia (Wipf, 1948; Biissow, 1956) and one of depressive psychosis (Wadsworth and McKeon, 1941) have been described in the literature. It is clear that in this polyglandular disorder rational treatment would consist of delicately balanced substitution therapy; DOCA, methyltestosterone, stilbestrol, thyroid, and cortisone have been used to this end (Beck and Montgomery, 1957). Nevertheless, it appears that cortisone alone, in small doses, produces marked improvement in a high proportion of cases, Relatively few patients need a combination of both cortisone and thyroid (Sheehan and Summers, 1954; Whittaker and Whitehead, 1954; Fourman and Horler, 1954). Five of the 7 patients treated by Beck and Montgomery (1957) were relieved of their symptoms, including intolerance to cold, by cortisone alone; the other two required the addition of thyroid, which abolished their cold sensitivity and restored their energy. Even symptoms which might be thought to be more closely related to gonadal insufficiency can be favorably influenced by cortisone: libido may be restored in the female and erections return in the male. The menstrual cycle is never restored. High maintenance doses of cortisone (75100 mg a day) have the same deleterious effects as in certain cases of Addison’s disease, patients becoming depressed or deluded ( Sheehan and Summers, 1954). The addition of methyl testosterone

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(Whittaker and Whitehead, 1954) produces a greater restoration of body hair and, it is reported, sometimes an increase in libido and sexual performance in the male. A few days after the institution of cortisone treatment, return of interest and energy is experienced subjectively, and all authors stress the psychological improvement. Of 16 patients observed by Kind, 10 lost all their psychological symptoms after prolonged treatment; the other 6, although improved, still showed some degree of apathy and lack of drive. It was concluded that the effects of treatment on the psychological symptoms are less satisfactory if symptoms have been severe and of long duration and also if the patient’s previous personality was abnormal. The response to cortisone is illustrated by a case from the Maudsley Hospital, diagnosed as hypopituitarism when admitted in coma, 10 yr after a postpartum hemorrhage. After failure of lactation and, later, of menstruation she had complained of loss of energy and initiative, absent libido, and failing memory. When stabilized on a maintenance dose of cortisone (25 mg daily) a return to apparently full health occurred-libido was restored, energy and cheerfulness returned, and the patient was able to take an active part in the family business. The surprised husband remarked that after a lapse of 10 yr his wife had again requested sexual intercourse. 3. Conclusions to Clinical Studies

It is evident from the studies that have been reviewed above, that the experience of emotion by healthy subjects is associated with a rise in the plasma level of 17-hydroxycorticosteroids,and that this is due to an increase in adrenocortical activity. This takes place both when the emotional arousal occurs spontaneously, as the result of environmental stimulation, or when induced experimentally within the laboratory. In general, environmental stress is more effective than experimental procedures in provoking steroid rises, except in the case of hypnotically induced anxiety. The evidence derived from experimental studies is valuable, however, because it clearly indicates that it is the intensity of the subject’s emotional response which correlates with the increased adrenocortical activity and not the intensity or type of the stressful stimulus. One is led to conclude that “emotional arousal” is the effective stimulus rather than the particular type of emotional response evoked, e.g., anxiety, fear, or sad-

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ness. This view is confirmed by the findings of the Walter Reed group who also stress the importance of the degree of general emotional involvement in a situation. Much of this work has been concerned with emotional responses which are clearly of a distressing nature. But it has also been demonstrated that exposure to new situations, which might be regarded as slightly threatening but certainly not as extremely stressful, is highly effective in stimulating the adrenal cortex. Even the anticipation of a pleasurable or exciting experience may have the same effect. One is reminded here of the effectiveness of first experiences in causing steroid rises in monkeys. As Hamburg (1959) has pointed out, all of this work is concerned with the delineation of those psychological states which are associated with an increase in adrenocortical activity, while at present little is known about psychological conditions which may have the reverse effect. Whether these changes in adrenocortical activity have a beneficial or protective function is not clear, but it is possible that they may at times be harmful. Although the emotional disturbances produced experimentally induce only transient rises in plasma steroids, this may not be the case when the emotional stress is prolonged. The morning levels in subjects experiencing emotional stress average about 21 pg/100 ml and are much lower than the levels which can be produced by ACTH administration. Nevertheless, they are as high as in some patients with Cushing's disease but, in the former group, the normal diurnal variations still occur and so the tissues are not exposed to a sustained high level of steroid. This level of cortisol production, if accompanied by increased production of adrenalin, could be responsible for an increase in blood cholesterol levels and hence be a factor in the genesis of atheroma and hypertension. It has already been mentioned that sustained emotional stress may be a significant factor in the development of Cushing's syndrome which has been regarded by some authorities as essentially a hypothalamic disorder. Absence of the normal diurnal variation in plasma steroids, which is the central endocrine abnormality in this syndrome, has also been produced in monkeys with bilateral hippocampal ablation and fornix section, and has recently been described in human subjects with dif€use brain-damage ( Eik-Nes and Clark, 1958) and also with localized damage to the temporal lobe and midbrain ( Krieger, 1961). The effect of emotion on adrenocortical activity has been amply confirmed by studies upon psychiatric patients. Considerable in-

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creases in plasma steroids are the rule in anxious and depressed subjects and in acute schizophrenic patients showing emotional turmoil. However, chronic schizophrenics displaying no disturbance of affect have steroid levels within the normal range, Recovery from depression appears to be accompanied by a decline in adrenocortical activity but too little information is available at the moment to allow a firm statement to be made about mania. In general, however, it can be concluded that, in psychiatric disorder, it is the emotional state itself rather than the particular type of illness which determines the level of adrenocortical activity. There are, of course, individual exceptions to this generalization; anxious or depressed patients are found with low blood steroid levels and recovered depressives may still show levels that are higher than normal. Nobody would claim that the emotional state was the principal regulator of plasma steroid levels but patients behaving exceptionally in this respect certainly merit further study. The effect of mood upon the adrenal cortex is paralleled by the effect of adrenocortical disease upon mood. The adrenocortical hyperfunction of Cushing’s syndrome is almost always accompanied by mood change, mainly depressive in type and also by emotional lability and overreactiveness. Mood changes are frequently induced by ACTH and cortisone administration, although euphoria is more common than depression. Acute psychotic episodes, resembling functional psychoses, occur in both conditions. Mood change is equally common in the adrenocortical hypofunction of Addison’s disease and hypopituitarism, where it most often takes the form of apathy and indifference. In the two latter conditions, delirium and disturbances of consciousness due to metabolic crises are common while functional psychoses are rare, In both hyper- and hypofunction appropriate treatment of the endocrine condition can be expected to restore the psychological state to normal or to produce marked improvement. It has been reported that adrenalectomized patients, maintained on adequate, fixed doses of steroids, experience a more serene emotional life and less fluctuation in mood than before their illness. This suggests that the changes in adrenocortical function which accompany emotion may play a part in determining the emotional experience. This notion is supported by the fact that patients with Cushing’s disease are disturbed by the intensity of emotional e v e -

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rienoes and by the loss of emotional response which is a usual, if not invariable, feature of adrenocortical hypofunction. While all the evidence overwhelmingly points to an intimate relationship between mood and the adrenal cortex, it is equally clear that no single cause for psychiatric illness can be found in its disordered activity. II. The Thyroid and Psychiatry

A. THYROIDAL FUNCTION IN MENTALDISORDER 1. Studies Based upon the Basal Metabolic Rate A possible association between thyroid activity and some cases of schizophrenia was suggested by the reported success of thyroxine treatment in periodic catatonia (Gjessing, 1939), as well as by the discovery that a low basal metabolic rate (BMR) was common among schizophrenic patients (Bowman and Fry, 1925; Hoskins and Sleeper, 1929; Lingjaerde, 1933). Moreover, some of these patients were insensitive to very large doses of thyroid extract by mouth. The BMR will not be considered in detail because it depends on many factors other than thyroid activity. It is sufficient to mention the results of an investigation by Bowman et al. (1950). These workers found a rather low mean BMR of -6.5, and a range of -31 to -1-7, in a series of 26 schizophrenics. An admittedly much smaller group of 7 psychoneurotic patients, however, showed an even wider range of -32 to +14 and a lower mean of -12.3. It seems clear that most schizophrenic subjects have a BMR within the normal range, particularly when allowance is made for malnutrition and lean body build. In a minority of schizophrenics a low BMR of -20 to -30 is found, but much the same is true of the general population. In recent years attention has also been directed to patients presenting vague ”neurasthenic” complaints who were found to have a low BMR, and it was suggested that they were suffering from “metabolic insufficiency” or “non-myxedematoushypometabolism.” This disorder, if it exists, was characterized by a low metabolic rate in spite of a normal radioiodine uptake and was said to respond to treatment with triiodothyronine but not with thyroid extract (Kurland et al., 1955). A careful study of 22 patients who conformed to the criteria of the syndrome was made by Levin (1960). He found that their complaints were suggestive of psychoneurosis rather than myxedema and that their MMPI scores indicated a general maladjustment. A

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double-blind trial failed to show any difference in the effects of triiodothyronine, thyroxine, and an inert placebo on either symptoms or metabolic rate. A similar controlled study by Sikkema (1960) on 20 patients showed that improvement occurred as often with placebo as triiodothyronine. It seems safe to conclude that a number of psychoneurotic patients have very low metabolic rates, but this is not evidence of a causal connection, since, as we have seen, the same situation exists in the general population. 2. Studies with Radioiodine a. Radioiodine Uptake in Schizophrenia

The introduction of radioiodine made possible a more direct estimate of thyroid function and several investigators have measured the radioiodine uptake in schizophrenic subjects. Bowman et al. (1950) reported a mean 24-hr uptake of 34% in 26 patients, compared with 28%in 38 control subjects. Cranswick (1955) similarly reported a mean uptake of 33%.These differences are small and difficult to interpret. In at least one investigation of psychiatric patients rather high uptakes were shown to be due to iodine deficiency in the hospital diet (Kelsey et al., 1957). This criticism does not apply to the Norwegian patients of Lingjaerde et al. (1950), whose diets were rich in sea fish. The mean uptake was 3% in 27 schizophrenics. This was not significantly different from the mean uptake in other diagnostic groups. There were no control data available for comparison but the authors considered that these results confirmed the findings of Bowman et aE. (1950) and Cranswick (1955). When the clinical state of the schizophrenics was considered, however, it was found that the mean uptake in 6 patients with active schizophrenia was 28%,compared with 42%in 16 patients in an inactive phase of the illness. These results are in the opposite direction to the plasma steroid findings in schizophrenics [see Section I. B. 1. c(4)]. Stevens and Dunn ( 1958) however reported low-normal radioiodine uptakes in their group of 28 schizophrenics. It is clear that no general conclusions can be drawn from these investigations, perhaps because the iodine uptake is so susceptible to changes in iodine intake and also because one group of schizophrenics may be very different from another in symptomatology, emotional state, length of illness, and so on.

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b. Application of the I, Index to Schizophrenia A rather more complex index of thyroid function, the I, value, derived from the initial rate of uptake of radioiodine by the thyroid and the actual uptake at 24 hr, was used by Reiss and his colleagues (Batt et aE., 1957). They found that the I, value was within their rather narrow normal limits in two-thirds of their schizophrenic group. Of the schizophrenic men, 19%had decreased and 15%increased thyroid activity; the respective figures among the women patients were 14%and 218. Very similar findings emerged from an investigation of chronic schizophrenics in Zurich using the same method (Stoll and Brack, 1957): 20%decreased and 1 m increased thyroid activity in men, 8%and 25%in women. The significance of these findings depends very much on the reliability of the method and on the accuracy of the normal limits. It has been shown, with the same toroidal counter used by Reiss, that considerable variations ‘occur when the iodine uptake is measured on repeated occasions in schizophrenic patients (Hare and Haigh, 1955; Crammer and Pover, 1960). Reiss (1954) also noted variations on repeated testing but reported that they correlated with changes in clinical status. A change towards normal from either direction was associated with clinical improvement, while persistence of thyroid activity outside the normal range was associated with lack of improvement. (These results applied to other diagnostic group also, and were independent of the type of treatment given), Stoll and Brack (1957) carried out a second estimation of the It value in 29 cases but were unable to demonstrate any relationship between clinical improvement and normalizing of thyroid activity. They also used the original I, values as a basis for treatment, as suggested by Batt et aE. (1957); thyroid hormone was given to 24 “hypothyroid” schizophrenics and antithyroid medication to 31 “hyperthyroid” schizophrenics. Improvement occurred in only 4 and 11, respectively. They concluded that the apparent anomalies of thyroid function were of theoretical interest but of no practical importance. Stoll and Brack‘s patients have been followed up by von Brauchitsch (1961) who found that those with higher levels of thyroid activity tended to have the better prognosis, When other diagnostic categories are considered there is no clear evidence of abnormality in iodine uptake, although Reiss (1954)

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reported a wider than normal range of It values in both aflective and neurotic disorders. Other investigators concluded that anxious patients (Dongier et d.,1956) and depressives (Gibbons et d.,1960) have uptakes which do not differ significantly from controls. The same conclusion about depressives can probably be drawn from the data of Lingjaerde et al. (1960), although they use a psychiatric classification system which is not easily translated into Anglo-American terminology.

3. Protein-Bound Iodine a. Psychiatric Disorders

The other main index of thyroid function is the serum protein bound iodine, which is less easily influenced by dietary variations. Starr et a,?. (1950) reported normal values in psychiatric patients generally while Brody and Man (1950) found a mean PBI of 5.4 pg/lOO ml in 57 schizophrenics, 5.5 pg in 125 psychiatric patients of other types, and 5.3 pg in euthyroid normal subjects. Bowman et al. (1950) reported a slight decrease in mean PBI when 11 depressives were compared with normal controls (5.5 pg compared with 6.2 pg). Gibbons et al. (1960), however, found no significant difference between 19 depressives and normal controls. As the patients recovered, slight changes in PBI occurred and the mean change was a small decline of 0.5 pg, which was just statistically significant but hardly so clinically. However Board et al. (1957) had found a similar decline in PBI in improving depressive patients, and their retarded patients had a rather higher PBI than the nonretarded depressives. Board et al. (1956) measured PBI levels in 30 patients newly admitted to hospital, The PBI in the whole group was not significantly different from the control value but that in their “psychotic”patients, 10 of whom were depressed, was significantly different (6.4 pg versus 4.9 pg) , The last three investigations suggest that prolonged severe emotional disturbance, such as depression, is associated in some cases with an increase in the serum PBI level, and that the PBI may decline as recovery occurs. The changes are small, however.

b. Effect of Psychological Stress Experiments in animals have shown that “psychological” stress may cause a profound change in thyroid function. Is there any evi-

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dence for such an effect in man? Hetzel et d.(1952) aroused strong emotion by means of stressful interview in 3 euthyroid and 3 hyperthyroid subjects and found that after the interview the PBI rose to well above control values. So rapid a rise in the blood level of a hormone which has a biological halflife of 7 days or more is a rather unexpected finding. Tingley et al. (1958) reported a significant rise in the PBI level of medical students on an examination day. On the other hand, Dongier et al. (1956) were unable to affect the blood level of thyroid hormone by stressful interviews, Volpe et al. (1960) studied medical and postgraduate students preparing for examinations as well as professional footballers in training and during games. The PBI fluctuations remained within the expected range and they concluded that there is no evidence that thyroid function in normal subjects is readily affected by mental stress. Nor were they able to detect correlations between fluctuations in PBI and the ordinary stresses of living. The evidence, then, is equivocal. One may perhaps conclude that emotional tension is associated with slightly increased thyroid activity as assessed by measurement of the PBI. B. PSYCHIATRIC CHANGES IN THYROID DISEASE 1. Hypothyroidism In established myxedema, psychological symptoms are the rule; loss of interest and initiative, slowness of response, failure of memory, and a general dulling of the personality are all well-known features of the disorder. The characteristic mood is apathy rather than depression, and such symptoms are readily understood as the psychological counterpart of the lowered metabolic rate. That the brain is affected in the general physical disturbance is clearly shown by the changes seen in the electroencephalogram, namely, a slowing in the dominant frequency and a reduction in voltage (Ross and Schwab, 1939; Browning et al., 1954). There is also a reduction in cerebral blood flow and in cerebral oxygen utilization (Scheinberg et al., 1950). Moreover, the psychological symptoms and both the abnormalities in the electroencephalogram and in cerebral metabolism are improved by substitution treatment with thyroid hormone. Although no specific neuropathological changes have been described in myxedema, it has been stated that severe and prolonged untreated

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illness can give rise to gross dementia because of extensive irreversible brain-damage (Peters, 1951; Bleuler, 1954). In milder cases of hypothyroidism, the physical signs are less obvious and the psychological symptomatology may be less characteristic, so that depression, irritability, or even excitement may take the place of apathy (Richard, 1952). Evidence of intellectual impairment in these milder cases was presented by Reitan ( 1953), who examined a series of patients by means of the Rorschach test and found that their results fell midway between those of a group of brain damaged patients and those of a neurotic group. These milder symptoms can also be expected to clear with adequate substitution treatment. The incidence of severe mental disorder in hypothyroidism has been a subject of discussion for many years. British and American writers have been more impressed by their frequency than continental authorities; the report by Asher (1949) may be contrasted with the scepticism of Bleuler’s (1954) monograph. The famed Committee on Myxoedema, set up by the Clinical Society of London in 1888, reported the presence of delusions in 18 out of 46 cases, hallucinations in 16 out of 43, and “insanity” in 16 out of 45. The insanity took the form of “acute or chronic mania, dementia or melancholia.” A marked predominance of such symptoms as suspicion and selfaccusation were noted. Admittedly, this was in the days before thyroid treatment, when advanced myxedema was more common. Numerous case reports have since been published, and Browning et al. (1954) found 100 cases of myxedema in association with psychoses reported in 30 papers. It has been suggested that this association may be a chance one since both conditions are liable to occur in women of the same age group (Davies, 1949). Almost all of the cases described in the literature have been women. Myxedema in association with severe mental illness does not appear to be common in the mental hospital population. The wide-spread survey with radioiodine in Zurich revealed only 3 instances of such an association (Bleuler, 1954). Only 6 cases were reported in the University Psychiatric Clinic in Heidelberg in 50 yr. On the other hand, Asher (1949) saw 14 cases in an observation ward in London in a considerably shorter period, and the present authors have seen 7 cases in the last 5 yr. In 2 of these cases, the psychiatric illness had preceded the onset of the hypothyroidism.

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Granted that the incidence of severe mental disorder in myxedema varies from one country to another, it remains to consider what type of psychosis occurs and the effect of substitution therapy upon it. Browning d al. (1954) reviewed 100 cases from the literature and were impressed by the variety of psychiatric phenomena reported: “Behaviour may range from excitement to languor, the mood from mania to severe depression, the thought content from suspiciousness to paranoid delusions, which may include the presence of hallucinations .’’ Significant improvement or complete recovery usually followed adequate treatment with thyroid. They reported 7 personal cases, all of whom they considered to be examples of delirium, although organic features were obvious in only two. The main features were mania in 1 case, anxiety and bizarre hypochondriasis in another, agitated depression in 2, and depression with paranoid ideas in 3. All improved with thyroid treatment and the electroencephalogram returned towards normal. They concluded that the cerebral metabolic defect of hypothyroidism was responsible for an organic delirium and that the secondary psychological features depended on individual and personality factors. This conclusion depends very much upon the validity of their criteria for delirium, Support for their view is provided by a careful reading of ‘the 14 cases reported by Asher, 8 of which showed obvious disorder of consciousness. In 6 cases, however, the sensorium was clear; 5 of these had a predominantly paranoid illness, and the remaining case was severely depressed. Full recovery occurred in all but one of these cases with thyroid treatment. Of the series of 14 cases, 2 died, 3 failed to improve, and 9 recovered completely. Asher (1949) concluded that thyroid treatment was responsible for the mental irnprovement and suggested that failure to respond might mean that irreversible changes had occurred. A typical case of mania, occurring in a cyclothymic young woman after the onset of hypothyroidism, as well as 2 cases of depression, was reported by Kind (1953). In these cases, recovery occurred when the hypothyroidism was treated. No evidence of organic psychosis was found in these patients. These cases of predominantly affective disorder may be compared with the 6 cases reported by Huber (1956), in all of which the symptoms were considered to be schizophrenic, again without any definite evidence of organic features. In five cases psychological recovery occurred in

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2-6 weeks after the institution of thyroxine treatment. Huber concluded that these cases were examples of symptomatic psychoses. This more recent contribution supports the observation of Browning et al. (1954) and of Asher that adequate substitution therapy usually results in mental recovery. But a cautionary note may be added here because, of the 5 cases seen recently at the Maudsley Hospital in which psychiatric symptoms followed the development of hypothyroidism, 2 cases remained unchanged and only 1 case definitely recovered from her psychological illness during the period of physical improvement produced by thyroxine. Similar observations have been made by Pitts and Guze (1961) and Snyder (1961). 2, H y p e r t h y r o i d h The psychological symptoms of hyperthyroidism are well-known and are present in almost every case of the disease. Typically they include emotional lability and overreactiveness with predominant anxiety and tension. Some patients are depressed and a few are euphoric. A very small group are indifferent and apathetic, showing the so-called apathetic form of hyperthyroidism ( Bleuler, 1954; Hare and Ritchey, 1946). Appropriate antithyroid treatment abolishes or greatly reduces the psychological symptoms. It has been contended that many of these patients have shown marked psychological symptoms prior to the onset of the physical illness, symptoms related to emotional conflicts or to personality disorders which are felt to be important in the genesis of hyperthyroidism (Mandelbrote and Wittkower, 1955; Bennett and Cambor, 1961). The large literature on the psychosomatic aspects of hyperthyroidism cannot be reviewed, except to say that there is wide agreement that psychological factors may determine the onset of the disorder (Means, 1948; Lidz, 1949; Ham et al., 1951; Racamier, 1951). Many believe that the psychotherapeutic handling of hyperthyroid patients is equally as important as adequate drug treatment, Robbins and Vinson (1960), who do not support this view, administered “objective” psychological tests, including the Maudsley Personality Inventory, to 10 hyperthyroid patients and to similar groups of normals and psychiatric patients in various diagnostic categories. The hyperthyroid patients were tested before and after successful treatment. It was deduced from the pattern of results that the untreated hyperthyroid patients were similar to a brain-damaged group, and that adequately treated

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patients resembled nonnal subjects. They concluded that the importance of personality factors had been overstressed and that the toxic effect of the hyperthyroidism on the brain was responsible for the psychological symptoms. If the Maudsley Personality Inventory results are considered alone, however, it is seen that the untreated patients had a very similar “neuroticism” score to patients with somatization reactions ( 15.5 and 15.9, respectiveIy) , After antithyroid treatment, this score had fallen to 9.6, whereas their normal group scored only 5. More severe mental disorders are also seen in hyperthyroid patients. Before the introduction of antithyroid drugs, psychoses of the acute organic type were frequently encountered in association with severe cases and with thyroid crises. These accounted for 102 of the 134 cases of hyperthyroidism with mental disorder seen at the Mayo Clinic (Dunlap and Moersch, 1935). Occasional cases are still reported, but the delirium may nowadays be due to antithyroid drugs (An& and Titeca, 1951). The incidence of other psychoses in hyperthyroid patients has been in dispute and the subject has recently been reviewed by Bursten (1961). At one extreme, Lidz and Whitehorn (1949) detected evidence of psychosis in 20% of thyrotoxic patients attending an out-patient clinic, while Kleinschmidt et d.(1956) considered that 204: of their 84 thyrotoxics were schizophrenic or borderline psychotics. Such high percentages seem to be dependent upon a readiness to diagnose schizophrenia from minimal symptoms, and contrast with the general statement that overt psychosis is uncommon ( Katzenelbogen and Luton, 1935; Mandelbrote and Wittkower, 1955). From 1955 to 1958, Bursten (1961) found only 10 patients with psychosis and concurrent thyrotoxicosis among 8000 patients admitted to a New York mental hospital. On the other hand, 10 of 54 thyrotoxic patients admitted to a general hospital in Buffalo were diagnosed as psychotic, although of 34 admissions to another hospital, none were so diagnosed. Since the former hospital received most of the psychotic patients from the community, there was obviously a bias towards a high incidence of psychosis and, interestingly enough, the incidence was not significantly greater than that of psychosis among diabetics admitted to the same hospital. Bursten felt able to conclude, however, that the concurrence of overt psychosis with thyrotoxicosis was not a clinical rarity. The type of mental illness that is most often associated with hyperthyroidism has also been in some dispute. Dunlap and Moersch

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(1935) found manic depressive illness, particularly depression, to be the most common (26 of 32 cases of functional psychosis), and most psychiatrists are familiar with the concurrence of hyperthyroidism and agitated depression. Mania has been described occasionally (Tusques, 1941) and other authors have been more impressed with schizophrenia-like illnesses ( Kleinschmidt et ul., 1956). The 10 cases of Bursten’s were divided as follows: 3 with organic psychosis, 1 with psychosis of undetermined type, 1 with depression, and 5 with schizophrenia. The relationship between the physical disease and the mental disorder is not always clear. Bleuler (1954) felt that the apparent association with frank schizophrenia might be due to chance, on the grounds that the association is rare and that the physical and psychological states vary independently. The present authors are familiar with cases of agitated depression and hyperthyroidism in which antidepressive treatment is needed for the depressive symptoms in addition to antithyroid treatment for the relief of the hyperthyroidism. Here, too, a chance association is a tempting explanation, but when the mental disorder follows the onset of hyperthyroidism and ameliorates when the latter is treated, it suggests that a causal relationship exists. There are, however, those interesting cases in which the course of the illness suggests that the hyperthyroidism is itself a consequence of the mental disorder. For the present, one cannot but agree with Bursten’s conclusion that it is impossible to describe a relationship of thyrotoxicosis to psychosis which will apply satisfactorily in all cases where the disease processes appear concurrently. 3. Conclusions: Thyroid Disease and Psychiatry It seems clear from the evidence reviewed above that, despite the findings of earlier workers, thyroid activity as measured by the blood level of thyroid hormone is essentially normal in the major psychoses. The most that can be said is that the blood level of hormone may be slightly raised in conditions of severe and sustained emotional disturbance. Although there is general agreement about the serum protein-bound-iodine levels in psychiatric disorders, findings derived from measurements of radioiodine uptake are somewhat discordant. It is possible that these differences will be resolved when methods for the measurement of serum stable iodide and, therefore, of absolute thyroidal iodide uptake, can be applied. Further work in this area must depend upon the application of methods

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of indisputable accuracy and is more likely to be rewarding if confined to the intensive investigation of a series of individual cases. There is overwhelming evidence, however, that both hyper- and hypothyroidism are associated with profound emotional changes, and good evidence that they can give rise to severe mental disorder. In a similar way, excessive or deficient adrenocortical activity causes emotional changes and psychiatric disturbance. Although it is clear that psychological events can rapidly influence the activity of the adrenal cortex, there is no evidence, as yet, of an analogous effect by mental events upon short-term changes in thyroidal function. REFERENCES Addison, T. (1855). “On the Constitutional and Local Effects of Disease of the Suprarenal Capsules.” Samuel Highley, London. Aird, R. B., and Gordan, G. S. (1951).J. Am. Med. Assoc. 145,715. Albright, F. (1942).Harvey Lectures 38, 123. Altschule, M. D. (1953a).Intern. Record Med. 166, 190. Altschule, M. D. (1953b).“Bodily Physiology in Mental and Emotional Disorders.” Grune & Stratton, New York. Anand, B. K., and Dua, S. (1955).1. Physiol. (London) 127,153. Anderson, E., Bates, R., Hawthorne, E., Haymaker, W., Knowlton, K., Rioch, D. M., Spence, W. T., and Wilson, H. (1957).Recent Progr. in Hormone Research 13, 21. ’ Andrk, M. J., and Titeca, J. (1951).Acta Neurol. Belg. 51, 806. Asher, R. (1949).Brit. Med. J . 2, 555. Astwood, E. B., Raben, Y. S., Payne, R. W., and Grady, A. B. (1951).J. Am. Chem. SOC. 73, 2969. Barrett, A. M., Hodges, J. R., and Sayers, G. (1957).J . Endocrinol. 16, xiii. Batt, J. C., Kay, W. W., Reiss, M., and Sands, D. E. (1957).J . Mental Sci. 103, 240. Beck, R. N., and Montgomery, D. A. D. (1957).Brit. Med. J . 1,441. Bell, P. H.(1954).J. Am. Chem. SOC. 76,5565. Bennett, A. W.,and Cambor, C.G. (1961).Arch. Gen. Psychiat. 4, 160. Bergen, J. R. (1951).Am, J . Physbl. 164,16. Bergen, J. R., and Hoagland, H. (1951).Am. J. Physiol. 164,23. Bergen, J. R., Hunt, C. A,, and Hoagland, H. (1953).Am. J . Physiol. 175,327. Blau, J. N.,and Hinton, J. M. (1960).Lancet 1, 408. Bleuler, M. ( 1954). “Endokrinologische Psychiatrie.” Thieme, Stuttgart. Bliss, E. L., and Branch, C. H. H. (1960).“Anorexia nervosa.” Hoeber, New York. Bliss, E. L., and Migeon, C. J. (1957). J. Clin. Endocrinol. and Metabolism 17, 766. Bliss, E. L., Migeon, C. J,, Nelson, D. H., Samuels, L. T., and Branch, C. H. H. (1954).A.M.A. Arch. Neurol. Psychht. 72, 352. Bliss, E. L., Migeon, C. J., Branch, C. H. H., and Samuels, L. T. (1955).Am. J . Psychid. 112, 358.

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NEUROLOGICAL FACTORS IN THE CONTROL OF THE APPETITE By And& Soulairac Psychophysiological Laboratory, Facultd des Sciences, Paris, France

I. The Regulating Nervous Structures

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304 304 308 311 314 A. Different Types of Information 314 B. Various HypotneSes Regarding Control Mechanisms . . . . 322 111. Tentative Explanatory Hypothesis Regarding the Control of Appetite 332 A. Control Through “Interior Environment” . . . . . . . 332 B. The Sensory Role in Feeding Control . . . . . . . . 336 IV. General Conclusions . . . . . . . . . . . . . . 339 References . . . . . . . . . . . . . . . . 342

. . . . . . . B. The Rhinencephalon and Feeding Behavior . . . . . C. Neocortical and Striated Structures in Feeding Behavior . . 11. Nervous Mechanisms Controlling Feeding Behavior . . . . A. Hypothalamus and Feeding Behavior

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Experimental research in the last few years has shown indisputably that the need to eat and the amount of food ingested are under the direct control of the central nervous system. The need to eat determines what is commonly known as hunger. In certain cases, even the alimentary needs become more specialized; these are the specific appetites. In the course of this work, reference will be made indiscriminantly to “hunger,” “appetite,” and even to “feeding behavior” (the comprehensive psychophysiological reactions manifested as hunger), save in certain cases, when we shall consider some more specialized qualitative control systems. Feeding behavior presents a particularly complex psychophysiological problem, for it implies a very close integration of basic homeostatic controls, of nervous integration at all levels, and finally, the reflection of this as an exteriorization of a specialized and completed neuromotor drive. It is not surprising that a great number of 303

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theories have been advanced to explain the mechanism responsible for hunger. Recent data from contemporary neurophysiology gives glimpses of possible solutions. Thus, although some kind of nervous control is certain, a great deal of inaccuracy and numerous lacunae in our knowledge still remain regarding the nature of the regulating mechanisms themselves that initiate the functioning of the nerve structures. After a rapid survey of the current state of knowledge of the nervous structures concerned in feeding behavior, we shall examine the theories suggesting an explanation of its basic controls, and shall attempt to offer a tentative hypothesis covering the overall psychophysiological regulation of such behavior. I. The Regulating Nervous Structures

The higher nervous control of feeding behavior seems basically to devolve on diencephalic structures (hypothalamus) which apparently are controlled by a whole series of telencephalic higher structures (rhinencephalon, neocortex, and possibly the striatum). A. HYPOTHALAMUS AND FEEDING BEHAVIOR

Historically the first experiments were on the hypothalamus and these contributed greatly to the orientation of later research. 1. The E3ect of Lesions By 1933 Keller et d.,and Krieg (1938)noted that certain hypothalamic lesions increased the appetite in animals. But Hetherington and Ranson (1940) were the first to find, using stereotaxic techniques, that lesions of the ventromedial nuclei of the hypothalamus’ caused obesity, Brobeck et al. (1943) showed that animals became obese because they ate more than normal animals and used Ylypothalamic hyperphagia” to designate the essential disturbance of the hypothalamic lesion. Hyperphagia normally takes place in two distinct phases: 1. The first is a dynamic phase immediately following the operation, during which the animal eats 2-3 times more than it eats normally and rapidly takes on weight. 2. The second is a static phase during which intake of food returns practically to normal but weight remains at a high IeveI. It should be noted that both phases depend directly on the hypotha-

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lamic lesions. In the case of extensive lesions, some of the animals remained in the dynamic phase until death. The physiological signifwance of a return of normal appetite once a certain obesity is reached is not yet known, but as we shall see it has given rise to hypotheses regarding the regulating mechanism (see Section 11. B ) . The nervous lesions that cause these disturbances, are located in the tuberous and posterior levels of the hypothalamus, The most effective of these are localized in the ventromedial nuclei and particularly in the ventrolateral portions. In the rat, the destruction of the ventromedial nuclei need not be total for appreciable hyperphagia to be observed. Hetherington and Ranson (1939) suggested that this may be due to the destruction of neuraxons leaving these nuclei along the ventrolateral portions. The same fibers could, therefore, be interrupted at more posterior levels with similar results. In the monkey, Ruch d d.(1942) caused obesity by much more posterior lesions of the ventral portion of the hypothalamus, of the anterior region of the mesencephalic tectum, and of the H fields of Forel. -These findings lead to the conclusion that the neurons of the ventromedian portion send their neuraxons to the remote posterior portions along an inferior lateral path and then along a superior lateral path. In the course of systematic research on the hypothalamus, Soulairac and his co-workers (1947-1957)came to similar conclusions on the localization of effective hypothalamic lesions as the cause of hyperphagia. It should be pointed out, however, that isolated lesions of the anterior hypothalamus (preoptic region, and supraoptic nuclei), of the upper middle hypothalamus (paraventricular nuclei and dorsomedian nucleus) and of the posterior hypothalamus (mammillary complex) never produce any notable disturbance of feeding behavior. During 1951 Anand and Brobeck noted that, in the rat, bilateral hypothalamic lesions in the lateral regions of the ventromedial nuclei led to a discontinuance of feeding and thence to death in a few days. The authors concluded they had evidence of a “feeding center,” the destruction of which caused the stoppage of the mechanism of hunger, In point of fact, the reality was soon shown to be much more complex than the authors had thought. Soulairac d d,(1954)soon showed that the specacity of such lesions was by no means so precise. Bilateral destruction of the lateral hypothalamic areas often resulted in both aphagia and adipsia, and for rats thus lesioned, food

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and drink appeared to have lost all biological significance. Morgane (1961a-d) also reported that different types and degrees of the adipsia-aphagia syndrome could be produced, depending on the location of the lesions, from the lateral hypothalamic area up to the ventromedial level. The author confirmed the earlier results of Soulairac et ul. (1954) on the production of a “motivationalinertia” syndrome in respect to water and food, during which the animals lost all interest in these substances. Actually, all manner of intermediate results are also possiblefrom lesions causing death in 4-5 days due to total and irreversible aphagia and adipsia all the way to lesions causing this syndrome for a few days, after which progressive recuperation takes place. Furthermore, the localization itself of effective lesions was examined by Soulairac et d.in 1954. Lesions situated more medially, at the external limit of the ventromedial and dorsomedial nuclei, and extending dorsally up to the zona incerta, often destroying one or other of the fornices, frequently cause also total and definitive anorexia, often accompanied by adipsia. In such cases, the so-called “feeding center” does not have lesions but the important feature seems to be a lesional separation of the medial and lateral structures. Teitelbaum and Stellar (1954) noted similar facts and stressed the possibilities of recovery under certain experimental conditions. Montemurro and Stevenson (1956, 1957) also found that lateral lesions could cause adipsia without aphagia. Morrison and Mayer (1957) came to somewhat comparable conclusions indicating that effective lesions could involve appreciably greater regions than those noted by Anand and Brobeck (1951). It would appear from these data that it is not possible to speak of a “feeding center” in the anatomical or functional sense of the term, but rather of nervous mechanisms, the functioning of which intervenes during the integration of information for feeding behavior. 2. Effects of Stimulutim of H y p o t h a b i c Centers Experimental stimulation of the structures previously noted as having a role in the control of feeding behavior has enabled some of their functional aspects to be specified, Delgado and Anand (1953) noted that electrical stimulation of the lateral hypothalamic areas in the cat appreciably increased daily food intake. Larsson (1954) also obtained hyperphagia in goats with elec-

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trical stimulation of the same region as well as with intrahypothalamic injections of hypertonic solutions. Some of Larsson’s findings were rather striking. In certain animals, stimulation of the lateral hypothalamic areas may initiate considerable increase of masticatory movements, deglutition and rumination, with no appreciable modification in the amount of food ingested. It is also worth pointing out that Larsson also obtained hyperphagia in the goat by electrically stimulating the nucleus of the vagus motor in the bulbar region, but the animal’s alimentary behavior was greatly disturbed qualitatively. Morgane (1961a) undertook a detailed study of the electrophysiology of the hypothalamic structures concerned with alimentary control. Electrical stimulation of the lateral hypothalamic region causes food intake in satiated animals and also enables the animal to cross electrified barriers for a food reward. Stimulating the medial lateral region has the same effect on consumption but does not motivate the animal to the electdied grating. Lesion or stimulation of the anterior and posterior medial forebrain bundle (MFB) at the level of the “feeding centers” does not modify basic alimentary behavior. After lesions in the MFB, however, external lateral stimulation never causes the animals to cross the electrified grating. External lateral stimulation and simultaneous posterior periventricular stimulation will inhibit the crossing of the electrified grating and even stop such an act instantaneously if it is being executed. Simultaneously stimulating the external lateral area and the ventromedial nuclei in satiated animals only induces basic feeding, In animals feeding while responding to electrified grating tests after external lateral stimulation, the addition of ventromedial stimulation slows down but does not entirely suppress either of the 2 activities. Ventromedial stimulation alone never causes feeding or crossing of the grating, but if the stimulation is interrupted a ‘febound” feeding is obtained in the satiated state. These findings by Morgane are of interest since they show that, even at the level of the hypothalamic structures, it is necessary to dissociate a system responsible for food consumption proper and a system governing motivation of hunger. Morgane assumes that the feeding system proper may be situated in the external lateral area in the diffuse territories of the pallidofugal system. The hunger-motivation system corresponds anatomically to the more medial components of the MFB. Thus, stimulation of the external lateral area

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would energize the mechanism of feeding by direct facilitation of the motivational portion of the alimentary system at the ventromedial level. Furthermore, the latency of the basic alimentary response when the ventromedial area is stimulated first, followed by stimulation of the external lateral area, implies that the lateral mechanism has to produce sufficient potential to supplant what is probably a satiation brake with a constant resistance. The motivational system of the MFB intervenes both in starting and in stopping the aIimentary drive. The findings by Morgane do not c o h those of Wyrwicka and Dobrzecka (1960)on the goat in which combined stimulation of the ventromedial nuclei and of the external lateral area totally suppress feeding behavior. It would also appear that the primary feeding responses, and possibly other forms of bisic behavior also, may be anatomidy and physiologically dissociated from the motivational repertory linked to each basal need. Need and motivation are linked in Krasne’s experiment (1960)in which the rats not only stopped eating when the ventromedial region of the hypothalamus (“satiation center”) was stimulated but also learned to press a level to avoid being stimulated. If a great deal of uncertainty still persists in connection with the precise delimitation of the inner structures of the hypothalamus, it would seem that as a first approximation one may assume the existence of 2 mechanisms operating at once-antagonistically and synergistically, The medial structures which are represented essentially by the ventromedial nuclei constitute the satiation mechanism, whereas the lateral hypothalamic areas constitute the hunger-activation mechanisms. It is still too early to decide whether one mechanism dominates the other, as experiments are contradictory. It is likely, however, that apart from the two basic mechanisms there may be other structures having a specific integrating function, which might represent the first level of actual motivational control of feeding behavior. It is possible, as we shall see later, that such an integrating system is linked directly with the mechanisms controlling vigilance.

B. THE RHINENCEPHALON AND FFXDING BEHAVIOR The importance of rhinencephalic formations in regulating a variety of instinctive behavior and the majority of somatovisceral

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activities has naturally led to studying their presumed role in the neurophysiology of feeding behavior. We shall here relate only those studies directly concerned with the true rhinencephalon, and shall reserve for a later section the role of certain neocortical formations usually associated with the physiology of the rhinencephalon. It should be stressed, first of all, that there is some difficulty in interpreting experimental results since as a rule, apart from more or less specific motor reactions, stimulating the rhinencephalic s t r u o tures causes extremely complex behavioral reactions, in which are closely interlinked reactions of fear, anxiety, flight, or stupor simultaneously with elements of oral and/or sexual behavior. The Kliiver syndrome and associated phenomena, of which Kliiver and Bucy (1939) have given a classic description, should be mentioned. These initial findings indicate that the results generally obtained during such research show the highly integrated character of the nervous control, Starting with the hypothesis of Bard and Mountcastle (1948) that the amygdaloid complex in the cat acts as an inhibiting center in rage reactions, certain authors have studied the effects of ablation and stimulation of the amygdala on feeding behavior. Some clinical research (Sawa et al., 1954; Terzian and Ore, 1955; Alajouanine et al., 1957) had already indicated certain cases of obesity in man following rhinencephalic ablation. In the cat Morgane and Kosman (1957,1959) found that following bilateral lesions of the amygdaloid complex mainly covering the associative area between the lateral and basal nuclei, as well as certain regions of the pyriform cortex, a very distinct hyperphagia appeared. Food intake increased appreciably and produced obesity, for which the authors were able to describe a dynamic phase and a static phase. Similar findings were also noted by Green et al. (1957), Wood (1958), and Fuller et a2. (1957). These authors, however, do not always agree on the amygdaloid structures responsible for this disturbance. On the whole these results invalidate the initial findings of Anand and Brobeck ( 1952), who noted no quantitative modification of hunger following bilateral lesion in the amygdaloid nuclei in the rat. Morgane and Kosman (1960) combined amygdaloid lesions with lateral and ventromedial hypothalamic lesions in the cat, and noted both hyperphagia and an appreciably greater obesity (about 3 times more rapid in development) than following amygdaloid

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lesions alone. The theoretical importance of these results will be discussed later and an attempt will be made to analyze the links between the rhinencephalic and hypothalamic control structures. Recent experiments by Fonberg and Delgado (1961) have carefully examined the effects of amygdaloid stimulation on feeding behavior in the cat. Stimulating the basolateral amygdaloid nucleus inhibits the intake of food in starving animals. The reaction is immediate, and if the cat is in the process of eating he will stop masticating as soon as stimulation starts. The inhibition usually subsists for several minutes after the stimulation is stopped. The animal refuses to eat even if food is introduced in his mouth. In addition, no fear, aggression, or hyperactivity was noted. If the stimulation be repeated daily, the repetition increases the duration of the resulting inhibition. The authors found in one animal that the refusal lasted several hours or even several days following a 10-sec stimulation. According to the same report, learning of instrumental feeding reactions is also entirely blocked by amygdaloid stimulation. The authors, furthermore, stress that direct stimulation of the basolateral nucleus of the amygdala can reinforce an inhibitory conditioning of feeding. Chronic stimulation (stimulation by a chronic electrode using a remote controlled transistor stimulator, i.e., without any intervention by the experimenter) of the basolateral portion of the amygdala also results in a considerable decrease of food intake during 6 hr following stimulation, but overall consumption over 24 hr is only partially reduced. It appears therefore, that the majority of experimental results demonstrate the inhibiting role on food intake of the amygdaloid nuclei. The amygdala does not appear to intervene in triggering pain or fear. Experiments by Morgane and Kosman (1960) show furthermore, that the hyperphagia caused by lesions of the amygdala is not modified by destruction of the ventromedial or lateral hypothalamus. This invalidates the concept that the amygdala acts via the hypothalamus. The role of the amygdala on hunger would therefore be independent of the hypothalamic structures. We shall see later that certain authors have assumed that it acts via the caudal nucleus and septal areas. These working hypotheses assume two relatively independent food control systems, one being the ventral and lateral hypothalamic system and the other being the amygdaloseptocaudate system, Finally, Fonberg and Delgado (1961) have lately shown that a

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reduction of food intake through stimulation of the amygdala could be produced not by indirect inhibition of hunger itself, but by changes in the taste and odor of the food. They report observation of a cat which, after a few seconds’ stimulation of the amygdala, ceased eating for 3 days until a new kind of food was offered, e.g., fish instead of meat. When the amygdala was again stimulated, appetite for fish disappeared and the cat refused to eat until a new kind of food, e.g., sugar concentrates, was offered. A further stimulation of the amygdala curtailed his interest in condensed milk. We shall learn below (Section C ) of similar results following the destruction of neocortical structures. C. THEROLEOF NEOCORTICAL AND STFUATAL S m u c r u ~ IN ~s FEEDING BEHAVIOR

Among the neocortical structures studied in connection with their presumed role in food intake, a distinction must be drawn between formations attached to the enlarged rhinencephalon or “visceral brain” (limbic system, anterior temporal pole, gyrus cingularis, and septal region) and the specifically neocortical structures, unattached to any rhinencephalic formation (e.g., frontal cortex, motor and sensory cortex). It is known that stimulating the anterior g y r u s cingularis causes cessation of all motor activity ( Delgado’s arrest reaction, 1952). The phenomenon may also be observed during feeding behavior but it is difficult to attribute any specificity to it. It is sometimes possible in the case of limbic stimulation to determine feeding automatisms, as in amygdala stimulation, but however marked the responses obtained, no increase in daily food consumption is observable, Stimulating the septal region also produces particularly distinct and wellordered oral activity, greatly resembling the spontaneous activity. Such reactions are obtained even after complete ablation of the neocortex (Schaltenbrand and Cobb, 1931; Rioch and Brenner, 1938). It is known that integration of complex segmental behavior, by associating parasympathetic reflexes and activity of the striated musculature, is particularly noticeable at the level of the septal region. Thus, the importance of the septal areas may be assumed as the location where oral activities are integrated. Recent research has stressed the role of these structures in emotional and affective behavior ( Brady and Nauta, 1953,1955). The role of the cortical regions and of sensorimotor and fronto-

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orbital regions in alimentary behavior is samewhat dif6cult to specify. Delgado (1952) electrically stimulated the anterior and posterior sigmoid gyrus in the cat and observed that in well nourished animals, who refuse food even when forced, stimulating the medial portion of the presylvian sulcus determines licking activity. The animals then eat an additional amount of food and continue to do so for as long as the stimuIation lasts, It would seem, however, that up to the present the most noticeable results have been obtained by ablation experiments, Pribram (cited in Fulton, 1951) observes that in the baboon the ablation of the whole orbitoinsulotemporalregion initiates an increase in food consumption. Richter and Hawkes (1939) had earlier reported that ablation of the frontal poles in the rat produced a state of hyperactivity and an appreciable exaggeration of food intake. Cobb (1944) also reported that interrupting the connections between the frontal lobe and the hypothalamus in man occasions an increase in appetite, Other experiments by Pribram and Bagshaw (1953) showed that ablating the insular region mainly affected the gustatory mechanisms. Andersson and Larsson (1956b) found that prefrontal lobotomy in the dog occasioned a slight and irregular diminution of food consumption and inhibited the action of amphetamine on the appetite. Soulairac (1952),following lesions of the cerebral cortex in the rat, observed varying effects depending on the location of the injury, This, in the middle and posterior areas, did not produce any appreciable, quantitative results. Variation is essentially qualitative when the animals are on a spontaneously chosen diet. Caloric intake does not vary, but carbohydrate consumption diminishes in favor of standard f0od.l Later Soulairac and Soulairac (1958, 1958) were able to specify the role of the anterior cortical areas. Bilateral lesions covering areas 6 and 10 occasioned a somewhat peculiar caloric intake. With a standard diet the operated animals do not change their food intake, but if choice is permitted between standard food and glucose, the amount of ingested standard food does not vary, but the animals consume in addition a certain amount of glucose, which has the effect of increasing overall caloric ‘The self-selection method is used: The rats can choose between a 10% 100 dextrose solution and the “standard” food, the composition of which is the following: corn meaI, 480; cod liver oil, 10; brewer’s yeast, 20; casehe* 250; margarine, 20; dry milk, 200; CaS04, 10; NaCl, 10; plus vitamin mixture, twice a week.

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intake. What seems to happen is that the somewhat strict caloric control in these animals is disturbed by nonrecognition of the calories furnished by the glucose. Actually, the experiments of Soulairac and Soulairac (1958) indicate that ablation of areas 6 and 10 determines an appreciable increase in the gustatory threshold for glucose, which rises from 1to 3.5%.When such neocortical lesions are accompanied by lesions of the hippocampus or the gyrus cingularis, food consumption increases. These experiments show that anterior cortical lesions occasion disturbance of the perceptive recognition of glucose which is reflected in raising the gustatory threshold. In none of the animals however were any of the cortical areas of gustatory projection touched, which according to Benjamin and Pfaffmann (1955), are located in the lower portion of area 2. This change in gustatory sensitivity following lesions in areas 6 and 10 should not be attributed to any disorder of the sensory capacities themselves, but to disturbance at a higher level bringing about a reduction in the significant value of the stimulus. Such reductions may legitimately be attributed to a lowering of the level of general vigilance, and the neocortex would thus intervene directly in food regulation by assuring the integration of activating stimuli originating in the basic centers of the hypothalamus, the essential role of which would thus be to trigger specialized states of vigilance. Delgado (1957, 1960) had reported that electrical stimulation of the head of the caudate nucleus in the monkey produced a notable inhibition of food consumption. Recent research by Morgane (1961~)stresses the role of the globus pallidus and more particularly the pallidofugal systems in regulating hunger. Lesions in the internal portions of the pallidum or combined lesions of the ansalenticularis and the lenticular bundles produced results very similar to the anorexia syndrome occasioned by lateral hypothalamic lesions. Even if tube-fed, most of the operated animals die, which would indicate not merely behavioral but also metaboIic disturbances. ACcording to Morgane any lateral portion of the hypothalamic alimentary system is strictly dependent on the pallidofugal circuits, which detracts from the validity of the claim that this hypothalamic region is a “feeding center.“ From the evidence it can be concluded that a whole complex of nervous structures appear to participate in the mechanisms con-

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trolling hunger. The rhinencephalic and neocortical mechanisms would appear the best suited, hierarchically, to control the hypothalamic mechanisms, and one could reasonably consider them as basic homeostatic structures. Nevertheless, it is still difficult to detect the interconnections in this nervous complex. II. Nervous Mechanisms Controlling Feeding Behavior

The theory of nervous structures capable of ensuring the control of hunger assumes the possibility that they can be continually informed of the alimentary needs of the organism by physiological informatory mechanisms. A number of theories have tried to describe the exact nature of these information mechanisms, but on the whole it appears that all the current theoretical explanations derive from that postulated by ROUX,who wrote in 1897: “It’s the cry of our organism demanding nutritive substances when the inner environment becomes impoverished. All the cells in our organism are interrelated and this interrelation is rendered necessary by the multiple functional specializations, by the division of labor. When a cell feels a need which it is itself unable to fulfill on account of this specialization, it calls the other cells through the agency of the nervous system. Such is the origin of all nutritional reflexes, and in the sensation of hunger there is nothing but a cortical reflex, a reflex incompletely adapted giving rise, as an epiphenomenon on this ground, to an act of consciousness: the sensation of hunger, in the old sense of the term.” Keeping in mind the nature of the information from the inner environmental itself, we shall try to analyse in detail how this information ensures the nervous regulation af hunger.

A. DIFFERENT TYPESOF INFORMATION A great number of studies have attempted to determine the nature of the physiological information enabling an individual to control his food consumption suitably, and numerous theories have been evolved to provide a generally valid explanation.

1. Mechanical Gastric Information The theory of Carlson (1916) on the relation between gastric contraction, sensation of hunger and food consumption is well known, but at the present time it seems no longer to have any but

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historical value. Nevertheless, the existence of these gastric contractions is indisputable and even if the mechanism may no longer be held responsible for hunger, any general explanation will have to take it into account. It is, in fact, known that insulin, which increases food consumption, increases gastric contractions, while glucose which is a factor in satiation reduces them appreciably. It appears, however, that the contractions represent only an epiphenomenon and not a determinant cause, but in this capacity they possess an indisputable importance, as was shown by Paintal in 1953.The presence of tension receptors in the stomach wall would, depending on the degree of distension or emptiness and depending on gastric inertia or hypermotility, give rise to a discharge of nerve impulses, the efferent path of which might be the vagus nerve. I n reality, this phenomenon is very complex and, conceivably, one should dissociate gastric distension from contraction. Food substances held in the stomach do not inhibit hunger contractions and only duodenal penetration of food determines the inhibition of these contractions, even after total denervation of the stomach. These findings led Quigley (1955)to assume the existence of an enterogastrone hormone as the origin of the inhibitory reactions. In any case, gastrectomy experiments eliminate the possibility of the origin of hunger being solely gastric, Soulairac (1950)observes that gastrectomized rats not only continue to feed but manifest an increased appetite following insulin administration. 2. General Metabolic Information The generally recognized fact that animals consume food as a direct function of their organic needs and that a strict balance exists between intake and energy expenditure, has led a good deal of research to the study of the metabolic control of hunger. Richter was one of the first to stress the great self-regulating capacity of the organism in respect to food and to include this among the general homeostatic processes. Using his technique of “self-selection” he was able to demonstrate both quantitative and a qualitative control by specific appetites (Richter et al., 1938). The effect of various physiological disturbances and endocrine variations was studied in depth by Richter (1936),and Richter and Eckert (1937).In his general conclusion Richter assumes that a nutritional deficiency causes metabolic changes throughout the organism in-

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cluding the mechanisms of taste. These changes in the sensory threshold would lead to research into well-defined substances, One arrives at the concept, which is not easy to uphold, of a mosaic-like control of food intake. Hormonal control of hunger undoubtedly is a fact and we need only mention the extremely important roles of the pancreatic, thyroid, corticosuprarenal, hypophysis, and sex hormones of which a detailed study was made by Soulairac (1947a). Such hormonal regulation, however, in no way prejudices the existence of an information mechanism which does not itself appear to be hormonal, but possibly rather the effect is one of a hormononeural mediator. Attempts have been made to define the nature of this. Among the metabolites through which the hormones might act, blood glucose occupies a foremost place in all theories. Originally, Bulatao and Carlson (1924)put forward hypoglycemia to explain gastric contractions, and for many years this theory of glycemic control of hunger remained the accepted one, As time went on, however, numerous authors failed to observe any correlation between variation in blood glucose level and gastric contraction, or with food intake. Research by Soulairac (1947a) stressed both the absence of any direct relation between the level of blood glucose and hunger and between the direction of change of blood sugar level and the action of a given hormone, Insulin administration determines hypoglycemia and an increase of hunger, but alloxanic diabetes is associated with hyperglycemia and increase of hunger. Thyroidectomy reduces the blood sugar level and reduces food intake, and the same relation holds good in the case of corticosuprarenalectomy. It is no longer admitted that variation in the blood sugar level is the only mechanism regulating hunger or satiation. Thus, Mayer (1953) suggested glycemic control of hunger based not on simple variation of the blood sugar level but on the differential content of glucose in arterial blood and venous blood. This difference or delta glucose index, according to Van Itallie et d.(1953) is the determining factor, and is quite unrelated to the absolute content of glucose in the blood. An increasing arteriovenous difference would indicate the presence of adequate carbohydrate reserves in the organism and would give rise to a state of satiation. Conversely, a reduced difference would give the necessary information in carbohydrate deficiency and would determine feeding behavior. We shall see below (Section 11. B) how Mayer postulates the existence of specialized nervous regulators in support of the glumstatic theory.

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A number of authors have rightly stressed that it would be surprising if glucose alone ensured the control of hunger, whereas proteins and lipids are indispensable factors to all the higher forms of life. Relatively little is known as yet about protein control of hunger, but it is known that the rapid administeration of amino acids reduces hunger and that the anorexia is accompanied by hypoglycemia (Mellinkoff et d.,1955). In some experiments, appetite is shown to be at its minimum when the blood sugar level is minimal and hunger recovers as the blood sugar level goes up and at the same time the serum amino acids are reduced. It was Kennedy (1953) who mainly stressed the possible role of lipid deposits in the control of appetite, Hunger would thus be partly regulated by the level of lipids permanently held in the metabolic circuit. If the lipids are put in reserve in the form of deposits, thus causing a sharp drop in the lipids in circulation, an increase in alimentary needs would result. Kennedy checked this hypothesis on both obesity and hypothalamic hyperphagia in the rat and stressed that under certain transitional physiological conditions (e.g., pregnancy or lactation) these might be the physiological mechanisms that were brought into play. Finally, it should be mentioned that among the general metabolic sources of information a very important role, according to Brobeck (1948),is played by thermal control. Animals eat to conserve their heat and cease eating to prevent hyperthermia. Since it is known, however, that the amount of food ingested is not directly proportional to energy expenditure, the author assumes that the important factor in alimentary control is not the energy value of the food but the amount of extra heat liberated through its assimilation. The mechanism is regulated in accordance with oxygen consumption determined by the CO, and pH concentration in the blood. Without going further into the arguments presented by Brobeck (1948) and by Strominger and Brobeck (1953),it would appear that this type of information would not cover the whole explanation of alimentary control, It fails to explain gastric contractions of hunger and the frequency of food intake, and the effect on hunger of disturbances of carbohydrate metabolism (hyperinsulinism or sugar diabetes) during which no thermal disturbances occur, Finally, Brobeck's hypothesis would lead one to expect anorexia during hyperthyroidism, which is conducive to hyperthermia, and hyperphagia during hypo-

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thyroidism, which reduces the central temperature. Experimental data show contrary results (Richter, 1933; Warkentin et al., 1943; Soulairac, 1947a).

3. Information of Digestive Origin

A great number of experiments, dispersed through the available physiological literature, have shown the indisputable role of the small intestine in regulating appetite. The establishment of a close correlation between the direction of food modification under the action of various factors and intestinal absorption of gIucose led Soulairac in 1947 to consider the process of intestinal absorption itself as one of the major factors in the control of appetite. The hypothesis was backed by two sets of experiments: a. Various physiological conditions modifying carbohydrate intake (and often overall intake) modify the value of intestinal absorption of glucose in the same direction; b. Any modification in intestinal glucose absorption, whether experimentally or pathologically produced, brings about a variation in the same direction of the animal's food intake. Any hormonal disturbance modifying food intake effects the intestinal capacity for glucose absorption. This absorption factor, developed according to the Cori (1925) technique, enables a fairly constant value to be obtained in a normal animal, since it is computed as a function of body weight or body area. Administration of desoxycorticosterone, thyroxine, or insulin increases both intestinal absorption and food intake, Adrenalectomy, thyroidectomy, hypophysectomy, and anterior pituitary extracts lowered intestinal absorption and also reduced the amount of food ingested. Sugar (alloxan) diabetes, which appears to be an exception to the hypothesis of simple glycemic control, causes an appreciable increase in intestinal absorption as well as in food intake, but it is significant that the initial period of development of the diabetes through alloxan (lasting about 8 days and marked by an important but momentary reduction of the appetite especially for carbohydrates) is accompanied by a momentary reduction in the intestinal absorption factor for glucose ( Soulairac, 1947b; Soulairac and Desclaux, 1948). Production of hyperphagia by hypothalamic lesions occasions a greatly increased intestinal absorption of glucose ( Soulairac, 194713) . Although not found by Bogdanove and Lipner (1952), this intestinal

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absorption following hypothalamic lesion was confirmed in the rat by Mayer and Yanonni (1956) although their interpretation differed from that of Soulairac. The glucagon which Mayer and his coworkers showed to be a factor in reducing food consumption reduces the intestinal absorption of glucose appreciably and immediately while at the same time producing hyperglycemia (Ponz et al., 1957). Such reduced absorption cannot be attributed to the hyperglycemic state since intravenous administration of glucose in rats produced no change in the intestinal absorption of glucose. The question raised by such observations, and which have been mostly discussed by Mayer ( 1955b), is whether such changes in intestinal absorption of glucose are the consequence of the modified alimentary activity or, on the contrary, are determinants in alimentary control. Soulairac (1947b) reported a number of experiments which seem to show that modification of intestinal absorption is capable by itself of modifying the animal's food intake. Phloridzine is a glucoside whose intense and reversible polyuric and glycosuric action is well known. Research by Nakasawa (1922) and others had shown that this substance is capable of reversibly inhibiting the absorption of physiological hexoses by the small intestine, and that it inhibits phosphorylation processes at the level of the intestinal mucous membrane. Lundsgaard ( 1933) also demonstrated the specificity of phloridzine. In the rabbit and cat, 0.02 M phloridzine fails to affect the intestinal absorption of glucose or glutamic acid. In both cases, simultaneous absorption of glucose is distinctly inhibited by phloridzine. According to Verzar and Laszt (1935) the intestinal absorption of lipids is also inhibited. In the rat, Althausen and Stockholm (1938) and Soulairac (1947a) observed that intestinal absorption of glucose passes during 1hr from a normal of 173 mg to 36 mg following phloridzine (or from 216 to 52 mg, depending on the technique used). Dosage of phloridzine given intramuscularly to mice and rats resulted in a distinct and sometimes very appreciable reduction in overall food intake and carbohydrate consumption, The drop is rapid from the start of the injection, lasts during the entire treatment, and stops suddenly at its termination (Soulairac, 1947a). Another experiment by Soulairac ( 1947a) showed that, conversely, any increase in the intestinal absorption of glucose causes an increase in food intake. Lactoflavin phosphate occasions a significant rise in intestinal absorption at the same time as a marked enhance-

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ment in the appetite especially for carbohydrates. The increase is immediate and persists 1 or 2 days after stopping the treatment. Somewhat similar facts, too, had been noted by Griffith and Graham (1929) when using B2complex. Taken together, these facts appear to show that the level of intestinal absorption is one of the factors in regulating food consumption, and seems to come into action fairly rapidly in order to ensure partial short-term control. Very numerous studies have demonstrated that intestinal absorption is not a passive phenomenon but depends on selective activity of the intestinal mucous membrane, more particularly on its capacity for phosphorylation. Soulairac (1947~)presented the results of some experiments analyzing neuro-endocrine control of the intestinal absorption of glucose due to the regulation of the phosphorylation processes. Later histochemical research showed the importance of the activity of phosphatases localized at the level of the intestinal mucosa, and these phenomena of glucose absorption enabled a histological index to be deduced, which is particularly useful in the morphological study of such phenomena ( Soulairac, 1948a, b, c ) . The mechanisms of intestinal phosphorylation and regulation of food intake (already demonstrated in experiments using phloridzine and lactoflavinphosphate) have now been investigated with repeated studies using simultaneous dosage of an hormonal hunger-activating substance and of an inhibitor of phosphorylation (phloridzine), or, on the contrary, by producing a state of experimental hypophagia associated with the dosage of an activator of phosphorylation (lactoflavin phosphate). If doses of deoxycorticosterone acetate and phloridzine are given, or of thyroxin and phloridzine, the increase in food intake normally noted under the action of the hormone alone is no longer produced; there is a reduction in the food ingested. Conversely, administration of lactoflavin phosphate to thyroidectomized or adrenalectomized rats causes an increase in the rate of food intake, the action being much more appreciable with thyroidectomized animals than with adrenalectomized ones ( Soulairac, 1947a). The intestinal metabolic aspect of food control has been little studied, has frequently been criticized, and naturally raises a number of problems, the chief of which is the possible transmission of such metabolic information to the central nervous system. Hill et al.

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(1952) determined the effects of certain nutritive and nonnutritive substances on food consumption, by introducing the substances directly into the jejunum of a dog by means of a fistula. Introducing a 10.8%glucose solution (at the rate of about 150 cc for a 7 kg dog) directly into the intestine 1hr before presenting a meal, reduced the amount of food ingested by 31%.The same volume of a 33.3%glucose solution half an hour before the meal reduced food intake by 9 E . The same amount of saline solution taken in the same conditions produced no change in the food consumption. If the glucose solution is replaced with a nonnutritive substances in suspension (200-300 cc of substance containing 50 gm of nonnutritive fibers for a 12 kg dog), a reduction in the ingested food is observed, though the reduction is much less (of the order of 15-18%less). This seems to show that 2 factors intervene here: first and foremost is the hypertonic solution of glucose, the effect of which seems predominant; second, the intestinal distention caused by the volume of the administered solution which may trigger reflexes which at least temporarily reduce the desire to eat. Dosage of tyrosine under the same conditions (namely 8.1 gm and 21.6 gm, 1 hr or 4 hr, respectively, before the meal) causes no change in the amount ingested despite the known effects of tyrosine on specific dynamic action. Findings of the same order had already been made by Quigley et al. (1941, 1942) by introducing various substances at the level of the duodenum. It remains to be discovered by what mechanism the presence of nutritive substances and, in particular, glucose at the level of the intestinal mucous membrane are able to regulate food consumption. The importance of enzyme processes on intestinal absorption is well known. Verzar (1936, 1937) in particular stressed the role of the phosphorylation processes underlying the active absorption of sugars and lipids. In the course of histophysiological research, Soulairac (1948a) showed a close correlation between the activity of alkaline phosphatase in the duodenal mucous membrane and the active absorption rate of hexose. Enzyme activity is strictly localized in the villous mucous membrane, and substances that inhibit absorption, such as phloridzine and monoiodacetic acid, also inhibit the activity of phosphatase. Hormonal action (hyper- or hypoactivity ) modify food consumption, intestinal absorption and the activity of alkaline phosphatase in a strictly parallel manner. Even in the case of sugar diabetes, which has raised so many problems in the control of

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hunger, a similar parallelism is evident (Soulairac, 1948a,b, c; SOUlairac and Desclaux, 1948). However, although the action of the nervous structures on certain aspects of tissue metabolism is beginning to be understood, we do not know how the peripheral enzyme processes transmit information to the central nervous system. It does not appear that, in itself, the activity of phosphatse would act as a stimulus capable of being transmitted through the nervous system. Yet this specialized enzyme activity is accompanied by other metabolic activities which set up certain chemical mediators. We shall revert to this important concept in Section 11. B.

B. VARIOUS HYPOTHESES REGARDING CONTROL MECHANISMS The central nervous system has structures which appear to control the triggering of alimentary responses in a rigorous manner. Direct action on these nerve structures modifies very rapidly, in fact almost immediately, food consumption quantitatively and qualitatively, and such reactions may be obtained experimentally in the absence of prior metabolic modification. The most difficult problem to solve is how these two systems of metabolic control are integrated. Whatever the theory held, it is essential that it should explain how the diffuse cellular information of metabolic origin is transmitted to the central nervous system, how the nervous mechanisms of feeding behavior are triggered, and how this behavior ceases often well before the metabolic balance, whose initial disturbance occasioned the reaction, is re-established. This gives some idea of how complex the problem is and how difficult it is to find an unequivocal explanation. A very great number of factors undeniably intervene, i.e., metabolic, humoral, nervous, and conceivably psychological, in the Pavlovian sense of the term.

1. Alimentary Controll by Nervous Glucoreceptors; The Glucostatic Theory of J. Mayer (1953) We have seen earlier to what type of information (delta-glucose index) Mayer (1953) attributes the major role as conducting information of the alimentary needs of the organism. For the central nervous system to make use of such information, it must be assumed that specialized structures exist at the level of the hypothalamic centers; these Mayer designates “glucoreceptors.” They are sensitive to glucose changes in the blood. According to his theory, the regula-

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tory hypothalamic structures are chemosensitive, and this sensitivity is dependent on the extent to which the nerve cells are able to use the glucose. The existence of glucoreceptors is a postulate based on a number of observations, which, however, is supported only by somewhat indirect evidence. Mayer insists on the importance of carbohydrates in the metabolism of the nervous system, on the relatively low carbohydrate reserves of the organism and on the absolute necessity for the latter to be kept informed of the carbohydrate reserves for the purpose of homeostatic control, one of the most important being hunger. Zunz and La Barre (1927) showed that in dogs with their heads isolated from the rest of the body, but with innervation intact, hyperglycemia of the head region occasions hypoglycemia of the body. This was recently confirmed by Duner (1953). Yet these experiments bring little support to arguments in favor of very specialized chemoreceptivity, since a great number of responses are known which are obtained by the isolated head or cross-circulation methods showing the action of extremely varied substances on physiological control. To assume such specialized sensitivity for each of the responses would lead straightaway to the concept of an immense mosaic of neurones, frequently within the same hypothalamic structure. This concept at the present moment is not verified by facts. Mayer (1953) attempted to give evidence of such glucoreceptors inside the ventromedial nuclei of the hypothalamus. These, as we have already seen, would then be sensitive to glucose to the extent that this glucose can penetrate such cells, and their metabolism would be more similar to that of peripheral metabolism than to that of the brain in general. Certain experiments have shown that in fact glucose consumption by the brain is insensitive to the action of insulin (Himwich et al., 1941; Van Itallie et al., 1953). This makes it necessary to assume these glucoreceptors are very specific. Although themselves neurons, they would no longer behave like normal neurones on the metabolic plane, This point is difficult to reconcile with the findings of Forssberg and Larsson (1954),on which Mayer partly bases his concept. Mayer believes that at the glucoreceptor level the transformation of the chemical phenomenon, i.e., availability and utilization of glucose into a neuro-electrical phenomenon is due to the passage of potassium ions accompanying the glucose phosphate from the exterior to the interior of the hypotha-

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lamic receptors ( Mayer, 195%). The glucostatic mechanism would thus act like a governor brake, inhibiting the continuously activated mechanism of food intake, Thus, satiation, not hunger, would actually be controlled. One of the histophysiological arguments, to which Mayer refers in order to show the anatomic existence of these glucoreceptors, is furnished by a study of the hypothalamus in mice made hyperphagic and obese by means of aurothioglucose. This product destroys a great number of cells in the ventromedial region of the hypothalamus, the cell destruction being less dense with increasing distance from the nuclei. Aurothioglucose has a kind of specificity in the production of edema and cytolysis of the hypothalamic centers, while other related substances presenting the same toxicity (such as aurothiomalate, aurothiosulphate, aurothioglycerol, aurothiosorbitol, etc.) have no such reaction, and produce neither hyperphagia nor obesity. Mayer thinks that gold, because it is attached to the glucose, is transferred to the cell which it destroys; and it would be legitimate, therefore, to consider the ventromedial hypothalamic region as intensely glucoreceptive ( Mayer and Marshall, 1956). Such research into the modification of the nerve structures related to changes in the carbohydrate metabolism had already led Morgan et al. (1937) to observe, in subjects with sugar diabetes, fairly localized lesions in the cells of the paraventricular nuclei, with, in particular, chromatolysis and neuronophagia. According to these authors the paraventricular nucleus has the capacity of being stimulated by chemical changes in the blood and more particularly by substances involved in glucose metabolism. Biochemists have also attempted to find characteristic changes in the cerebral metabolism during states of hunger. Kerr and Ghantus (1936)observed that the amount of cerebral glycogen remains unaltered during a fast. Relatively little is known, however, regarding the metabolism of different parts in the nervous system, especially under physiological conditions. Bore11 and Orstrom ( 1945),while studying the incorporation of radioactive sulfur in different regions of the nervous system of rats and rabbits, noted appreciable differences which they found difficult to systematize. Soulairac and Desclaux (1951)showed that administering large amounts of insulin, liable to produce coma, brought about metabolic changes related to carbohydrates throughout the nervous system including the spinal cord. Alkaline phos-

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phatase activity almost entirely disappeared, and similar experiments by Shimizu ( 1950) and Shimizu and Kumamoto (1952)on cerebral glycogen confirmed this result. Forssberg and Larsson ( 1954),and Larsson ( 1957) studied the chemical composition and distribution of tracer substances in the hypothalamic region in starved and nourished rats, with particular reference to creatine phosphate and adenosine triphosphate. They studied the incorporation of P32(in the form of Naz HPO,) and of C" (in the form of C14-glucoseand NaHCO,) in the hypothalamic feeding center and in neighboring hypothalamic areas. A control study was made on the blood, liver, muscle, and brain. In starving rats the samples, which included the feeding centers, showed a preferential consumption of indicating an increase in the physiological activity in proportion to the state of satiation, Conversely, in well-fed rats the activity of the neighboring regions was increased, whereas that of the alimentary region was reduced proportionately. Overall activity in the hypothalamus remained the same, as well as that in the blood and brain, showing a specificity in the behavior of the hypothalamic feeding region. Intrahypothalamic variations in ATP and in creatine phosphate, from a state of hunger to a state of satiation, appear to give a constant curve, showing a differential sensitivity in the state of satiation, with the ADP :ATP ratio playing the major role. Experiments with labelled glucose gave somewhat similar results. In starving rats the feeding center is more receptive to glucose than other hypothalamic areas and is also more sensitive than that of satiated animals. The liver, too, shows a higher incorporation of and C1" than in a state of hunger. These results are somewhat difficult to interpret, since little is known regarding the exact significance of such metabolic changes. Every nerve cell consumes appreciable amounts of ATP and glucose, and phosphorylation processes are particularly intense during intermediate metabolism. The supply of energy enabling the synthesis of chemical mediators to occur calls for the factors studied by Forssberg and Larsson (1954).Various studies attempting to determine the histochemical location of the enzyme activities of the nervous system have pointed to privileged areas, of which the hypothalamus forms a part. It should be stressed furthermore that, for instance, in the hypothalamus the distribution of phosphatase activity is not affected in a uniform manner (Soulairac and Desclaux, 1951; Eranko, 1951; Cohn and Richter, 1956).

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Abrahams et al. (1957) obtained similar results in connection with the distribution of cholinesterases in the hypothalamus of the dog. Finally, biochemical techniques using radioactive elements do not allow of the problem of cell density to intervene. Although it may no doubt be interesting to know that metabolic changes are produced at the level of certain regions of the nervous system during various physiological states such as hunger and satiation, it is actually extremely difficult to draw any conclusions regarding the existence of glucoreceptor structures. As stressed by Larsson (1955), there is in the starving rat an increase in the amount of blood furnished to a relatively large area of the hypothalamus, including the “feeding center,” as well as in the amount (some 8 times more) of ATP and creatine phosphate, Such phenomena are difficult to relate to the postulate of a relatively small number of glucoreceptors. Rather, it would appear that there is an overall rise of activity in a given region which not only comprises the “feeding center” but adjacent parts of the hypothalamus. In such a glucostatic hypothesis of the control of appetite one arrives at the notion of a chemosensitive nervous structure the stimulation of which causes satiation. Any appreciable arteriovenous difference would stimulate the glucoreceptors of the hypothalamic ventromedial nuclei, and to Mayer these medial formations represent an active factor in feeding control. What is controlled is satiation, and the intermittent disappearance of this state would trigger the mechanisms of food intake. In any case Mayer did not formulate any very precise hypothesis regarding the nervous mechanisms by which the hypothalamic pulses determine alimentary behavior. 2. Alimentary Control through Thermoregulation

As we saw earlier (Section 11. A. 2 ) , Brobeck (1948, 1960) pointed out the intimate correlation between body temperature and food consumption and advanced the thermostatic theory of hunger control. He suggested that when food consumption is inhibited, heat acts either on thermosensitive neurons of the anterior hypothalamus and of the pre-optic region, or directly on neurons of the appetite center, The specific dynamic action of foodstuffs acts directly on cells of the anterior region of the hypothalamus and determines cutaneous vasodilatation, and this is accompanied by a central inhibition of the appetite and an induction of satiation. Recent experi-

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ments by Anderson and Larsson (1961) appear to bring some support to the argument in favor of the part played by thermoregulation in feeding control. Lesions in the pre-optic region destroy the “heatloss center,” and when such lesions are at all extensive they cause not only permanent adipsia but hypophagia or even aphagia. Central projections arriving from the hypothalamic feeding centers would pass through the pre-optic region to reach the rhinencephalon, and its connections would be essential in developing a conscious need to eat and drink. Cooling the pre-optic region and the anterior hypothalamus determines hunger in the nourished, normothermal animal. Thus, it would appear that stimulating the anti-cold mechanism would activate the hypothalamic feeding center, either directly or by inhibiting the ventromedial satiation center. Conversely, local heating of the same structures would inhibit food intake in a previously hungry animal and simultaneously determine the ingestion of a large amount of water, even if it had previously showed no sign of thirst. In these experiments, the short latency of the effects on feeding of central cooling or heating, and the rapid reversibility of such effects, eliminate the possibility of any chemical change in the internal environment being able to act directly on the hypothalamic feeding centers. According to these authors, the close temporal relationship between the triggering of hunger and that of peripheral vasoconstriction on the one hand, and the end of food consumption and the start of peripheral vasoconstriction on the other hand, is evidence of the close functional correlation between the thermocontrol mechanism and the hypothalamic “feeding center.” It is a little early still to link these experimental data with our present knowledge of the mechanisms of hunger control. Actually Han and Brobeck (1961) noted that ventromedial lesions of the hypothalamus did not result in any primary alteration of thermal regulation, although their effects on feeding behavior are well known. 3. Feeding Control by the Digestive Tract

Earlier we stressed (Section 11. A. 3 ) how important is the role seemingly played by phenomena of intestinal absorption and the whole digestive metabolic system as a source of information for the central nervous system in regulating appetite. As a working hypothesis, it might be advanced that the continuous operation at a variable intensity of the digestive tract gives rise to a steady production of

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nervous impulses and of chemical mediators. Among the latter, it would seem that particular attention should be given to the production of acetylcholine, noradrenaline, histamine, and Euler’s substance P. It is known (Douglas d al., 1951) that there are large amounts of histamine and P substance in the intestinal mucous membrane, and their concentration decreases from the duodenum to the colon, the gradient being identical with that noted by Soulairac (1948a) for alkaline phosphatase activity. Numerous authors have also shown that the different layers of the intestine contain acetylcholine, even after sectioning of all the extrinsic nerves. It is assumed that it is manufactured in the ganglionic plexus although it may quite possibly be synthesized in the smooth muscles and in the gland cells ( Feldberg and En, 1949a). As regards isolated intestinal loops, d-tubocurarine, nicotine, and cocaine, in concentrations which paralyze the ganglia or intrinsic nerves (as is shown by the disappearance of peristaltic reflexes), do not prevent the production of acetylcholine. Perfusing efferent intestinal vessels in it, an eserined Locke solution shows the presence of acetylcholine, in quantities that are not diminished by addition of cocaine even in a concentration as high as 1:800. Acetylcholine synthesis, therefore, is particularly intense in the mucous membrane, and may be responsible for the secretion of enteric sugar (Feldberg and Lin, 19491,). It is generally considered that acetylcholine stimulates the intestine; yet Burn and Vane (1949) also showed its inhibiting action. In a bath containing pieces of rabbit intestine, they add increasing amounts of acetylcholine. With each addition there is an immediate increase in contraction, followed by relaxation, the intensity of which grows with the concentration. Finally, adding acetylcholine produces an inhibition resembling that of atropine, which persists until the bath is changed, after which normal rhythm is re-established. The required concentration for total inhibition is 3 x lo4. Another phenomenon can be noted once the intestine has thus been inhibited by a fairly high concentration of acetylcholine and the bath is changed. Acetylcholine becomes inhibitory for the organ in question at lower concentrations which previously had been activating. The mechanism is not well understood. It might be due to the effects of excess acetylcholine blocking the acetylcholinic receptors; this would fall within the general concept, familiar to biochemists, of the

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inhibitory effect of an excess of some substance in an enzyme-substratum ratio ( Burn, 1950). It seems, on the other hand, that the amount of the contents of the intestinal lumen favors, but does not cause, the action of acetylcholine on peristalsis. Chujyo (1952), furthermore, has shown the role of passive extension of the intestinal wall in the production of acetylcholine. Hayama and Ikeda (1959) also showed variation in acetylcholine as a function of mechanical action on the intestinal wall. Stretching the intestine circumferentially immediately reduces the acetylcholine rate by 10-20%, and it goes back to normal again 15 sec after stretching is stopped. This experiment demonstrates the very rapid synthesis of acetylcholine in this tissue. The possible role of adrenaline, and doubtless also of noradrenaline, should likewise be envisaged. Normally, it inhibits intestinal motor activity, and it also reduces intestinal absorption of glucose together with alkaline phosphatase activity of the small intestine (Soulairac, 1947b). It does not appear, however, that it is produced directly at the level of the intestinal mucous membrane, and its major effect here would be to inhibit very readily any motor effects of acetylcholine, as happens at the level of the motor end-plate of the skeletal muscle ( Bulbring and Burn, 1942). This general concept, that intestinal tissue activity is locally controlled by substances such as histamine, acetylcholine and adrenaline, is especially interesting in connection with the hypothesis of a digestive control of the visceral information mechanisms of hunger. The interrelation of these 3 substances would make a delicate and fine equilibrium possible between stimulation and inhibition, Stimulation and inhibition might be produced by acetylcholine, or perhaps by histamine; adrenaline would intervene via facilitation mechanisms. Each tissue would, thus, have a kind of receptor by which stimulation and inhibition would be produced, the latter resulting from an excess of the mediator. In the intestine, such inhibition would result from the presence of large amounts of acetylcholine (Burn, 1950). Results which seem to fit perfectly into the general scheme we have just outlined were obtained by Brown et a2. (1958). In the cat, stimulating the nerves of the small intestine, without giving any agent to block the sympathetic, does not give rise to significant amounts of noradrenaline in venous blood. But when a blocking

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agent is used such as dibenzyline (10 mmg/kg), the response is abolished and noradrenaline appears in the efferent blood. Thus, in the intestine the liberated mediator is inactivated after combining with the receptors. From another angle, Kurotsu et al. (1955) showed the role of the hypothalamus on histamine metabolism. Electrical stimulation of the hypothalamus in normal and in adrenalectomized rabbits, causes changes in the histamine level in the blood. Stimulating the ventromedial nuclei reduces the histamine in the blood, whereas stimulating the lateral nuclei increases it. Following adrenalectomy, stimulating the ventromedial nuclei causes the histamine level to rise, while stimulating the lateral nuclei causes it to fall. Thus, the reactions are entirely reversed with respect to the normal. This production of complex chemical mediators, due to the functioning of the intestine itself, cannot in the majority of cases extend beyond their site of production on account of their instability. It would nevertheless appear that tissue metabolism has a part in the production of these mediators. Their roles, locally, would be to occasion the emission of pulses due to the various muscle responses that they produce in situ. It appears, in fact, that throughout the digestive tract there are tension receptors (Paintal, 1954a, b), which are sensitive both to more or less permanent peristalistic movements caused by the liberated mediators, and to tension exerted on the walls by the passage of nutritive substances. Apart from these tension receptors which are important mainly in the stomach, Paintal notes the existence of intestinal chemoreceptors which are insensitive to distension. These structures, on the other hand, are very sensitive to certain drugs injected arterially, such as 5-hydroxytryptamine, adrenaline, and nicotine. This type of chemical stimulation is primary, not secondary, to changes in the smooth muscles. Paintal furthermore notes that, whereas such receptors are sensitive to a wide range of variation in blood glucose levels, very large doses of glucose must be given in order to obtain this result, and it seems improbable that small physiological variations are able to stimulate these structures in their normal state. Vagal fibers from such receptors show, following distension of the gut, a discharge of impulses during the phase of slow re-adaptation, and there appears to be a linear relationship between the extent of gastric distension and the number of impulses per second along the fibers. During meals or after drinking water, impulses are pro-

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duced in numbers increasing with the intake of the substance. Maximum stimulation occurs when the stomach is completely distended. Later on, the number of impulses, which continue to reach the brain, remains constant for some time, and diminishes only slowly on account of the slow re-adaptation of the receptors as the stomach starts to empty. Paintal suggests that aside from their role in different reflexes, the receptors act in the immediate sensation of satiation, both in hunger and in thirst. When the number of impulses reaches a certain rate, ingestion of food or water ceases. But such a mechanism would operate only partially in a permanent state of satiation. These findings agree fairly well with experiments of Janowitz and Grossmann ( 1949) and Share et al. ( 1952). In experiments they distended the stomach of the dog both before and during a meal, and obtained data by changing the volume without altering the calorific content of food. But as Towbin (1949) suggests, such an afferent mechanism could be responsible only for an immediate and temporary satiation, but not for any permanent satiation following intestinal absorption. Paintal suggests that the afferent vagal fibers end at the level of the hypothalamic ventro-medial nucleus; this would activate such structures and would trigger the inhibitory mechanism of food consumption. Yet it also seems that numerous metabolic events modify the, intestinal motility and may thus determine the nervous impulses, which we stressed as important in the nervous control of hunger. Sudsaneh and Mayer (1959) showed that intravenous injection of glucagon, of epinephrine or of norepinephrine inhibits gastric contractions in the rat. These 3 substances are known to inhibit food consumption very strongly. Now, it is also known that glucagon and epinephrine affect carbohydrate metabolism, and produce hyperglycemia (although by different mechanisms); and it is known that norepinephrine does not do so. Norepinephrine inhibits food consumption even on fasting rats just like the other 2, substances. It would appear that the only link between the action of the 3 substances is their effect on intestinal motility. Also known is the possibility of altering intestinal motility by hypothalamic stimulation (Masserman and Haertig, 1938). The degree of intestinal inhibition or of increase of peristaltic action following this stimulation corresponds approximately to the intensity of the other triggered autonomous and emotional responses. Mayer and Sudsaneh (1959) noted that gastric contractions in

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hypothalamic, hyperphagic or aphagic rats are similar to those of normal rats. Adrenalin inhibits these contractions in both normal or in operated animals. These data seem to show that such gastric contraction does not depend on the integrity of the hypothalamic structures regulating hunger, but are likely to function independently. Sharma et al. (1962) furthermore have shown a relationship between gastric activity and the functioning of the hypothalamic centers recorded by means of deep electrodes. Gastric distension would increase the activity of “satiation centers” but gastric contractions produce no change in the hypothalamic structures. This gastric distension which is known to inhibit hunger gives rise to impulses which are probably conveyed via the vagal fibers arising from the gastric tension receptors (Janowitz, 1958). Thus, the operation of the digestive tract, mainly intestinal absorption, can be assumed to have an important role as an information mechanism keeping the central nervous system advised of the metabolic balance of the organism. The link between the intestinal absorption processes and the central nervous system would be a series of chemical mediators, which are synthetized in greater or smaller quantities within the mucous membrane itself in the course of its metabolic and/or motor operation. The most important of the mediators appears to be acetylcholine. The effects on the centers may be envisaged under two forms, either ( a ) a relatively specific effect on the hypothalamic structures, in which we have postulated the existence and role of feeding control (i.e., hunger or satiation centers), or else, ( b ) a more general effect on the mesencephalic-hypothalamic structures intervening during regulation of the mechanisms of waking and/or vigilance. This latter aspect will be stressed in the following tentative hypothesis of the mechanisms regulating appetite. 111. Tentative Explanatory Hypothesis for the Nervous Control of Appetite

A.

CONTROL THROUGH ‘‘(INTERIOR ENVIRONMENT”

Prominent among the metabolic factors in the control of hunger are the chemical mediators produced by the digestive functions. Whether transformed into afferent nervous pulses of vagal or sympathetic nature, or conveyed by humoral or other as yet unknown intermediate media, they must reach the central nervous control

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structures. We have seen that these presumed carriers of information are mainly acetylcholine and epinephrine, and one cannot but be struck by the analogy existing between these hunger regulating factors and the factors known to control the arousal mechanisms. Conceivably, the nervous control structures at the hypothalamic level are divided between cholinergic formations and adrenergic formations. This being so, and knowing that epinephrine produces anorexia, it could be assumed that the ventromedial nuclei, or “satiation center,” are adrenergic, whereas the “feeding center” (lateral area) is cholinergic. Such specificity in the nervous structures would be somewhat surprising and in this connection the experiments by Pickford (1939,1947), and Duke and Pickford (1951) show a direct action of acetylcholine on the vegetative centers of the hypothalamus governing diuresis and the secretion of posthypophyseal hormones. Now, acetylcholine inhibits diuresis, as does the stimulation of the hypothalamic supra-optic nuclei. Histochemical research by Koelle (1954) and Soulairac and Soulairac ( 1962) show the existence of powerful cholinesterase activity in the lateral hypothalamic areas and particularly in the pallidofugal system, whereas the ventromedial nuclei are totally devoid of such. It seems nevertheless that the information mechanisms, aside from their extremely localized specific action, are in some nonspecific manner also able to take part in the control of vigilance. It should be stressed furthermore that this concept does not eliminate the possibility of other information through control mechanisms (e.g., deltaglucose) being present, which, by acting on specific structures, would contribute their part to the activation of adrenergic and/or cholinergic systems. Certain experiments will now be discussed that show the importance of nonspecific regulation of vigilance in the control of feeding behavior. Two vigilance systems are known to exist, the operation of which may be summarized as follows. There is a basic system essentially governing the waking state and sleep, the nerve structures of which are represented by the mesencephalic reticular substance and whose humoral control is mainly adrenergic. There is a second system superposed on the first which enables the focussing of attention. The nervous structures responsible for the second system would comprise the whole of the thalamic reticular system and by certain rhinencephalic formations and would be especially

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sensitive to stimulation of a cholinergic nature. An initial series of experiments by Soulairac (1947a) and Soulairac and Soulairac (1959) enabled us to bring forward evidence for the effect of adrenergic and cholinergic stimulation on the feeding behavior of the rat. Adrenaline has a very distinct anorexiant effect on the rat and mouse. If rats are chronically dosed with amphetamine (0.6 mg/24 hr/8 days), which has a typical adrenergic action, a very significant reduction in food intake takes place as well as an overall caloric consumption, Administering atropine (10 mg/% hr/8 days) also causes a very significant reduction in overall caloric consumption and in food intake. These first data showed that it was possible to cause hypophagia either through experimental induction of an hyperadrenergic state (e.g., with amphetamine), or of a hypocholinergic state (e.g., with atropine). This would indicate that, in feeding control, the adrenergic mechanisms have an inhibitory role opposed to the facilitating action of the cholinergic mechanisms. Other experiments helped further to define the mechanism. It is known that insulin induces an appreciable rise in food intake, and for a long time it was considered that this action was essentially hormonal by inducing hypoglycemia. Certain data however call for the matter to be reconsidered. Thus, simultaneous administration of insulin and atropine suppresses all food consumption, whereas the hormonal effects of insulin remain ( Soulairac and Soulairac, 1959). It should therefore be assumed that insulin acts on feeding control mainly through its vagal effect, since an anticholinergic agent inhibits the action. The same authors showed that simultaneous administration of insulin and amphetamine also prevented hyperphagia. What happens seems to be that the vagal action of insulin is cancelled by the adrenergic action of amphetamine. This would assume a fairly strict state of balance between cholinergic and adrenergic stimulation in the normal control of the appetite. These data are theoretically important, as they show that there is no direct relation between the blood-sugar level and food consumption, and that the effects on appetite of a hormone, like insulin, are due not so much to their hypoglycemic action as to their cholinergic properties. This experimental differentiation between the hypoglycemic action of insulin and its alimentary effects has in fact been checked experimentally. Insulin with amphetamine no longer has any effect on the appetite although the drop in blood sugar is appreciable (.080

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gm/ml). The same holds good for the simultaneous action of atropine, which is nil on the appetite but results in a drop in blood sugar of .062 gm/ml ( Soulairac et al., 1961a). Other research confirms the inhibitory role of the sympathomimetic amines on hunger control. Administering reserpine and serotonin results in very significant reduction in food intake. Now, it is known that reserpine acts on the nervous system through simultaneous liberation of serotonin and catecholamines. Exogenous serotonin alone causes similar effects, although in this case it might be asked whether at least part of the effect is not of intestinal origin, as it acts on the smooth muscle of the gut. Experimental inhibition of monoamine-oxidase through iproniazid also shows that producing an excess of endogenous catecholamine is enough to cause hypophagia (Soulairac and Soulairac, 1960). Furthermore the simultaneous administration serotonin and iproniazid potentiates the effects of the two substances. Other experiments have shown that most of the substances modifying the vigilance mechanisms act directly on feeding control. An instance of this is LSD-25 which appreciably inhibits food consumption, the effect being relatively proportional to the dose administered ( Hamilton and Wilpizeski, 1961) . One problem, however, appears difficult to solve. How do such adrenergic modifications act in order to regulate feeding? One may envisage either some general action on the vigilance mechanisms rendering the central regulatory mechanisms more or less sensitive, or else some direct action on the regulatory structures themselves. The experiments of Epstein (1959, 1960) seem partly to contradict the results of Brobeck et al. (1956) which show an increase in the electrical activity of the ventromedial nuclei following amphetamine injection. Epstein notes that amphetamine produces anorexia in both normal animals and in animals made hyperphagic through lesion of the hypothalamic ventromedial nuclei. These data should be compared with those reported by Andersson and Larsson ( 195613). These showed that frontal lobotomy in the dog appreciably reduces the inhibitory effect of amphetamine on appetite. Epstein comes to the conclusion that the ventromedial nuclei cannot be considered as the only control mechanism of the appetite and that the effects of sympathomimetic amines may be due to an increase in motor activity accompanied by suppression of the eating response. It would appear that the effect of destroying the

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ventromedial nuclei in producing hyperphagia is more readily explainable in animals by the production of an overresponsiveness at a number of locations that stimulate the feeding response. This is the thread of the experiments by Smith et al. (1961). The reactions to food by normal rats are compared with those of rats rendered hyperphagic by ventromedial lesions, all the animals having previously been given either water or hypertonic solutions, or nonnutritive bulk. The results indicate that the 2 types of animals react in a similar manner and that, of the two, hypothalamic rats eat less. The authors interpret the results as evidence that hyperphagia in hypothalamic animals is the result of an exaggerated affective reaction rather than of any derangement of the feeding control mechanism itself. The concepts we have reported above present a tentative explanation of the nervous control of feeding initiated by information arriving from a lower medium. In psychological nomenclature these elements would represent factors of internal motivation. Unquestionably, however, sensory phenomena occasioned by exogenous stimuli interact directly in any nervous control of hunger. B. THESENSORYROLEIN FEEDING CONTROL The two characteristic senses that are concerned are smeIl and taste. The first research work attempting to check whether states of hunger or of satiation would occasion gustatory changes are those of Richter (1939) on adrenalectomized rats. The animals showed a very appreciable increase in consumption of sodium chloride and a study of the taste threshold for this substance revealed a very different recognition value in the adrenalectomized animal (0.035%)from the normal animal (0.55%).This represents a 15x reduction with respect to the normal threshold, Richter concluded that adrenalectomized rats ingested more salt, not because they learn that salt relieves their deficiency but on account of chemical changes in the mechanism of taste, giving rise to a better discrimination for salt. Experiments by Soulairac (1947a-d) also showed that endocrine disturbances causing changes in food consumption bring about a modification of the taste threshold for glucose. The average taste threshold for glucose is 0.77%in the mouse, and 1.08% in the rat. Deoxycorticosterone, which raises glucose consumption, reduces this threshold, which in the case of the mouse falls to 0.40%.Adrenalectomy, which reduces glucose consumption, brings the threshold

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in the rat down to 0.40%.Thyroxine raises glucose consumption and lowers the threshold in mice down to 0.3%; insulin, which has an important hyperphagic action, raises the threshold in the rat to 1.77%. There is, nevertheless, no direct relationship between the taste threshold and the direction of the modification in eating behavior, and the only conclusion to be drawn from such experiments is that varying the food consumption is accompanied by gustatory disturbance. Thus Richter’s conclusion is thereby invalidated. Furthermore, an experiment by Meyer (1952) on man shows that during deprivation of food lasting 34 hours, successive tests every 3 hours of the threshold levels of sugary, salty and bitter agents indicate that a reduction in organic reserves does not occasion any changes in the taste threshold of glucose, nor indeed in the other thresholds. As against this, depriving young subjects of sleep (Furchtgott and Willingham, 1956) occasioned an increased taste threshold for acids, which was particularly evident after deprivation of 48 and 72 hours. This would lead one to envisage the intervention of general fatigue and a lowering of attention in the mechanisms of taste, as indeed in a great number of sensory reactions (Kleitman, 1939; Goodhill and Tyler, 1947). The taste phenomena observed are not therefore as simple as had originally been thought by Richter, and numerous workers have attempted to reintroduce them into the general framework of feeding control. MacLeary (1953) in particular studied whether feeding preferences were based simply on taste factors or whether, depending on individual taste, specific amounts are ingested on account of their effect after ingestion. Thus, for instance, introducing glucose solution in a rat stomach may modify its preference for glucose solutions having different concentrations, whereas introducing a 20%glucose solution increases the ingestion of water; this falls during the 15 min following ingestion of 20%glucose solution by 5.3%.Thus, an effect may be observed on the gustatory receptors of a food substance introduced into the stomach directly without passing through the buccal cavity. Fructose has an even more clearcut effect. A study of the time interval required to this effect shows that postingestive changes occur in under 6 min, which could conceivably be interpreted as showing the existence of such a mechanism concerned in normal feeding behavior. Intragastric dosage of saccharose does not have the same effect, but on the other hand isoosmotic solutions of

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glucose, urea, and sodium chloride give identical results in reducing ingestion of a glucose solution. Water and the electrostatic balance of the animal are thus able to influence its preferential behavior; but, if intraperitoneal administration of glucose increases water intake it does not modify the consumption of a 5.3%glucose solution, Intravenous injection of sodium chloride also reduces consumption of a glucose solution at concentrations between 5.3 and 20%.These experiments would appear strongly to favor very centralized control of the mechanisms of taste and therefore would not enable one to envisage such peripheral changes as those of the sensitivity of a gustatory receptor itself. Electrophysiological research by Pfaffmann and Bare ( 1950) provided some detail on the significance of gustatory changes on feeding behavior. Starting again with the phenomenon observed by Richter regarding increased gustatory sensitivity for sodium chloride following adrenalectomy, the authors attempted to find out whether the sensory threshold was altered as was thought by Richter. They registered the action potentials produced in the chorda tympani of the rat by applying brine solutions of known concentrations on the anterior portion of the tongue. In normal and adrenalectomized rats, the action potentials appear at the same concentration for sodium chloride and are of equal amplitude, which implies that taste sensitivity at the lingual receptor levels is unaltered by adrenalectomy. The authors noted, however, that the electrophysiological threshold of taste in the normal animal is appreciably below its preference threshold and is virtually the same as that of the adrenalectomized animal. Thus, the normal animal recognizes concentrations of sodium chloride not preferable to water, whereas after adrenalectomy, once the taste threshold is reached, it chooses salt. This experiment is particularly important since it places the problem of taste in regulating feeding behavior at the level of the central nervous system. It is no longer a question of sensory variations but of the significance of a given stimulus in a given physiological situation. Observations by Fuller and Jacoby (1955) and of Teitelbaum (1955, 1961) support these findings. The authors found that in hereditarily obese and hyperphagic mice and in rats with hypothalamic lesions causing obesity and hyperphagia, a different sensitivity was found from that of normal animals having sapid qualities in the diet. Whether sapidity alters positively or negatively the animal's natural

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preference, the reaction of a hypothalamic animal given an adulterated diet is always most important. Teitelbaum, furthermore, notes that a normal animal passing from a standard diet to a diet containing 50% glucose reduces its food intake, whereas an animal with hypothalamic lesions increases it significantly. Soulairac and Soulairac (1958) found the same phenomenon in rats with lesions of the anterior neocortex (areas 6 and 10) having neither hypothalamic lesions nor obesity. It may therefore be assumed that the taste mechanisms merely reflect the central nervous changes. Taste sensitivity in a given animal appears to remain homogenous and what varies is the possibility of making use of and interpreting the sensory messages at the level of the integrating nervous centers. This means that the essential regulating factor of such peripheral sensitivity is represented at the various levels of attention. IV. General Conclusions

Control of feeding behavior takes place in three essential and interdependent phases, the first of which is essentially metabolic, whereas the other two are typically nervous (Soulairac, 1958). The metabolic phase has been sufficiently dealt with and need occupy us no longer; we need only recall that it covers the basic homeostatic phase during which energetic, humoral, and hormonal controls take place. This metabolic phase represents the essential element of information in the central nervous system. The importance of information of digestive origin has been stressed sufficiently. The second phase is represented by nervous control at the level of the diencephalic centers after information of metabolic origin has been received. This information appears to be conveyed by both a nervous route, which is normally the most important, and by a humoral route, as is attested by vagotomy experiments. The control structures extend all the way from the bulbomesencephalic formations to the hypothalamus, but the main role seems to be played by the middle hypothalamic formations. The ventromedial nuclei especially receive impulses occasioned by sympathetic stimulation and/or liberation of adrenalin or sympathomimetic amines. These appear to be sensitive specifically, and stimulating them would occasion a state of satiation,

meaning a state of homeostatic balance during which the animal does not need to ingest nutritive substances. The lateral hypothalamic areas would receive impulses of parasympathetic origin, and would be especially sensitive to acetylcholine; stimulating them would initiate new impulses whose role would be to control the action of higher integrating structures. When such stimulation is occasioned, as a consequence of depletion of energetic substances, the metabolic homeostasis is overwhelmed and new mechanisms have to be called into play to restore metabolic balance. This concept of the nervous control of hunger and satiation has the advantage of not postulating any very specialized structures, such as glucoreceptors, the existence of which has yet to be demonstrated. The reciprocal physiological operation of the hypothalamic centers of hunger and satiation through the agency of adrenergic and cholinergic mechanisms fits better into the framework of the present state of our knowledge of the general activity of the central nervous system. The participation of general chemical mediators in the mechanisms of hunger also more readily explains the means of transmission to higher nervous structures and the action of specialized attention mechanisms such as hunger, thirst, and sexuality, the effects of which should be exerted on all fundamental behavior. The feedback action of the hypothalamic structures on the metabolic control mechanisms are exerted either by direct nervous routes, for which there is much evidence, or via a humoral route. The importance of the hypothalamic structures on gastrointestinal motility and on the control of the anterior hypophysis is known. The latter would govern the operation of the various peripheral endocrine glands, whose importance was shown earlier during the phase of metabolic control. During the metabolic phase, which is liable to have a relatively independent continuous homeostatic control, the association of a nervous mechanism creates a new control circuit. Under certain conditions the hypothalamic centers, through nervous and hormonal action, permit the achievement of homeostasis in states when metabolic balance cannot be corrected at the periphery. Information reaching such general control structures would trigger the actual mechanism of feeding behavior only when a simple play of the peripheral diencephalic control no longer suffices. The third phase would consist of an integration of all the stimuli into a spatiotemporal scheme which would result in the feeding be-

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havior proper. Experimental results in evidence of nervous structures responsible for this are relatively fragmentary as yet, but the cardinal role played by the rhinencephalic formations have been stressed, and it is easy to conceive that such a mechanism could act via some of the structures belonging to Papez’s (1937) circuit. This is the integrating phase during which nervous modifications would occur enabling the animal to come into specialized contact with his environment and to act thereon through specific activities. Such contact would depend mainly on the possibility of attaining a state of specific vigilance; this would be effected only by substituting a momentary cholinergic dominance to a permanent adrenergic activation during the waking state. This phase therefore calls for archaic nervous structures (rhinencephalic) for the automatic attainment of behavior-generally speaking, oral activities and adequate sensory variation-together with control structures offering the possibility of inhibition and/or facilitation, represented by the neocortical formations. It is due to this higher phase that the two lower preceding phases are integrated into an overall and unified activity, calling for action by the individual as a whole; this represents real psychological behavior. In case of disturbance this phase also becomes the weak point in the mechanism, since its constituent controls are then able to function on one side or the other of actual homeostatic requirements. This study of neurological factors controlling appetite leads to the concept that, whatever the physiological elements of motivation, in all behavior there are variations characteristic of the vigilance level. Putting the animal under tension, sometimes known as the appetitive phase, would be governed by the mesencephalic vigilance level, hence of the adrenergic type. In order that specialization and completion of activity may take place, ensuring specific and adapted behavior (consummatory phase), it is essential that in place of this ”adrenergic” vigilance level, a vigilance of the cholinergic type be substituted, whose nervous mechanisms are conceivably located at the level of the reticular thalamus and of the rhinencephalon. We thus find a pattern closely approaching what is known regarding conditioning mechanisms in which, after an initial or irradiation phase, a concentration phase must inevitably follow which is the final phase in conditioning. This concept may throw some light on certain neurotic disturbances of feeding behavior, as for instance in the case of anorexia nervosa.

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SOME BIOSYNTHETIC ACTIVITIES OF CENTRAL NERVOUS TISSUE’ By R. Y. Coxon University Laboratory of Physiology, Oxford, England

I. Introduction

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11. Methods of Study

111. IV. V. VI.

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A. The Whole Animal . . . . . . . . . . . . . B. Isolated Tissues . . . . . . . . . . . . . . C. Permeability Barriers and Intracellular Compartments . . . Fatty Acid Synthesis in Brain . . . . . . . . . . . Protein Synthesis in Brain . . . . . . . . . . . . Glycogen Turnover in Brain . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . .

349 352 354 356 363 371 379 383

I . Introduction

Classical accounts of the chemical activities of living organisms have tended to classify them broadly into the contrasting categories of anabolic and catabolic (see Starling, 1905). More recent researches into the chemical events in cells have centered particularly upon the energy exchanges which, by coupling together synthetic and degradative reactions, make possible the use of energy liberated in the degradative for the performance of the synthetic reactions. An awareness of the importance of such thermodynamic relationships in biology is not in itself an innovation-Sir William Bayliss (1924) wrote that “the phenomena peculiarly characteristic of vital changes are those associated with the actual processes of transfer or transformation of energy.” Nonetheless, it is probably more true today that mass and energy are closely intermingled in the thinking of biochemists (just as they have been for some time in the thinking of ‘The author wishes to acknowledge the willing help of Mrs. B. Lazlett, M.A., Mrs. A. Spenser, B.Sc., Miss L. Bailey, and Miss A. G. Smith in the preparation of this manuscript and figures.

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physicists). A central problem which confronts contemporary investigators is the unravelling of the details of the processes to which Bayliss referred. So far as anabolism is concerned two partially separable aspects of the process can be distinguished: In the one, there is actually an increase in the mass of the organism; in the other a so-called “steady state” is maintained. In the latter, the anabolic processes simply keep pace with certain catabolic tendencies which seem inalienable natural features of protoplasm. Since perpetual motion is not thermodynamically permissible within an isolated system, the maintenance of a steady state other than equilibrium in a cell, or assemblage of cells, requires the expenditure of energy. Increase in mass is associated with an increase of internal energy and, in the case of an ordered structure such as a living cell, it also involves a reduction in randomness, or entropy, in the system of which the organism forms a part. This relationship was pithily expressed by the distinguished physicist Schrodinger ( 1945), when he asserted that “what an organism feeds upon is negative entropy.” It has been known for over 100 yr (see Bischoff and Voit, 1860) that living animals derive their energy from the combustion of a limited number of classes of chemical substance, viz. proteins, carbohydrates, and fats, but during the past few decades much effort has gone into the study of the individual reactions of these materials in the course of degradation to their metabolic end products, carbon dioxide and water, From these studies has emerged the striking fact that throughout a vast range of living organisms there exists a final common path for the catabolism of all these major types of foodstuff, through an integrated sequence of enzymic reactions involving a series of special cofactors and linked very intimately through them to the generation of stores of utilizable chemical energy, in the form of phosphorylated derivatives of the organic base adenosine, This chain of oxidative reactions was formulated by Krebs and Johnson (1937) as the tricarboxylic acid cycle and is sometimes known as the Krebs cycle; together with the energy-storage system employing nucleotide phosphates-the wide significance of which was recognized by Lipmann in 1941-it has been demonstrated (with occasional trivial modifications ) in practically every multicellular organism studied and many unicellular ones as well (see Krebs and Lowenstein, 1980).

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In contrast with the very widespread distribution of these catabolic systems in living matter, some at least of the anabolic systems must be, in the last analysis, peculiar to a given organism since by their operation they confer upon it whatever individuality it may possess. One may anticipate, however, from the broad lines of evolutionary theory that there will be certain underlying similarities in the anabolic processes found in many types of cell, although superimposed upon these there will be variations conferring the specificity which differentiates one kind of cell from another. Applying these general considerations to the central nervous system of animals, we may expect to find the constituent cells endowed with chemical mechanisms for carrying out catabolic energy-yielding reactions very similar to those found in other cells; on the anabolic side, we may expect to find some reactions taking place which also occur in other tissues, along with others peculiar to neural cells. It should be reiterated that catabolic and anabolic reactions are very closely linked in that the energy liberated from the former is utilized in the latter. Indeed, the rate of the catabolic reaction may depend upon the rate of the anabolic in some circumstances. The synthesis of energy-rich nucleotide phosphates, which may be coupled to both aerobic and anaerobic catabolic processes, is clearly of central importance in the economy of cells since the energy trapped in such compounds is utilized in the formation of the complex lipids, proteins, carbohydrates, and nucleic acids which make up their protoplasm. An important achievement of modern biochemistry has been the development of methods by which the chemical mechanisms of complicated synthetic reactions may be elucidated. Before entering into a discussion of any particular biosynthetic systems, a few general comments will be made on the methods now available and on their limitations when applied to the central nervous system of higher vertebrates. II. Methods of Study

A. THEWHOLEANIMAL

The time-honored method of the nutritionist is to deprive a young animal (or plant) of a particular nutrient and then follow the effect of the deficiency on the continuance of its growth. By this means the vitamins were discovered (Hopkins, 1912),but the ap-

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proach is basically a negative one calculated to reveal the absence rather than the presence of biosynthetic capacities; comparative studies of this kind between species (or larger subdivisions of the living world) may, however, reveal potentialities which are not present in one but are present in another and which may prove of value in studying a biosynthetic mechanism. An example of such a difference in biosynthetic capacity is to be found in the case of ascorbic acid, which is produced by the rat but not by the guinea pig. Sometimes isolation from the animal’s urine of a metabolite derived from an orally administered precursor may throw light on a synthetic mechanism. Thus, the synthesis of hippuric acid from benzoic acid, which has been used as a clinical test for liver function (Quick, 1936), also provided a model for the study of peptide bonds in the test tube using enzymes prepared from liver cells (Chantrenne, 1951). In its crude form, this technique gives no useful information regarding the contribution of particular organs to the health and growth of an animal, but it may be extended by applying it to animals deprived of organs by surgery. In this way the role of the liver in the synthesis of urea was demonstrated (Bollman et al., 1924) and likewise the role of symbiotic intestinal bacteria in the supply of certain vitamins to their host (see Najjar and Barrett, 1945). Up to a point, the interdependence of certain organs can be established by this type of experiment, but the interpretation of the findings is not always simple. For instance, the importance of the liver in maintaining the health of the brain derives in part from its capacity to detoxicate ammonia as well as its capacity to supply glucose from its glycogen stores. A further type of interaction between organs is that seen in the endocrine system where the link depends on the production not of raw materials for growth but of regulatory substances. In the case of the brain, the absence of thyroid hormone at a time when growth of the body as a whole is going on (though at a subnormal rate) will lead to a selective failure of development of cerebral function. No instances are known to the writer in which dietary deficiency of the precursors of a bulk component of the central nervous system leads to a selective arrest of its development although, according to Platt (1961) a chronically reduced intake of protein may lead, terminally at least, to a symptom-complex including neurological manifestations and to histological changes in spinal neurons. On the other hand, in the case of the water-soluble vitamins a failure

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to absorb the very small amounts required for health may lead to gross damage, as seen in subacute combined degeneration of the spinal cord. Here, however, biochemical activity is impaired indirectly rather than by an actual shortage of raw material. Studies of synthetic processes in the steady state (i.e., in a condition of nutritional balance) are only possible in the intact adult animal by using some form of “tracer” technique, permitting the replacement of some atoms or molecules by chemically similar ones which may be detected in the absence of a net change in the total numbers present. Some “turnover” studies will be discussed later. It may be recalled in the present context that some information may be obtained indirectly by disturbing the steady state of nutritional equilibrium through partial or complete starvation. In such circumstances, the progressive decomposition of many organs can be followed, but the gross effects of undernutrition on the bulk of the central nervous system are in fact conspicuously slight (see Lusk, 1921, p. 105). An instance where the transfer of material from one organ for incorporation into another during inanition is furnished by the migrating salmon in which muscle cells lose about 50% of their protein (without being diminished in number), the material liberated being incorporated in the enlarging gonads (Miescher, as quoted by Lusk, 1921, p. 102). A similar situation exists in the postpartum female mammal when fatty acids may be transferred from the adipose depots to the lactating mammary gland, In both these cases, the observed change could be explained by a breakdown of large molecules to smaller ones in the donor organ, with a subsequent reassembly of some of these units in the recipient organ, probably in different proportions from those obtaining in the tissue of origin. Such transfers can be followed and their mechanisms analyzed by measuring arteriovenous differences in the concentration of intermediate “currency” compounds and also by the use of labelled intermediates (the label being at the present day usually a radioactive atom, but stable chemical groupings have also been used as labels with success, as for example elaidic acid employed by McConnell and Sinclair (1937) in studies of the synthesis of cerebral lipid). The existence of the blood-brain barrier is often an encumbrance in such studies in the whole animal since, to a considerable extent, it isolates the central nervous system from the internal environment to which other body cells are exposed, namely an inter-

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stitial fluid, which is in equilibrium in many respects with the blood plasma. In some instances, however, the existence of the blood-brain barrier with its highly selective permeability can be exploited to localize certain chemical events to the central nervous system. An example, is the work of Sporn et ul. (1959) on the synthesis of urea where their argument rests on the fact that they were able effectively to restrict the availability of their precursor to the central nervous system by injecting it into the cerebrospinal fluid rather than into the blood stream. The importance of ensuring access of precursor to the central nervous system is illustrated in the work of Gaitonde and Richter (1956) on the incorporation of labelled amino acids into cerebral proteins of living rats, to which further reference will be made later.

B. ISOLATED TISSUES 1. Nonneuronul Structures in Nervous Tissues The interpretation of biochemical data on whole organs and on various preparations made from them should always be tempered by an awareness of inhomogeneity in cytological makeup. This inhomogeneity becomes increasingly a problem as one proceeds in the allocation of function from the macroscopic to the microscopic and submicroscopic level. For example, if a perfused liver can be shown to convert compound A into compound B during passage through its substance, it is reasonable to regard this as compatible with the idea that this transformation may well occur in the intact animal when compound A is presented to the gland in the blood entering from the aorta or portal vein. The same, mutmtis mutundis, is true when the perfused brain takes up glucose, as in the experiments of Geiger (19%). If the tissue (whether hepatic or cerebral) be fragmented, the fragments incubated with some reactant, and the product subsequently isolated from the reaction mixture, this again is compatible with the postulate of the same sequence of events in vioo, and it may be possible to demonstrate a constant association between a particular reaction and the area of brain or gland from which the active fragments can be prepared. There is, however, a limit beyond which this narrowing process cannot be carried. Even in a relatively homogeneous tissue such as the liver there are connective tissue cells

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which might conceivably contribute to the events observed, and in the case of central nervous tissue this source of confusion is greatly magnified since there the variability between the constituent cells is enormously greater. Not only is there the gross subdivision into neuronal and neuroglial elements, but within these populations there are a great variety of types, ranging (among the neurons) from the giant Betz cells of the motor cortex to the tiny mitral cells of the cerebellum, and embracing within the glial group all gradations between the large astrocytes and the smallest microglia. By preparing slices, minces, or homogenates from specially selected areas in the central nervous system it may be possible to ensure the preponderance of one or another cell type as, for example, by using white matter (Korey and Orchen, 1959), where the only cell bodies present are of the glial series. Gray matter, however, contains neuronal and neuroglial elements so intimately entangled that their separation on a scale suitable for conventional biochemical experimentation is not practicable. The conventional method of producing a homogenate by grinding the tissue with a watery diluent between glass surfaces may introduce some degree of selectivity in that certain cells could be less mutilated than others in the process. But, generally speaking, most cell-membranes may be expected to undergo rupture during grinding, with liberation into the medium of the cellular constituents. The resulting homogenate will presumably contain both particulate and soluble contributions from all the cell types present in the original tissue, and, if the homogenate be fractionated by centrifugation, each fraction will contain organelles or solutes of similarly mixed origin. The contributions of the various cell types to the final product may be, to some extent, deducible from their known ultrastructure, and it is possible that further advances in electron microscopy and other morphological studies may lead to much more accurate identification of subcellular particles. According to FernBndez-MorBn (1957) mitochondria are prominent and numerous in neurons, and much less so in neuroglia, so that a mitochondrial suspension, even if not further subfractionated according to particle size and density might be regarded as predominantly (though not exclusively) of neuronal origin. However, against this must be set the fact that, according to Korey and Orchen (1959), only about 25%of cells are neuronal in cerebral cortex. It is interest-

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ing incidentally to compare this proportion with that found in liver, which is generally taken to represent a rather homogeneous tissue, for even then only 60% of cells are parenchymal. In this connection, a finding of H y d h and Pigon (1960) may be cited as indicative of future possibilities in the separate study of neuronal and neuroglial metabolism. Their results suggest that mitochondria from neurons may be amenable to study separately from glial mitochondria. If a sufficiently clear-cut distinction of this kind can be firmly established, it may in time be possible to determine the proportion of organelles from each source in a mixed population and to poison each type selectively. If differences between other subcellular particles arising from different cell types can also be recognized, then the same methods of identification might be applied to them also. Nurnberger and Gordon (1957) tentatively assign isolated nuclei to known cell-types of both the neuronal and neuroglial series on the basis of morphological criteria, but this is a simpler problem than that posed by the smaller cell inclusions. It is clear, however, for the present that the inhomogeneity of the cell population under study in both intact and fragmented brains must forbid the equating of their biochemical properties with those of neurons. Nevertheless, once this is recognized it is perfectly justifiable to relate the findings to the life history of nervous tissue provided one remembers that this is a nonuniform structural entity, the composite nature of which is probably essential for its proper functioning. C. PERMEABILITY BARRIERS AND INTRACELLULAR COMPARTMENTS

The importance of the blood-brain barrier in the design and interpretation of whole animal experiments has already been emphasized but it is no less important to recognize the effects of permeability barriers and other physical factors in the interpretation of the results of studies utilizing preparations of nervous tissue removed from the body. For example, it is not possible to demonstrate an in Vitro effect of diphosphothiamine in brain minces because the vitamin cannot enter the cells, whereas it is possible to demonstrate this with brain homogenates because the cell membranes no longer offer a barrier to its entry (Banga et al., 1939). Similarly, a dicarboxylic acid, fumarate, does not appear to enter slices or minces. Some unicellular organisms present such barriers (cf. Krebs and Lowenstein,

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1960) and they are even encountered in subcellular particles, as is shown by the impossibility of causing the reduction of intramitochondrial cytochrome with extramitochrondrial coenzyme I, although it appears that intramitochondrial cytochrome can readily be reduced by intramitochondrial coenzyme I (Lehninger, 1951). On the other hand, the spatial separation within an individual cell of the anabolic and catabolic enzyme systems may be turned to good account when carrying out studies with suspensions of cell fractions. Thus when investigating the synthesis of fatty acids (see Section 111) it may be advantageous to work with artificially constructed systems from which cell components which normally degrade these substances are deliberately excluded. In this instance, a favorable combination consists of water-soluble enzymes of the tissue together with the microsomes but without the mitochondria. Such a mixture will only function as a synthesizing system provided a source of free energy is made available as a substitute for the mitochondria which will perform this role in the cell; this can be done in the test tube by supplying ATP (see Abraham et al., 1960). In the intact cell (as has been pointed out earlier) both anabolic and catabolic reactions are continuously proceeding, and the overall economy of the cell demands the coupling of these processes insofar as energy derived from degradation is utilized for synthesis. The interposition of nucleotide phosphates and thiol esters, by providing a means of storing and transferring energy, permits the coupling of energetically antithetic processes it seems likely that such energystoring compounds may be capable, in some circumstances, of circulating in a ferry-like manner between their sites of production and their sites of decomposition. This may involve the movement of quite large particulate entities within a cell, and it has been suggested as underlying the known tendency of mitochondria to congregate near the nucleus in a cell at certain times, perhaps when protein synthesis is especially active (see Brachet, 1957). Finally, it should be noted that the artificial conditions under which slices of tissue are incubated in order to study their metabolism may lead to the breakdown of permeability barriers which may impair the functional integrity of the tissue. Coxon (1956) found that the enzyme aconitase leaked freely into the saline medium when brain-slices were shaken in it under the conditions in which in vitro experiments are customarily performed, while McIlwain ( 1960) has

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shown that the leakage of gangliosides from brain slices, which occurs during incubation, leads to a loss of sensitivity to electrical stimuli which, before such leakage occurred, had been capable of markedly aifecting the metabolism of the slices. In studies of fat metabolism in isolated tissues, special measures may be required to ensure the solubility of substrates and products in the medium and so make possible their transport in and out of the active cells. 111. Fatty Acid Synthesis in Brain

It was mentioned in the preceding section of this review that one of the great virtues of isotopic tracers is that they permit the study of the replacement of atoms or radicles in tissue constituents when no net increase is occurring in the quantity of these constituents present, In this way they enabled Schoenheimer (1942) to recognize the continuous changes taking place in apparently stable components of the animal tissues and to formulate his concept of the “dynamic state of the body constituents.” An early instance of the use of tracer techniques was in the study of the fatty acids of rat brain by Sperry et al. (1940). The procedure which they adopted was to feed to the animals by stomach tube a quantity of fatty acids in the form of linseed oil reduced with deuterium-enriched hydrogen. Since linseed oil consists largely of lscarbon acids (Deuel, 1951), it seems likely that the main product of the deuteration would be stearic acid. Subsequent analysis of the organs of these animals revealed that approximately 0.3%of the administered deuterium was incorporated into the fatty acids of the brain as against somewhere between &18%found in the liver fatty acids. These were adult rats and from the results it was concluded that the rate of turnover of fatty acids in brain was very considerably slower than in liver. The matter was further investigated ( Waelsch et al., 1940) by administering heavy water to rats over a period of some 7 days and it was found under these circumstances that the extent of labelling of the fatty acids of brain approached that in the liver and other regions of the body; the incorporation of isotope was, in fact, onethird as great in brain as in liver fatty acids. The conclusion from these two series of experiments was that the brain fatty acids in the adult rat are renewed at the rate of about 20%of their bulk per week and that the synthesis of the fresh molecules of fatty acid takes place in the brain itself. In t h i s way the

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comparatively slight labelling which followed feeding with preformed deuterated fatty acid as compared with the heavier labelling when the isotope was fed in inorganic form as water could be explained. The foregoing data relate to adult animals but the same group of investigators ( Waelsch et al., 1941) subsequently extended their experiments to cover also young rats in a phase of active growth. Again deuterated water was used as the tracer and it was then found that the brain fatty acids became labelled to a degree approximating that found in the liver fatty acids. In these young rats simple measurement of the change in total content of fatty acids in the brain left no doubt that accumulation of these compounds must have been going on, and a comparison of the time-course of the change in content with the time-course of myelination ( established histologically) enabled the effects of overall growth of the nervous system, on the one hand, and the myelination of pre-existing axons, on the other hand, to be distinguished to a certain extent from each other. It was found during the phase of growth unaccompanied by myelination that the fatty acids of the brain became labelled with deuterium to just about the same extent as those of the liver; this would be compatible with the possibility that the fatty acids in the brain had originated in the liver. However, by making a number of reasonable assumptions the authors were led to discard this possibility and advance the important hypothesis that the fatty acids are in fact synthesized in the brain. They calculated, using data from other sources on the turnover of fatty acids in the liver, that the rate of uptake in the brain would have to be impossibly fast for the fatty acids of this organ to attain the observed degree of labelling if transferred from the liver. It must be recognized, however, that this argument involves the assumption that the fatty acids of the liver are labelled to a uniform extent; if there existed among the fatty acids of the liver a fraction which, though itself very heavily labelled, was diluted by one or more fractions less heavily labelled, it might still be possible for the heavily labelled fraction to serve as precursor for the brain fatty acids and account for the experimental findings. On the other hand, it is known that a developing brain is unable to assimilate elaidic acid (which is an unnatural but very close analog of the fatty acids of animal tissues, and which is readily taken up by other organs); this supports the idea that the buildup and replacement of

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fatty acids in brain does take place by synthesis in situ rather than by uptake of preformed molecules from the body-fluids. Very similar results were obtained by Coxon and Chaikoff (1959), who administered orally to young rats the neutral fat tristearin in which the stearic acid was labelled with C'.' in the carboxyl group, stearic acid being a normal constituent of cerebral lipids. It was found that the cerebral fatty acids were only about one-tenth as radioactive as the corresponding compounds in the liver, The radioactivity was found predominantly in the fatty acids of the brain with a chain length of 18 carbons, but, since these are quantitatively among the most conspicuous, probably no special significance can be attached to this particular finding. The relative extent of labelling in the two organs would be compatible with either a precursor-product relationship between the liver and brain fatty acids or with an arrangement by which both liver and brain were drawing upon a common pool of precursors. In the latter case it would be inferred that the liver laid down a greater fraction of its fatty acids from the pool during the experimental period. However, the immediate precursors in the pool may well have been different for the two organs; thus, the liver could have been taking up stearic acid directly from the blood stream, while the brain, on the other hand, could conceivably have been synthesizing fatty acids from one or more of a number of other substances formed from the breakdown of the administered stearate in other organs and able to be transported in the blood. Among the likeliest of these would be acetate, which Paoletti et al. (1961) found to be capable of supplying radioactive carbon for incorporation into cerebral stearic acid when injected intraperitoneally into young rats. Acetyl radicles derived from long-chain fatty acids might also be transported as acetoacetate. In these experiments it was hoped to isolate stearic acid from the brains of the young rats in sufficient yield and with sufficiently heavy labelling to compare the position of the radioactive carbon atom in the molecule with that in the starting material. But, this has not yet proved practicable. In principle, such an experiment is feasible and should provide a definite answer to whether maturing brain takes up fatty acids from the body fluids or synthesizes them entirely in situ. Whatever uncertainty may still exist regarding the actual process in the living animal, experiments with isolated brain preparations have shown clearly that the young organ does possess the poten-

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tiality of synthesizing acids with chain lengths of at least 6 carbons from a precursor no more complex than acetate. Grossi et al. (1958, 1959) report that slices from the brains of rats, age 10 days, will incorporate radioactivity from a ~ e t a t e - 1 - Cto~an ~ appreciable extent. The same observers found that this ability was 40 times less in slices prepared from rats, age 60 days. They also found some ability to incorporate acetate-carbon into fatty acids in cell-free homogenates of the brains of young rats and this ability was, curiously enough, more marked in the brain than in the liver of such young animals. Acetate appears not to be readily utilizable from the blood by the adult brain. McMillan et al. (1957) found that, although intracisternal injection of this substance resulted in the appearance of its carbon atoms in the cerebral lipids of mature rats, little effect was obtainable by intraperitoneal administration. However, Kabara and Okita (1961) point out that some incorporation of C14 into brain cholesterol can take place when the isotope is administered intraperitoneally either as acetate or as mevalonic acid and that the extent of such incorporation as compared with that in other organs is rendered less conspicuous by the large total quantity of cholesterol present in brain. This amounts to about 5 times that found in the liver (Campbell, 1961) and could lead to extensive dilution of the newly introduced labelled atoms. Klenk ( 1957) has reported that relatively little incorporation of C14into brain fatty acids took place when labelled acetate was given by mouth to rats but that brain slices incubated in vitro proved comparable to liver slices in their ability to incorporate radioactivity from C14-acetateinto their fatty acids, including the polyenic acids. Karnovsky et al. (1959), who studied the incorporation of C14-acetate into the total lipid of brain slices and isolated peripheral nerve, noted that this occurred many times more readily in tissues from 5-day old animals than in preparations from animals 50 days of age or more. In a further report Klenk (1958) mentioned that “subdurally” administered C14-acetate caused a considerable uptake of radioactivity into both saturated and unsaturated cerebral fatty acids. While there is no intention in this review to consider in great detail the individual chemical reactions which participate in the formation of cell constituents, it does seem expedient to call attention here to an important general point relevant to the elaboration

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of all the main classes of substance under discussion, that is, fatty acids, proteins and polysaccharides. Contrary to earlier ideas, there is good evidence recently that, for the most part, the synthesis of such substances is effected by chemical mechanisms different from those concerned with degradation of the same substances. Thus, biosynthetic processes do not simply consist in reversal of the reactions concerned with degradation, but may involve both different enzymes and intermediates. At &st sight this may appear as a more complicated concept of cellular biochemistry than the traditional view which predicted that, since enzymic reactions are in theory reversible (and can be shown to be so in practice under suitable conditions), they must be capable of proceeding in either direction on an extensive scale in living cells. In many respects, the newer picture is a simpler one in that it circumvents the considerable difficulty of explaining, e.g., how the radical shifts in the predominant direction of formally reversible reactions could be effected when, under physiological conditions of temperature and concentration, the equilibrium constants greatly favor decomposition. By utilizing a distinct chemical pathway for synthesis such unfavorable factors can be readily obviated and existing energy barriers overcome. The formation and breakdown of a hypothetical substance C may thus be envisaged as taking place by the operation of a cyclical system such as that crudely illustrated in Fig. 1, where the overall equi-

FIG.1. Schema showing overall reversibility of reaction between substances A and B giving C, proceeding by different intermediate complexes in the two directions. Overall equilibrium is indicated by dotted arrows. X and Y represent distinct adjuvant compounds on the synthetic and degradative pathways, respectively,

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librium is indicated and the distinct adjuvant compounds on the synthetic and degradative pathways are represented. In the case of the fatty acids, the catabolic sequence of reactions [reversal of which was originally thought to permit synthesis (see Lynen, 1953)] depends on the conjugation of the fatty acids with coenzyme A followed by successive oxidations at the carbon atom located in the ,&position relative to the carboxyl, the principal hydrogen carrier being nicotinamide-adenine-dinucleotide( NAD ) . On the other hand, the synthetic process by which long-chain fatty acids are built up from acetate radicles involves the combination of these with carbon dioxide to form the malonyl derivative of coenzyme A, while in the reductive steps the hydrogen carrier is nicotinamideadenine-dinucleotide phosphate (NADP) ( see Lynen, 1959). Brady (1960) has prepared from the brains of young rats an enzyme system which catalyzes the incorporation of C'" from malonyl coenzyme A into long-chain fatty acids in the presence of NADP as hydrogen-carrier, and it is of interest in this connection that Glock and McLean (1954) have found in brain enzymes capable of oxidizing glucose via hexosemonophosphate, which is a well-established means of generating reduced NADP, and so providing hydrogen atoms for fatty acid synthesis. Hotta (1961), although he is inclined to attribute a somewhat different functional significance to his results, has also produced evidence pointing to the operation of this NADPlinked oxidative system in brain. It is probable, therefore, that when fatty acid synthesis takes place in nervous tissue it proceeds by much the same steps as in other organs. In their experiments with growing rats (to which reference has already been made) Waelsch et al. (1941) studied not only the fatty acids but also the nonsaponifiable lipids. By reasoning similar to that applied to the data on fatty acids, it was argued that the nonsaponifiable lipids must also be synthesized in situ. Now, the nonsaponifiable lipids of brain include cholesterol. It is therefore important to set alongside the inferences drawn by Waelsch and his colleagues some more recent experimental findings reported by Davison et at!. ( 1959). These workers injected intraperitoneally into young rabbits on the seventeenth day after birth some cholesterol singly labelled in the number-4 carbon atom with radioactivity, They subsequently found that cholesterol isolated from the myelin of the rabbits' brains

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1yr following the injection was radioactive and-much more significant in the present context-the radioactivity was located almost exclusively in the number4 carbon atom. This retention of the labelled atom in its original position clearly indicates that some molecules of cholesterol must not only have entered the brain from the blood stream without degradation of their ring-structure but, having entered, must have remained in situ for over a year. By analogy, one might therefore predict that, notwithstanding the results obtained by Waelsch et al., some, at least, of the cerebral fatty acids might have entered the central nervous system by uptake as preformed chains from the body-fluids during the stage of active growth in early life. The animals used by Waelsch et al. (1941) were rats, while Davison d al. (1959) worked on rabbits, so that there may perhaps be a species difference also to be taken into account. The fatty acids of the central nervous system whether they are synthesized in situ-as seems to apply to the greater part of themor taken up from the blood stream, do not, for the most part, persist as free acids but are incorporated in the molecules of the much more complex lipids characteristic of nervous tissue. These include the phospholipids, for which the method of formation is now reasonably well understood as a result of the pioneering studies of Kennedy (1957) using liver preparations and the experiments of Rossiter d al. (1960) using brain enzymes. The metabolism of this class of compound formed the subject of a recent review in the present series (Webster, 1961) and will not be further discussed here except to recall that the biosynthesis of phosphatides from glycerides requires the participation of cytidine nucleotide. Fatty acids also appear in glycolipids such as the cerebrosides where they are linked to a monosaccharide sugar through a complex organic base. It wouId appear that the biosynthesis of cerebrosides is dependent upon uridine nucIeotide as a hexose carrier. Other glycolipids contain in their molecules the amino sugar galactosamine but little detailed information is available as to their mode of production. It should be noted that the fatty acids of brain include a number with highly unsaturated hydrocarbon chains which may contain as many as 6 double bonds. Some polyenoic acids are among those classified as “essential fatty acids” by nutritionists on the ground that they cannot be formed by such animals as the rat except from acids like linoleic which already contain at least 2 double bonds per mole-

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cule. Accounts of the effects of deficiency of “essential” fatty acids (Deuel, 1957) emphasize retardation of growth and dysplasia of the skin but give no special prominence to nervous disorders. This seems curious, unless there is some species difference, since from the results of Baker (1961) over 10% of the fatty acids found in human brain might be expected on the basis of their degree of unsaturation to depend for their production upon an adequate supply of linoleic acid. Possibly, the deficient rats devote their limited maternally derived stores of essential fatty acids preferentially to the laying down of neural tissue. The mechanism of conversion of linoleic acid to the longer chained, unsaturated acids does not appear to have been extensively studied in brain but in other tissues elongation of the carbon chain has been shown to result, as might be expected, from the addition of acetyl radicles (Steinberg et al., 1956; Mead and Howton, 1957). In living nervous tissue the lipids in which the fatty acids are combined are themselves incorporated into more complex molecular species either with other lipids or with proteins. The intimate nature of these complex materials, and of the bonds which hold together their components, are poorly understood as yet (see Le Baron and Folch, 1957), but their existence points to a link between lipid and protein metabolism. IV. Protein Synthesis in Brain

Since the advent of isotopic labelling, two groups of workers have studied in considerable detail the incorporation of labelled amino acids into the protein of nervous tissue. Richter and his colleagues in Great Britain, have concerned themselves particularly with the incorporation of S”-labelled methionine, while Waelsch and his co-workers in New York have studied a number of C1”labelled amino acids. Gaitonde and Richter (1956) have clearly demonstrated the incorporation of S”-methionine into brain protein and by injecting the substrate into the cerebrospinal fluid were able to induce a greater uptake into protein than was possible if the substrate only reached the brain via the blood stream. By assuming an equivalence of specific activity in the methionine available to the brain cells with that of the injected material (which seems justifiable under their experimental conditions) they calculated a rate of incorporation of the order of 0.41 ,ueq methionine/gm protein/hr. They

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point out that, if the rate of penetration of the substance were ratelimiting or if it were diluted to any considerable extent at the site of synthesis, this estimate would be rendered too low and that, therefore, it is to be regarded as minimal. On the other hand they did introduce into the cerebrospinal fluid something like 100 times the amount of methionine normally found in the available amino acid pool. This may conceivably have stimulated an increased rate of synthesis since, as Borsook et al. (1950) have shown, the rate of incorporation in isolated preparations of liver is elevated by increasing concentrations of available amino acid according to an approximately logarithmic relationship, The writer (Coxon, 1959) has found that the oxidation of some labelled amino acids in certain isolated organs is also sensitive to concentration and this is known to apply to the oxidation of other materials until saturation values are reached. This relationship is reflected in the Michaelis-Menten equation defining the affinities of enzymes and substrates but in the case of organized tissues may be modified by factors regulating the rate of penetration of reactants to the site of action of the relevant enzymes. It should be noted that as Gaitonde and Richter did not subdivide the proteins they extracted from the rats’ brains, their figures for the rate of incorporation represent an average rate: Some cerebral proteins may be renewed a great deal more rapidly and Gaitonde and Richter estimate that the half-life of some cerebral lipoproteins must be measured in seconds. Waelsch (1957) and his colleagues-in contrast with the British group-have relied to a large extent upon the administration of carbon-labelled amino acids injected in tracer amounts either directly into the blood stream or else into the peritoneal cavity from which transport to the brain would be expected to occur via the cardiovascular system (see Waelsch, 1957; Waelsch and Lajtha, 1980). Among others, the following two interesting observations emerged from these experiments. First, it was shown in the cases of leucine and lysine that there was a rapid entry of some molecules of these amino acids from the blood into the brain even though no bulk transfer could be demonstrated in earlier experiments in which a considerably elevated concentration of amino acid had been established in the circulating blood. In the case of lysine, it was estimated that the rate of the influx of the free amino acid into the brain was somewhere between 0.7 and 5 pg/gm brain/min and that

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the renewal of 50% of at least one protein fraction would take place every 3 days in the full-grown mouse. When different areas of brain were examined in the adult monkey, it was found that the highest rates of turnover of protein occurred in the white matter where most of the cell bodies present would be glial rather than neuronal in type. The authors, however, point out that although this finding could be taken to indicate a high protein turnover in the glial elements it could also be explained by uneven mixing of the labelled material, and they in fact favor the latter alternative. Waelsch‘s and Richter’s groups of workers agree in finding that, when the animals’ brains were homogenized and fractionated by differential centrifugation, maximal incorporation occurred, as in other tissues, in the microsomal fraction ( Waelsch 1957; Clouet and Richter, 1959) and there was evidence from the time course of the incorporation that the microsomal protein could function as precursor of the protein in other fractions. The microsomal protein so characterized was, in fact, a complex of lipid, protein, and nucleic acid and would thus correspond in general composition to granules described by Nissl (1892,1894) which are so characteristic a feature of histological preparations of nervous tissue. A totally different method of study which has been applied to the investigation of protein synthesis and also of nucleic acid synthesis is that used by Hyd6n (1958, 1960), which relies upon the analysis of single neurons dissected free from neighboring cells. This is technically very difficult, but a method of analysis has been worked out which gives reproducible results and permits the recognition of changes occurring as a result of induced functional disturbances in localized areas of the CNS. There are two principal methods used in Hyd6n’s work. Measurements of protein and nucleic acids and of total cell mass are made by absorption spectroscopy in the ultraviolet and X-ray regions of the spectrum. It has also proved possible to extract the nucleic acids from single cells and, following the extraction, the RNA may be digested with the specific enzyme ribonuclease, and the resulting mononucleotides separated electrophoretically and characterized (Edstrom and H y d h , 1954). The electrophoresis is carried out by an ultramicromodification of the usual filter-paper procedure in which a single cellulose fiber of 15 p diameter is employed. Analyses of single cells have also been reported by Lowry (1957)

R. V. COXON

and his results include assays of a number of enzymes. He measures the mass of the cell on a special type of balance (which in principle is similar to a torsion balance) sensitive to 0.00002 pg. The results are reproducible and, since all known enzymes are proteins, studies on the conditions governing the rate of formation of these highly specific proteins may be valuable in the study of protein-production generally within particular cells in the nervous system, since analogous studies on adaptive enzymes in populations of microorganisms have shed considerable light on certain general aspects of protein synthesis ( Monod, 1947; Spiegelman and Halverson, 1953). Hyd6n (1958) has gone so far as to propose a fairly detailed mechanism by which the synthesis of specific proteins (presumably by mechanisms involving the participation of ribonucleic acid, as in other situations) may constitute the physical basis of the elusive “memory-trace.” Having regard to the complicated structural network of the brain which has encouraged the formulation of theories of memory based upon interaction and facilitation within groups of neurons by modification of the ease of transmission of impulses between them, this seems at first sight to be heretical to the point of perversity. However, H y d h produces some cogent arguments to indicate that his proposed mechanism could operate with sufficient rapidity to be feasible. It must be admitted that attempts to specify in detail the nature of the memory trace in electrophysiologicalterms remain scarcely less speculative than his and are certainly not unobjectionable, by any means. Gaitonde and Richter (1956) and also Waelsch and Lajtha (1960) have similarly mooted the general proposition that protein-turnover may provide the organic basis for the storage of information, without, however, developing detailed speculations on the nature of the process. As regards the intimate enzymic mechanism of protein synthesis considerable progress has been made in recent years in elucidating its details. These details have been worked out on nonnervous tissues, especially mammalian liver (Zamecnik, 1959) and bacterial preparations (Gale, 1957) but such evidence as is available from less extensive studies on nervous tissue is compatible with the idea that the same general type of mechanism operates there also. It seems clear that, as with the fatty acids, the process of protein synthesis differs markedly from that concerned in their hydrolysis. It was for many years assumed that the production of proteins could

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be accounted for by a reversal of the familiar phenomenon of their digestion by proteolytic enzymes, yet repeated failure actually to demonstrate this, together with a growing awareness of the importance of energy-barriers in biochemical systems, have led to the recognition of the existence of a distinct biosynthetic pathway, in which energy-rich phosphate bonds and also the riucleic acids figure very prominently. An outline of a current scheme for the process of protein synthesis would regard amino acids as the immediate precursors and would exclude polypeptides as important intermediates. Each amino acid is activated by an enzyme specific to it by a process which involves the participation of adenosine triphosphate as an energy donor and results in the linkage of the amino acid with ribonucleic acid. When linked with ribonucleic acid, the amino acids are lined up appropriately so that, as they are released from the RNA, they unite to produce the amino acid sequence of the protein being synthesized. It is possible to conceive of codes by which the necessary information for selecting the amino acids in the correct sequence could be conveyed by the lineup of bases in nucleic acids, and in the case of deoxyribonucleic acid (which is found in the chromosomes), a mechanism for duplicating the coded information at the time of cell division has been proposed and is reasonably soundly based on experimental data (Crick et al., 1957). The concept of a deoxyribonucleic acid molecule as consisting of a double helix made up by cross-linkage between two complementary chains of nucleotides as proposed by Watson and Crick (1953) provides a workable mechanism for the transmission of genetic information at mitotic cell division. At this time the double helix may be visualized as uncoiling with the separation of its strands at one end, each strand then acting as a template for the formation from nucleotides in the medium of a new complementary partner. Thus, a helical particle of DNA of the daughter generation would consist of one strand inherited directly from the parent cell and one newlyformed strand and the observed distribution of isotopically labelled precursors incorporated into DNA during mitosis is in keeping with such a mechanism. Recent work on the synthesis of ribonucleic acid (RNA) in microorganisms has led to the recognition of a rapidly metabolizing fraction, now called “messenger RNA” ( Monod et al., 1962) which is thought to move between chromosomal DNA and the cytoplasmic RNA of the microsomes and to convey an imprint of the

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“instructions” coded in the DNA to the actual sites at which conjugation of amino acids to form protein is taking place. A graphic account of how this process may take place has been recently given by Hunvitz and Furth ( 1962). In the context of a discussion of protein turnover it may be appropriate to mention some findings of Geiger (1958) obtained by perfusing cats’ brains in situ with what he calls “simplified blood,” this being essentially a suspension of bovine erythrocytes in Ringer‘s fluid enriched with about 7% bovine albumen. It is found with this preparation that the brain will continue to consume oxygen and to exhibit electrical excitability for at least 1 hr. Subsequent analysis showed that about 40!% of the phospholipids and some 50% of the microsomes disappeared during an hour’s glucose-free perfusion, while free amino acids appeared in the effluent. It was found necessary in these experiments to maintain a more rapid flow of perfusate than was necessary in the presence of glucose and this was interpreted as being required to eliminate toxic products of metabolism. Secondly, it was shown that, when the brain was perfused with simplified blood containing glucose labelled with C14, some 60% of the glucose-carbon appeared to pass into compounds such as amino acids, only 30%being directIy converted to carbon dioxide. At the same time, the oxygen consumed by the perfused brain corresponded closely to that necessary to oxidize all the glucose taken up. Geiger, therefore, concludes that noncarbohydrate materials are oxidized simultaneously with glucose and part of the glucose taken up by the brain is used to resynthesize these compounds. Interpretation of CO, data in the whole animal when C1*-glucoseis metabolized is always complicated by the existence of pooIs not onIy of substrate and product but also of intermediates and by the differing rates of mixing in different organs. Thus, Coxon and Robinson (1959) demonstrated that the labelling of the expired CO, at any instant during which injected C14-glucosewas being metabolized was a resultant of intermingling of CO, of very variable radioactivity reaching the lungs from different regions of the body. Geiger’s cerebral perfusion system is admittedIy a great deal simpler than a compIete animal and the number of pools with divergent characteristics would be considerably less, so it is difficult to evade the inference that glucose was not being directly and exclusively oxidized. While it is uncertain to what extent conditions in the perfused brain differ from

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those in the normal (in particular the extent to which cellular compartmentalization is broken down), these observations do illustrate very forcibly the interplay of degradative and synthetic reactions. The question as to whether glucose is the exclusive “fuel” of brain according to classical ideas, hence, becomes in large part a matter of semantics. In the sense that, unless glucose is supplied in a quantity which will support the work of the brain the organ cannot maintain both its functional and structural integrity, glucose remains the brain’s unique fuel, since no natural substitute has been found. On the other hand, it would be naive at the present time to think of the glucose molecules as being exclusively converted to CO, and water; on the contrary, in the nervous system, as elsewhere, they enter a metabolic mixture from which a corresponding number of carbon atoms and hydrogen atoms are expelled as CO, and water, in each unit of time. Incidentally, the in &uo studies of Sacks (1956)and the in vitro experiments of Allweis et al. (1960) confirm that the immediate source of CO, in respiring brain can be material other than glucose. There are two respects in which the results obtained by studying the perfused cat brain are particularly relevant to discussion of protein synthesis in the organ. First, there is the question of the signscance of the appearance of amino acids in the perfusate when glucose is lacking. This might possibly result simply from a persistence of protein breakdown at a rate which was normal for the tissue but would be masked in the healthy brain by a continuous reincorporation of the liberated amino acids into fresh protein. In chemical terms, this would mean that the constant concentration of protein in nervous tissue is the result of an equilibrium state in which reactions leading to the synthesis and the breakdown, respectively, of protein are proceeding at equal rates. If the rate of the forward reaction falls, there will be an increase in the quantity of the precursors and if the velocity of backward reaction falls below the equilibrium velocity a buildup of products will take place. This may be formally represented as follows: Kl

Amino acids $ Protein Ka

(1)

where K , and K , are the rate constants of the forward and backward reactions. As indicated earlier (Sedion 111) this formulation is al-

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most certainly a gross oversimplification but in principle the argument is sound. Alternatively, the appearance of free amino acids in the eauent from a glucose-deficientperfused brain could be a consequence of the disintegration of some structural component within its constituent cells which, when intact, could-either by preventing the activation of certain intracellular enzymes capable of hydrolyzing proteins or by keeping such enzymes out of contact with their substrates or in some other way-ensure the preservation of the protein in a healthy brain. A second important result of the experiments of Geiger (1958) and his group on the metabolic activity of the perfused brain is their finding of a widespread distribution, among the chemical substances present in the brain, of radioactive carbon derived from glucose supplied to it. This brings into high relief the artificiality of too rigid a distinction between degradative reactions on the one hand and synthetic reactions on the other, The potentialities of the tricarboxlyic acid cycle as a synthetic pathway have been clearly recognized (see Krebs and Lowenstein, 1960; Weinman et al., 1957) and are inherent in the initial reaction of the cycle in which a 2carbon compound (acetyl coenzyme A ) is condensed with a 4-carbon compound (oxaloacetate) to yield citrate with 6 carbon atoms in its molecule. Amination of oxaloacetate or oxoglutarate among the cycle intermediates or of pyruvate arising via the glycolytic pathway will, of course, readily produce labelled amino acids from labelled glucose. Apart, therefore, from the coupling of synthetic with degradative reactions, which is necessitated by energy requirements, there is also an actual transfer of material, made possible by cross-over points in the sequences of metabolic reactions undergone by different types of substance. The fats and proteins which are the products of the biochemicaI reactions so far considered, despite the fact that some of them are undergoing moderately rapid renewal, may be classified as contributing, however ephemerally, to the structure of the central nervous system. Certain carbohydrates, of which the sugar moieties contained in nucleic acids and in galactolipids are examples, may also be designated as structural components of the CNS as may the amino sugars of the mysterious mucopolysaccharide “ground substance.’’ In addition to these, however, there occur in the brain small quantities of glycogen. In other organs, such as the liver and muscles

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where glycogen is a familiar feature, it provides labile stores of readily utilizable fuel for the rapid production of energy. Although the glycogen of the brain appears to be less labile than that in the liver and muscles, it does undergo renewal at a comparable rate and some studies bearing upon its turnover and synthesis will now be discussed. V. Glycogen Turnover in Brain

What may be termed the ‘‘classical” studies on cerebral glycogen were carried out by Kerr (1936, 1938), who isolated and unequivocally identified this substance in extracts of canine brain and, with his co-worker (Kerr and Ghantus, 1936, 1937), also determined its concentration in the brain of a number of other animal species. Kerr and Ghantus (1936) found in the brains of a randomly selected group of fed dogs a mean concentration of glycogen of 98 mg/100 gm,the individual values ranging from 77-130 mg/100 gm. Fasting appeared to increase initially the cerebral glycogen if it achieved anything; a high carbohydrate diet or the infusion of glucose for 40 min (with or without insulin) similarly produced no striking changes. Pancreatectomy was also without marked effect on cerebral glycogen, and depletion of the liver glycogen to about 50 mg/100 gm might occur without any noticeable change in its concentration in the brain. Similarly, phlorhizinized animals with blood-sugar levels in the region of 50 mg/100 ml had normal quantities of glycogen in their brains. Insulin, however, in convulsant or near convulsant doses did produce a considerable fall in cerebral glycogen levels. The lowest value observed was 30 mg/100 gm which was found in an animal ( a male rabbit) killed when the blood-sugar level was only 8 mg/100 ml after the administration of 20 units insulin/kg body weight ( presumably by subcutaneous injection) 2 hr previously. Kerr and Ghantus point out that Cori and Cori (1928) had shown the lowering of muscle glycogen following insulin to be a secondary effect of the compensatory output of adrenalin, but they regard this as an unlikely explanation in brain since, in their own experiments, adrenalin in combination with phlorhizin did not prove effectivein reproducing the action of insulin, The overall impression created by the work of Kerr and Ghantus was that the glycogen formed a relatively stable constituent of brain. However, Kerr and Ghantus (1937) also studied the rate of disappearance of glycogen

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from brain tissue post mortem and found that, unless the estimation was carried out really quickly after fixation, very low values were obtained, and there was a corresponding increase in lactate content. This clearly points to a very active mechanism for the destruction of glycogen by brain tissue, Hence, maintenance of the normal steady concentration found during life must imply either some constraint upon the system responsible for the postmortem decomposition or else the operation of a synthetic mechanism capable of effecting replacement of glycogen in the brain at a rate identical with that of its breakdown. More recent authors have concerned themselves with the glycogen metabolism of isolated brain slices rather than of the whole organ in aitu. Thus, LeBaron (1955) demonstrated that when slices of guinea pig brain were incubated in buffered saline containing glucose, synthesis of glycogen proceeded to the extent of some 18 mg/100 gm braidhr and that the final content of the slices was of the same order as that found in the brains of other species when these were rapidly removed from the living animal. LeBaron also showed that the synthetic process was independent of the phosphate concentration of the medium (though, of course, the dices contained some phosphate) but it was inhibited by the presence of dinitrophenol. The absence of influence by phosphate concentration together with an inhibitory action of dinitrophenol suggest that the buildup of glycogen requires high-energy phosphate esters rather than inorganic phosphate for its accomplishment. High concentrations of potassium, which have been found to promote the synthesis of glycogen in other tissues (cf. Buchanan et al., 1949) had very little effect. The maximal rates of glycogen synthesis in LeBaron’s experiments with brain were-it should be noted-only about onetenth of those reported in liver (Ostern et al., 1939) and diaphragm (Stadie and Zapp, 1947) and were appreciably less than the rate of autolytic decomposition of glycogen observed in brain post mortem (6.Chance and Yaxley, 1950). Kleinzeller and Rybova (1957) have also carried out studies on glycogen synthesis in brain slices and report that increasing concentration of potassium in the medium not only fails to stimulate the process but actually depresses it when a level of 40 meq/liter is exceeded. The rate of glycogen synthesis was also reduced by the addition of glutamic and aspartic acids. The effect of potassium was attributed by Kleinzeller et al. to its known influence (see Ashford

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and Dixon, 1935; Dickens and Greville, 1935) on the aerobic breakdown of polysaccharides. A related effect of potassium on cerebral glycogen was reported by Givinek (1958) in uiuo. This worker noted that the application of a 2%solution of KCI to the surface of the cerebral cortex of rats was accompanied both by a diminution in its glycogen content of some 15%and also by a wave of “spreading depression” in the electroencephalogram. It is also claimed that spreading depression evoked by mechanical percussion of the rat’s cortex was accompanied by a similar diminution (approx 25%)in cerebral glycogen content. The possibility cannot be excluded that the mechanical trauma affected the K+distribution across the cortical neurons. It may be relevant to these results that Heald (1960) has shown in vitm that raised K+in the medium leads to the breakdown of creatine phosphate. There appears also to be some correlation between the inhibition of conditioned reflexes ( KiivLnek, 1958) and the transitory fall in cerebral glycogen which follows potassiuminduced spreading depression. The restitution of the glycogen to normal after such an episode of depression appears to require something like 3 hr. Comparatively few studies of the turnover of cerebral glycogen in the intact animal have been reported. However, Palladin (1955) quotes Prokhorova (1954) as having found under certain conditions a rate of renewal equal to or greater than that found in liver. Prokhorova et al. (1957) also report that following the injection of C’” glucose the cerebral glycogen became radioactive and that its specific activity reached a maximum 1-2 hr following the administration of the isotope. They further demonstrated that the production of methemoglobinemia (and hence of hypoxia) by means of injections of sodium nitrite led not only to a decrease in the concentration of glycogen in the brain but also to a reduction in the extent of incorporation into it of C14 from glucose; this would appear to indicate a breakdown of the polysaccharide without replacement at the normal rate. These conclusions are based upon comparisons of radioactivity at intervals of % and 1 hour after the injection of isotope. The ratio of specific activity of the glycogen to that of the brain glucose was found to be one-third after an unspecified interval of time, and it is suggested that the turnover time of cerebral glycogen in the normal rat is, generally, of the order of 3 hr. This may be only a rough approximation since it ignores variations in the rate of change of the specific activity of both glucose and glycogen and

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also assumes a unique pathway for incorporation of C” from glucose into glycogen in the brain. One of the rare reports of increase above normal of the cerebral glycogen content is that of Bronovitskaya and Shapovalova (1957) who found that exposure of rabbits to raised oxygen tension produced such a change. Exposure for 1 hr to 4 atm of 0,resulted in a 50%rise in the cerebral glycogen. These authors cite Prokhorova (1954) as having found that the rate of renewal of the “total carbohydrate’’ of brain was less than the renewal rate for glycogen by 40 or more times. Timiras et al. (1956) found a rise of some 60%in the brain glycogen of rats treated with cortisol, and it is conceivable that the results of oxygen at high atmospheric pressures may be related to the accompanying stress and adrenocortical activation, although the latter is more commonly considered a concomitant of hypoxia rather than the reverse. In the writer’s laboratory, the incorporation of isotopic carbon into the cerebral glycogen of rabbits has been studied following the intravenous injection of several labelled substrates (Coxon and Henderson, 1958; Henderson, 1959). It was found (as indicated in Fig. 2) that, following that administration of uniformly labelled gluc u m

1400

Brain

ou -

o

7-

W

Muscle

c-

0

0‘ 750

9,

G

E

\

E o.

U

0

1 0

5 Hours

FIG.2. Time course of incorporation of C“ from uniformly labelled glucose into cerebral glycogen of rabbits. The curve shows radioactivity of glycogen at intervals after injection of 12 pc radioactive glucose. Some values for muscle are included for comparison (data of Coxon and Henderson, 1958).

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cose, the radioactivity of the cerebral glycogen increased slowly but steadily over the first 5 hr, after which a much steeper rise occurred during the succeeding 4 hours and was followed by a period, beginning at the ninth hour, when the values tended to rise only very gradually. From the first hour onwards, the glycogen of the brain was much more heavily labelled than that of a limb muscle and it is important to note in this connection that during the experimental period the rabbit was free to move about in the respiration chamber. It has since been demonstrated (Gordon-Smith and Coxon, 1961) that after the twelfth hour the specific radioactivity of cerebral glycogen derived from the injected C14-glucosebegins to decline. The general time course of the labelling is not greatly dissimilar from that found in some studies of the hepatic glycogen of rats (Stetten et al., 1954). Two further types of experiment have been carried out (Henderson, 1959) in order to examine some characteristics (directness, etc.) of the pathway by which the carbon of blood glucose is incorporated into glycogen. These experiments disclosed that, if radioactive carbon was supplied in the form either of bicarbonate or uniformly labelled glutamate, the extent of labelling of the cerebral glycogen sampled 5 hr after the injection was comparable to that observed after an equal dose of C14 given as glucose; and it is noteworthy that the glycogen of muscle did not become labelled from intravenous C14bicarbonate. These results suggest strongly that incorporation of carbon from glucose into glycogen in the rabbit's brain may take place through a series of intermediates and not directly and also that the cerebral glycogen in a conscious, unrestrained rabbit is being renewed more rapidly than that in its skeletal muscles. The problem of distribution amongst the cell types present arises no less with glycogen than with other products of biosynthetic reactions in brain. Of the histochemical studies designed to elucidate this point, that of Schimizu and Kumamato (1952) is amongst the most illuminating to have been traced. These writers studied the cellular localization of glycogen in the brains of rats and they controlled the specificity of their staining reaction by comparing sections prepared before and after exposure to ptyalin, which should hydrolyze glycogen but leave unaffected other carbohydrate materials such as those found in ground substances of the CNS. It appears from the results of Schimizu and Kumamato that glycogen can be located in

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both neuroglial and neuronal cells. In what precise state the polysaccharide is present, is open to question not only in regard to the brain but also in regard to other tissues. Stetten and Stetten (1960) take the view on this point that too little is known of the possible secondary structure of glycogen and its binding to various types of compounds to permit definite answers. Electron microscopic studies of glycogen isolated in particulate form by fractional centrifugation from guinea pig livers have been reported by Bondareff ( 1957), who recognizes 3 types of particle, distinguishable according to size. The smdlest granules are about 130 A in diameter and, in his view, probably correspond to the central cores of glycogen molecules. Larger particles of 600-1500 A are also visible and probably correspond to the structures isolated by earlier centrifugation studies, such as those of Claude (1954) and Lazarow (1942). Finally, there are larger aggregates of between 3OOO and 9OOO A in diameter which correspond to the granules familiar in stained sections viewed by light microscopy. Comparable studies on brain glycogen have not yet been reported and would, in all probability, prove technically difficult owing to the relatively small quantities present and the large variety of other material from which it would have to be separated. In terms of molecular weight, glycogen isolated from liver is markedly inhomogeneous, varying from 6 X 106--80 X lo6.Glycogen from muscle and liver is also metabolically inhomogeneous; in the case of that from liver, the smaller molecules must be more rapidly renewed that the larger since they take up radioactive carbon from glucose more quickly (Stetten et al., 1956). Curiously enough, the reverse appears to hold for muscle glycogen in respect of this particular characteristic but the behavior of cerebral glycogen in this regard does not appear to have been described so far. A somewhat different type of metabolic inhomogeneity has also been reported by Stetten and his co-workers (Stetten and Stetten, 1955) on the basis of experiments in which the glycogen of liver and muscle was subjected to an alternating succession of treatments with two enzymes, phosphorylase and amylase, which are chosen so as to attack the glycogen molecule at different loci. By this sort of “chemical dissection” of the glycogen, the radioactivity of different portions of the molecule synthesized from a radioactive precursor can be separately determined; examination of the results of such studies indicates that glucose moieties lying superficially in the macromolec-

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ular aggregate exchanged more readily with free glucose in the environment that did the glucose moieties lying more deeply imbedded. This picture seems to apply equally well to glycogen of both muscular and hepatic origin. If similar considerations apply to cerebral glycogen they will clearly affect the interpretation of data on the time course of incorporation of labelled atoms into its structure. Independent evidence of the inhomogeneity of glycogen of extracerebral origin comes from reports indicating that some fractions of tissue glycogen are more readily extracted than others, such as that of Willstatter and Rohdewald (1934), although there has been considerable discussion regarding the significance of such observations. The solubility of glycogen has been known for some time (cf. Kerly, 1930) to be altered by treatment with certain reagents, such as caustic potash, which are commonly used in isolating it from brain. Whatever uncertainties may remain as regards the molecular structure of tissue glycogen in general and cerebral glycogen in particular, the studies which have been quoted on the time course of incorporation of radioactive carbon into the latter leave no doubt that it is subject to a rather rapid turnover; in other words, a proportion of it is being continuously synthesized. The question then arises as to the chemical mechanism by which this synthesis is achieved. Reference has already been made in connection with the fatty acids and proteins to the frequent finding that, when a compound can be alternately deposited in and mobilized from a cell, synthesis and degradation tend to take place via different biochemical mechanisms. This rule seems to hold also for glycogen where there is a relationship with phosphate compounds involved in both synthesis and depolymerization. Thus, while glycogen seems to be degraded through conversion to glucose-1-phosphate by phosphorylase (as described by the Coris in 1937 using H,PO, as phosphate-donor) it now appears that the synthetic pathway must require the participation of uridine triphosphate. The utilization of uridine-phosphoglucose to form glycogen was discovered by Leloir and Cardini (1957) in liver but has also been shown to occur in brain by Breckenridge and Crawford (1959), who found that cerebral preparations from sheep and rabbits were capable of producing glycogen at the rate of approximately 100 mgJlOO gm brain/hr. The interrelationship of the mechanisms concerned with the buildup and break-

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down of glycogen has been schematized by Stetten and Stetten (1960) in the manner shown in Fig. 3. glycogen

UTP

d

FIG.3. Schema of the reactions concerned in the enzymic synthesis and degradation of glycogen (After Stetten and Stetten, 1960). UDP and UTP: uridine-diphosphate and uridine-triphosphate, respectively. UDPG: uridine-diphospho-glucose. -P: high-energy phosphate. PP: inorganic pyrophosphate. Pi: inorganic orthophosphate. c: UDPG-pyrophosphorylase. d: UDPG-glycogentransferase. e: UDP-kinase. f: phosphorylase. Solid-headed arrows indicate the likely directions of the predominant reactions.

Since uridine diphosphogalactose has been shown to act as an agent for incorporating galactose into cerebrosides in preparations of brain from albino rats (Burton et al., 1958), and since uridine nucleotides have been isolated from rat brain (Lolley et al., 1961) in quantities of the order of 0.05 pmoles/gm, it seems highly probable that the glycogen synthesis which has been observed in living animals takes place through the intervention of the uridine nucleotides. It is interesting to recall in this connection that Geiger and Yamasaki (1956) found that deterioration in the ability of the perfused cat brain to maintain its levels of phospholipid and to oxidize glucose was averted if uridine or cytidine were added to the bathing fluid, since the former is concerned in the incorporation both of glucose into glycogen and of galactose into cerebrosides, while the latter is a cofactor in the synthesis of phosphatides. It was noted early on in this account that insulin-induced hypoglycemia depletes the cerebral glycogen stores, and in connection with the general search for links between test-tube biochemistry and physiological events it is apposite to inquire into a possible correlation between the mobilization of reserves and the time of onset of functional failure of the brain following the withdrawal of the supply of glucose from the blood. From figures quoted by McIlwain

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(1959) it appears that mammalian brain contains about 20 mg/100 gm glucose and about 100 mg glyCogen/100 gm of which about 70 mg is probably mobilizable during hypoglycemia. Since the normal rate of glucose utilization is of the order of 6 mg/min/100 gm brain, the reserves might be expected to sustain activity for some 15 min. This argument assumes that utilization rate would be unaffected by the glucose concentration in the cells; in fact, cerebral utilization of glucose goes down as the blood sugar falls so that some utilization could probably continue for approximately 20 min but may not proceed at a high enough level to maintain satisfactory mental functioning nor, indeed, structural integrity in the nerve cells, for, as is well known, prolonged severe hypoglycemia may lead to irreversible brain damage in situ just as glucose withdrawal leads to loss of intracellular organelles in the perfused brain. VI. Conclusion

It was mentioned early in this review that the biosynthetic activities of highly differentiated cells must include certain rather specific mechanisms for the elaboration of compounds peculiar to these cells in addition to more general types of reaction sequences common to all living cells. The further generalization was made that on evolutionary grounds even the more specific processes might be expected to have certain affinities with chemical mechanisms found in other cells. It is, therefore, interesting to note that the enzyme system concerned in the production of the specific transmitter substance released by cholinergic neurons, choline acetylase, has been identiiied in nonnervous structures like the placenta and also in certain microorganisms (Hebb, 1957). Like other biosynthetic processes previously discussed, the production of acetylcholine depends upon phosphate-bond energy and coenzyme A. Acetylcholine has been designated (Burn, 1950) as a local hormone but another secretion of nervous tissue which has much more widespread humoral actions is vasopressin. A beginning has been made recently in the study of the biosynthesis of this hormone by Sachs (1961) who has isolated the material in a radioactive state after the administration to dogs of S35-labelledcystine. The chemistry of vasopressin is well understood from the classical studies by Du Vigneaud (1954) and his co-workers; the investigation of the mode of production of the compound derives additional and topical importance from its asso-

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ciation with the phenomenon of neurosecretion. Yet, though its details are not yet elucidated, neurosecretion need not be regarded as more than a special case of the secretory activity observed in many cell types at all levels of biological organization. In the case of those types of substance discussed in this review, their production is certainly not confined to neural cells. There is no reason to suppose that cerebral glycogen is formed by mechanisms markedly different from those encountered in liver and muscle, but the regulation of its concentration does appear to be differently effected. So far as proteins are concerned, no new principle need be introduced into the picture derived from studies of other cells but it is evident that in some way the “instructions” conveyed to the RNA of neurons must differ from those of the RNA of, for example, osteoblasts, although both are derived from the same fertilized ovum. In the case of the fatty acids, what is peculiar to nervous tissue is the subsequent metabolic fate of the carbon chains after their production through mechanisms not in any way specific to its constituent cells. Now that the basic and relatively nonspecific mechanisms have been uncovered, it is likely that progress will be steady in the elucidation of the more specific biosynthetic activities of neural and glial cells, although the exceptional chemical complexity of some of the components of the nervous system, such as myelin, will present formidable obstacles (see Folch et al., 1959). In touching briefly on the functional implications of the varied and vigorous biosynthetic activity demonstrable in neural cells, a quotation from Weiss (1950) seems especially apt. Commenting on the results of investigation of the life history of neurons, he notes that data obtained by morphological and biochemical methods are in good agreement and asserts that such studies indicate that “the mass of the neurone as expressed in the size of the cell body and the caliber of the axon is not a fixed static character but represents a steady-state equilibrium between continuous growth and concurrent degradation.” He pictures the production of “neuroplasm” in the neighborhood of the cell nucleus by the occurrence of protein synthesis in that vicinity followed by transport of the newly produced protoplasm peripherally along the axon, where it is then available for increase in length and width of the fiber. Evidence for the proximodistal movement of cell contents is provided by Weiss’s own well-known experiments (see Weiss and Hiscoe, 1948) in which

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axons of a nerve trunk were artificially constricted and it was subsequently demonstrated that axoplasm accumulated and distended the fibers behind the constriction. These observations were made on mature nerve fibers and it thus appears that the process by which the substance of a neuron is renewed must be controlled ultimately by the rate of production of protoplasm in the cell body but can be iduenced by conditions at the periphery, e.g., by section of the axon. In view of the intimate relationship between neurons and neuroglia and the claim that neuroglia may fulfill some adjuvant role in the nutrition of the neurons it is interesting to inquire whether the new formation in embryonic life or the regeneration of axons in maturity can take place in neurons isolated from neuroglia. There is a suggestion that in tissue culture the former process can occur. Gerard (1950) states that a regenerating neuron in some circumstances may synthesize several times its own volume of protoplasm per day at a time when it is reconstituting its axon, which in some cases may contain 1000 times as much material as the cell body. Some measurements of the rate of incorporation of C14-labelled cystine into the protein of the sciatic nerve of frogs ( Waelsch, 1958) supports the concept of a centripetal migration of protein along the axon since there is a wave of increasing protein radioactivity which passes distally. Some measurements of the appearance of enzymic activity at different points along a degenerating nerve are also consistent with the idea that the enzyme protein is synthesized in the cell body and migrates distally (see Hebb, 1957), although there are indications that cholinesterase may not behave in this way (Koenig and Koelle, 1961). Waelsch (1960) has put this concept into morphological terms by envisaging the possibility that in neurons “the membranes of endoplasmic reticulum are enlarged by their biosynthetic processes and that the force behind axonal flow is the growth of the membranes of the reticulum, channelled into the nerve axon.” Hydhn and Pigon (1960) have stated that “it seems reasonable to conclude, on the basis of data available, that the large neuron is an enormous gland-like structure, fulfilling its function as a steady and rapid producer of ribonucleic acid, proteins, and lipids.” While many neurophysiologists, accustomed to studying the electrical events in the CNS, would probably regard this as a very incomplete description of a nerve cell, it is, nonetheIess, a not altogether unfair

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one. In the extreme case of the neurosecretory cells of the hypothalamus, the microscopic picture is very similar to that of an exocrine gland (cf. Bargman, 1960) but in all neurons the end result of activation is probably the release of a “transmitter” substance into the extracellular fluid. In some instances the chemical nature of the transmitter is familiar enough as in the cases of the voluntary myoneural junction and the synapses of the sympathetic ganglia; in other situations, its nature is either speculative or wholly mysterious. However, in the case of the well-established transmitter acetylcholine, its site of formation appears to be indubitably witbin the cell from which it is released during the transmission of an impulse and it is to that extent an exocrine secretion from the neuron. Over and above the elaboration of this “external secretion,” the biosynthetic processes in a nerve cell, insofar as they determine its growth, must also control its functional characteristics, for, as Weiss (1950) has commented, “with the size of a nerve-fibre are correlated such physiological properties as threshold, sensitivity and conduction velocity.” Since the speed of saltatory conduction (see Stampfli, 1954) depends on myelination and since myelination in peripheral nerve is held to depend on the Schwann cells and in the CNS on oligodendroglia, it is evident that the biosynthetic activities of these cells must also contribute very significantly to the conducting function of their associated neurons, by which presumably their activities are in turn governed since the myelin disintegrates by Wallerian degeneration distal to a nerve section. Under any given conditions, the size of a nerve fiber is likely to remain effectively static; however, some of its constituents will be undergoing constant renewal and of this dynamic condition Weiss (1950)goes on to say: “Above all, the realization that the neurone is a system in a state of flux must have a profound influence upon our thinking about the integrative action of the nervous system including the phenomena of learning and memory which presuppose a certain amount of plasticity of the underlying substratum.” In addition to the possibility of minor changes within its substance as a byproduct of the dynamic state of the chemical structure of a neuron, Buller et al. (1960) have been led to postulate a “trophic” substance of a specific nature affecting the constitution of the muscle fibers innervated by the neuron. Such a postulate was needed to explain the dramatic results of some crossunion experiments in which muscles which normally exhibit the

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properties of “fast” contraction were converted into slow contractors when artificially joined to a nerve normally supplying a slow contractor. In man, it is likely (Burns, 1958) that the number of neurons in the cerebral cortex remains constant between birth and the age of 20 yr but between the twenty-first and seventieth years of life neurons are believed to perish at the rate of about 100,000per day. After birth, neurons of the CNS of mammals appear to have lost the power to reproduce by fission since no multiplication can be observed either during bodily maturation or following injury. They have, however, retained in a marked degree two other fundamental characteristics of living cells, namely excitability and capacity for growth, with which in biochemical terms may be included the capacity constantly to renew their substance. It would be exceedingly interesting and possibly instructive to discover why, with the passage of time, some neurons more readily lose the ability to adjust their biosynthetic activities to the decay rate of their constituents and so perish before their neighbors. hERENCES

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BIOLOGICAL ASPECTS OF ELECTROGOtNVULSIVE THERAPY By Gunnar Holmberg Psychiatric Clinic, Centrallararettet, Donderyd, Sweden, and Karolinsko Inrtitutet Medical School, Stockholm, Sweden

I. 11. 111. IV.

V. VI. VII. VIII. IX. X.

Elicitation of Therapeutic Convulsions . . . . . . Modifications of Treatment . . . . . . . . . Physiological Effects of ECT . . . . . . . . . Endocrine and Biochemical Changes Associated with ECT , Clinical Effects of ECT . . . . . . . . . . Psychological Effects of ECT . . . . . . . . . Physiopathology and Neuropathology of ECT . . . . Complications of ECT . . . . . . . . . . . Prognostic Test Procedures and ECT Mode of Action of ECT and Its Relation to Other Therapies References . . . . . . . . . . . . . .

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1. Elicitation of Therapeutic Convulsions

As early as the latter part of the eighteenth century, convulsions induced by large doses of camphor were used in the treatment of mental disorders (Oliver, 1785). It was not, however, until the 1930’s that this mode of treatment was revived by Meduna (1935), who more recently has published a comprehensive review of the development of convulsive therapy from camphor to Metrazol and its initial use in the treatment of schizophrenia (Meduna, 1956). In 1938, Cerletti and Bini advanced the technique of producing therapeutic convulsions by passing an electric current through the head. Animal experiments had earlier shown that this was a safe method of producing convulsions ( see Cerletti, 1956 1. Since then, the principal method of producing therapeutic convulsions has, for practical reasons, been by electric shock. In several clinics, however, chemical agents are still used, chiefly Metrazol, which is claimed to be more effective than electroshock by several 389

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workers (Zeifert, 1941; Meduna, 1956; Komora and Padula, 1957; Sicheneder, 1958). A convulsant drug combined with photic stimulation has been reported to be effective, provided grand ma1 seizures are evoked (Gastaut, 1950; Ulett et al., 1956). Other chemical agents used in convulsive therapy include megaphen ( chlorpromazine) (Lieser, 1956), hydrazides (Pfeiffer et al., 1956), hexafluorodiethyl ether (Esquibel et al., 1958; Impastato et al., 1960), and bemegride ( Ewalds, 1958). Combined Coramine (nikethamide) electroshock therapy has also been used (Fabing, 1948; Lighthart et al., 1956). The possibilities of varying the application of electrical currents to the brain have aroused keen interest. An important improvement would seem to be the modification of the original sine-wave stimulation into rectangular or other steep-wave unidirectional currents, which reduces the amount of current necessary to produce a convulsion (Friedman, 1942; Liberson, 1944; Strauss and McPhail, 1946; von Braunmiihl, 1951). A stimulus duration of 0.1-0.3 msec has been reported to be the most effective (Liberson, 1945; Beek and Stuart, 1953), while the optimal stimulus frequency has been variously given as 10 cps (Beek and Stuart, 1953) and 120-150 cps (Liberson, 1945). More recently, a stimulus duration of 0.7 msec and an interpulse interval of 1 msec has been suggested (Hovorka et al., 1960), values which closely approximate those found to induce minimal depression of behavior in animals ( Hovorka, 1958). The site of application of currents to the head has varied considerably (Gottesfeld et al., 1944; Friedman, 1958), but the “classical” bilateral temporofrontal electrode placement is that generally employed ( Hemphill and Walter, 1941). II. Modifications of Treatment

Originally, the sole aim was to produce an epileptic seizure but modified forms of electroconvulsive therapy (ECT), such as subconvulsive stimulation ( Gottesfeld et al., 1944; Alexander, 1950; Hirschfeld and Bell, 1951) and “electrocoma” with sustained stimulation (Leduc, 1902; van Harreveld and Kok, 1934; Frostig et al., 1944) have been reported and are used along with the orthodox type of convulsive treatment. However, subconvulsive stimulation has little or no effect in depressive cases, and the effect of electrocoma is no better than routine ECT, though the risks are greater.

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The convulsive phenomena of routine ECT can be moderated by inducing the seizure slowly by the “glissandoy’technique and sustaining an even tonic tension, with low-voltage stimulation, throughout the clonic stage (Tietz et al., 1949). This may somewhat reduce the risk of fractures. Although it is known that a low oxygen and high carbon dioxide level, a low blood sugar level, and reduced cerebral circulation will mitigate convulsions ( Holmberg, 1955), it is difficult to utilize these factors in clinical work. Drugs with central or peripheral actions are frequently used for this purpose. Sedative and anticonvulsive drugs reduce the intensity of the cerebral epileptic phenomena and thereby suppress the motor phenomena as well (Impastato et al., 1943; Tietz et al., 1949; Holt and Borkowski, 1951; Kerman, 1958). Bennett (1940) early started using curare in conjunction with ECT and reported excellent results. Curare preparations, however, produce prolonged apnea and may give rise to several complications (Montagu, 1953); this has limited its use in ECT. In fact, many fatalities have been caused by this drug (Impastato, 1957). Curare should evidently not be employed routinely by psychiatrists unskilled in anesthesiology. Several other neuromuscular blocking agents have been used with somewhat better results (Montagu, 1953). At present, succinylcholine preparations provide the greatest potentialities for muscle relaxation in ECT, producing a brief but effective neuromuscular block with no apparent side effects (Holmberg and Thesleff, 1952; Alexander, 1958). The use of this drug has practically eliminated fractures and other mechanical lesions. In rare cases, the postconvulsive apnea may be prolonged, owing to lack of cholinesterase in the body, but this hazard is eliminated by giving artificial respiration ( Montagu, 1953). Ill. Physiological Effects of ECT

A grand mal seizure induced by electric shock consists of an initial tension or jerk, caused by direct cortical stimulation, followed by a latent period of varying duration-inversely related to the efficacy of the current dosage (Toman et al., 1948)--and then by tonic and clonic convulsions. This is seen on inspection (Kalinowsky and Hoch, 1952) or in the electromyogram (Holmberg, 1953a). In the electroencephalogram, the tonic phase is characterized by a generalized intensive spike activity. During the clonic phase, there

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is spike-wave type of activity which is not synchronous with the clonic twitchings. The latter coincide with the electrical discharges in the brain stem (Jung, 1949). EEG studies during the convulsions can readily be done with the aid of muscle relaxing drugs (Piekenbrock et al., 1956; Piette, 1958). Immediately after the convulsion, the EEG shows a brief period of electrical silence, followed by a gradual return of activity which is at first sporadic and slow but later assumes its preconvulsive pattern. The application of the current to the brain, even in doses far below the minimum convulsive dose, produces immediate loss of consciousness (Impastato and Gabriel, 1957). The muscle reflexes are generally impaired during and immediately after the convulsion. ECT seems particularly to effect the telencephalic functions, and the recovery of normal reflex activity takes place in the caudo-oral direction ( MolnAr et al., 1956). During the convulsion, respiration is suspended, owing to spasm of the respiratory muscles and glottis which, together with maximal muscular and neuronal activity, produces an elevation of the blood carbon-dioxide tension and a considerable reduction of the oxygen tension (Altschule et al., 1947). The anoxia may be diminished by saturating the oxygen depots immediately before the treatment, and premedication with a muscle relaxant and oxygen insufflation will easily eliminate any asphyxia ( Holmberg, 1953b). This should guard against one of the more serious risks of ECT. It must be difficult, however, to completely eliminate cerebral tissue anoxia and hypercapnia during induced convulsions (Davis et al., 1944; Meyer and Gotoh, M O ) , even when peripheral conditions are perfect, since the increase in the neuronal activity of the brain cannot be met by a corresponding increase in the cerebral blood flow (Jasper and Erickson, 1941) . The central and peripheral events during convulsions cause considerable impairment of the general circulation (Perrin, 1961). The heart rate is often rapid and irregular (Bellet et al., 1941; Bankhead et al., 1950) with marked fluctuations in blood pressure (Silfverskiold and Amark, 1943; Brown et al., 1953b; Holmberg et al., 1954) , Premedication with atropine or other acetylchohe blocking agents will usually prevent irregularity of the heart rate and eliminate asystole (Hejtmancik et al., 1949). The increase in arterial pressure can be controlled, but not eliminated, by muscle relaxants;

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the heart rate is reduced and the venous pressure normalized when the Valsalva effect is abolished by muscle relaxants (Holmberg d al., 1954). The cerebral circulation is greatly increased during the convulsion (Brown d al., 1953a,b), but this does not fully meet the demands of the enormously increased brain metabolism (Jasper and Erickson, 1941) , which is the main reason for early termination of the convulsion ( Holmberg et al., 1955). The increase in salivary and bronchial secretion associated with induced convulsions can be counteracted by premedication with anticholinergic drugs ( Toman et al., 1948). ECT produces a variety of autonomic changes (Delay, 1948; Lunn, 1951), apparently due to involvement of autonomic regulatory centers in the epileptic discharge. Such autonomic changes, occasionally accompanied by more or less severe psychomotor restlessness, may predominate for some hours after the convulsion. Premedication with a short-action barbiturate will considerably diminish the postconvulsive restlessness and is also necessary to permit administration of a muscle relaxant ( Holmberg and Thesleff, 1952). Autonomic nervous system changes towards a greater sensitivity to epinephrine occur during the course of treatment (Funkenstein et al., 1948). IV. Endocrine and Biochemical Changes Associated with ECT

ECT produces a number of biochemical and hormonal changes (Kalinowsky and Hoch, 1961). A constant phenomenon is hyperglycemia of one to several hours duration (Gour and Bhargava, 1957), as well as hyperproteinemia and an increase in nitrogen compounds and serum potassium. The calcium and phosphorus content of the blood is likewise increased (Flach et d.,1960). Premedication with a barbiturate will eliminate blood sugar increase (Hann, 1958). An increase in blood pressure and cardiac rate-not eliminated by curarization, oxygenation, and barbiturate narcosis-and pupillary dilatation indicate a central sympathetic stimulation. An increase of catecholamines in the blood and urine has been demonstrated ( Weil-Malherbe, 1955; Sourkes et al., 1958). Since, however, the release of catecholamines is suppressed by barbiturates and muscle relaxants, it seems that this peripheral (adrenal) effect has nothing to do with the therapeutic action of ECT (Havens et al., 1959). ECT produces a peripheral liberation of serotonin connected with an antidiuretic effect (Valsecchi and Valzelli, 1957).

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The white blood cell count also undergoes characteristic changes (Kalinowsky and Hoch, 1952; Salde, 1952). This, together with the changes found in the fluid and electrolyte balance (Russell, 1960), indicates that ECT has a stimulatory effect upon the pituitaryadrenocortical system. Steroid excretion is increased after treatment (Ashby, 1949; Bliss et al., 1954). Guze et d. (1957) reported that in a depressed patient who had had bilateral adrenalectomy, ECT produced no increase in steroid production; this did not prevent a satisfactory effect on the depression. Menstrual disturbances, unrelated to the therapeutic effect, are common (Michael, 1956). Changes in body weight (Russell, 1960), sleep variations, and the aforementioned autonomic and hormonal changes lead to the conclusion that ECT stimulates the entire diencephalic-pituitary system. None of the more peripheral effects have been seriously claimed to be a necessary factor for the therapeutic efficacy of ECT, while a certain minimum of central stimulation appears to be a prerequisite (Roth et al., 1957). Transaminase levels in the serum are considerably elevated by ECT (Sanguinetti et al., 1958); the significance of this is unclear. The cerebrospinal-fluid enzyme activity is also increased, despite no major increase in permeability of the blood-cerebrospinal fluid barrier to these enzymes (Lending et al., 1959). Altschule (1959) reported an increase of the blood carbonic-anhydrase activity in most patients. He states that the violence and frequency of the convulsions determined whether or not an increase occurred but that these changes are not the cause of the therapeutic effect of ECT. The immediate biochemical changes in the brain associated with electric stimulation and epileptic discharge are too many to be recounted here. An interesting observation is that electrically induced convulsions increase the serotonin level in the brain (Poloni, 1956) and particularly in the brainstem (Breitner et al., 1961) but produce no change in brain amine-oxidase activity (Spilman and Badal, 1960). The increase in the serotonin level does not appear to be related to the intensity of the convulsions but rather to the fact that an electric stimulus has been given, It is also related to the duration of the sleep period after pentobarbital (Garattini et al., 1960). These effects may, however, be the result of increased permeability of the blood-brain barrier (cf. Weil-Malherbe, 1955; Clemedson et al., 1958; Rosenblatt et al., 1960). Potent substances, capable of

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producing excitatory or inhibitory effects, are present in the brain and their amounts are altered by electrically induced convulsions (Badal and Spilman, 1961); cf. the “acroagonin” concept of Cerletti (1956). The cholinesterase activity in the blood and nervous tissue is not increased (De Car0 and Altea, 1950). V. Clinical Effects of ECT

Convulsive therapy was initially used in schizophrenic patients but was later found to be of greatest efficacy in depressed patients (Verstraeten, 1937). It is now generally agreed that ECT is a most efficient tool in the treatment of depressive states. A series of treatments, varying in number from Z 8 or 10, usually 4 or 5, is needed to cure an endogenous depression. The probability of a complete cure is mostly reported to be between 80 and 90%, but higher and lower figures have been given. It is evident that ECT has a better effect on “pure” depressive syndromes, and that the presence of atypical traits, such as neurosis, schizophrenia, and organic disturbances, tends to give a poorer prognosis. One of the most favorable prognostic signs is the presence of rhythmic changes during the course of the day, i.e., early waking with anxiety and inhibition, and clearing up of the symptoms towards the evening with relative ease in falling asleep ( Sargant and Slater, 1954). Inhibition, even in the absence of severe depression, seems to be the symptom most frequently ameliorated by convulsive therapy. In depressed patients, ECT is frequently followed by a hypomanic reaction of a few days’ duration, which, in my experience, is benign and merely indicates that a complete and definitive cure has been obtained. Involutional depression, even in cases of long-standing, is generally considered to be at least as good an indication for ECT as is the depression of manic-depressive psychosis, Senile depression also responds well to ECT, although the effect is limited in the presence of senile and arteriosclerotic brain changes. Kalinowsky and Hoch (1952) state that “the responses of the various types of depressions are so similar that it is safe to assume a close relation or even the same underlying process in manic-depressive depressions, involutional depressions, senile depressions, and even the so-called psychoneurotic depressions; they all react to approximately four convulsions , . .” “Recovery from a depressive episode after only four or five treatments, sometimes earlier, is sufficiently stable to the

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extent of being helpful in differentiating between manic-depressive and schizophrenic psychoses.” With experience and restrictiveness in the selection of subjects, ECT is undoubtedly one of the most reliable and predictable methods of treatment in psychiatry. Electroconvulsive treatment is usually given 2 or 3 times a week. More frequent treatments have been advocated for a rapid effect (Frosch et al., 1951), but Sargant (1961) doubts the value of this and even considers 1 treatment a week to be enough. In general, more widely spaced treatments may allow relapse of the depression between the treatments, while too frequent treatments may result in cumulative confusion. ECT frequently results in a dramatic, but often brief, relief of catatonic stupor. This is interesting, in view of the inhibitory nature of the stuporous state, which somewhat resembles depressive inhibition. On the other hand, in many cases of acute schizophrenia with anxiety and latent psychotic symptoms, ECT causes intensification of the anxiety and aggravation of the psychotic features. Thus, in “borderline” schizophrenia, ECT may be used as an aid in diagnosis ( Halpern, 1949). In schizophrenic patients, from 2040 treatments are required to obtain beneficial results of any significant degree and duration ( Kalinowsky and Hoch, 1952). This indicates that another, less specific action of ECT is probably operating here. A large number of treatments, or a “block” of treatments administered over a short period of time, produces a definite ‘‘pacifying‘‘ or sedative effect in psychoses, e.g., manic states (Brussel and Schneider, 1951; Sharp d al., 1953). Extremely intensive, so-called “regressive shock therapy” or “annihilation treatment” has been used to erase predominant psychotic patterns, at least for a short period (Rothschild et d., 1951; Glueck et al., 1957). ECT has been extensively used for sedative purposes in mental hospitals throughout the world but appears now to be largely replaced by ataratic drugs. There is reason to believe that the cumulative effects of intensive ECT may lead to irreversible brain changes, while more moderately spaced treatments, even in large numbers, are rarely reported to cause any permanent damage, Electroconvulsive treatment has also been employed in various nonpsychotic conditions, mainly those of psychoneurotic or psychosomatic nature. Very few controlled studies have been done, but

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it is likely that suggestion plays an important role. ECT is frequently applied in obsessive-compulsiveneuroses. However, only the depressive component, if present, is relieved, while the classical obsessivecompulsive syndrome is very resistant to, and sometimes exacerbated by, ECT. Only in neuroses with a depressive component or inhibition is there a fair expectation of a beneficial result (Pilkington, 1957; Roberts, 1959a). Where neurotic anxiety, fear, depersonalization, and labile autonomic reactions dominate the clinical picture, ECT produces poor results and frequently leads to exacerbation of the symptoms, indicating that such stimulatory treatment should not be given where autonomic and emotional overactivity already exists. Strong fear of ECT should be considered a contraindication. In such cases, ECT generally results in severe disturbances of memory and concentration (Gallinek, 1957; Mitchell et al., 1960). Prernedication with a barbiturate or other suitable sedative will generally reduce the fear. In summary, ECT appears to have at least 3 types of clinical effect: ( 1) ameliorates specifically depression and inhibition; ( 2 ) frequently increases neurotic and schizophrenic (“pseudoneurotic”) anxiety and may provoke overt psychotic symptoms in patients with latent schizophrenia; ( 3 ) sedates psychotic unrest when administered intensively. VI. Psychological Effects of ECT

Reports by psychologists on the positive, mood-elevating, and anti-inhibitory action of ECT are infrequent, while there exist many reports on the adverse effects, such as memory impairment and decreased learning ability. During a therapeutic grand-ma1 convulsion the patient is completely unconscious. In rare instances, he opens his eyes and looks around immediately after the convulsion has ceased, but as a rule he looks up briefly first after 5-15 min and then, unless disturbed, falls asleep again for an hour or more. Recovery of consciousness and orientation is reported to take place gradually after about an hour (Lunn and Trolle, 1949; Wilcox, 1956; Mowbray, 1959). Tests applied 6 hr after treatment showed decreased learning and reproduction ability (Cronholm and Molander, 1957). One week after a series of treatments, a residual defect in memory retention could still be detected (Cronholm and Blomquist, 1959).

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It is difficult to determine whether ECT has any longlasting effects on intellectual capacity, owing to interference, on the one hand, from the disease process and, on the other, to the beneficial effect of the treatment. Memory changes of long duration have been reported in some cases (6. Brody, 1944), but the findings are difficult to evaluate. Other reports indicate no apparent adverse effects on memory even after 100-250 treatments (Perlson, 1945; Rabin, 1948). It is my impression that the adverse effects are often emotional, generally of a phobic-neurasthenic nature, rather than intellectual, and difficult to determine by testing. In elderly, arteriosclerotic subjects, however, severe confusion and memory defects may occur; such subjects often take a considerable time to return to normal. Very intensive treatment probably may cause irreversible brain damage with resultant intellectual impairment, even though some authors deny that such risk exists (Glueck et al., 1957). In animals, electroconvulsions were found to reduce fixed behavior in a maze (Murphree and Peters, 1956) and to diminish a learned avoidance response for a period of more than 2 weeks (Carson, 1957). Masserman and Jacques (1947) were able to show that in cats experimentally produced neurotic patterns are disintegrated by electroconvulsions. However, the therapeutic effect in mentally ill patients is apparently unrelated to memory defects (Meyer, 1951; Kalinowsky and Hoch, 1961). A reduction of the strength and duration of the convulsive stimulation has been reported to lead to less memory disturbance and confusion (Liberson, 1948). This has been confirmed in animal experiments ( Hovorka, 1958). The degree of impairment of delayed reproduction of learned associations is related to the amount of current applied as well as to the intensity of cerebral convulsive activity (Ottosson, 1960). Nonconvulsive electrostimulation, applied immediately after an eIectroconvulsive shock, has been claimed to reduce the postshock memory defects (Alexander, 1953). This could not be confirmed by other workers (Cronholm and Ottosson, 1961). The most probable explanation is that the subconvulsive stimulation has a certain excitatory effect, which is also shown by its power to arouse patients from insulin coma ( H o h a n and Wunsch, 1950) and from barbiturate coma ( Hawkins et al., 1954). The arousal from insulin coma is not dependent upon an increase in blood sugar but is probably due to a nonspecific, peripheral action of the electrical current

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(Jones et al., 1955). Chemically induced convulsions are frequently stated to cause less memory disturbance and confusion, and again, an excitatory effect of the convulsant drug may be responsible for the quicker arousal and reduced initial confusion. In summary, ECT may be said to have the following psychological effects, in addition to those mentioned earlier: ( 1 ) a brief postictal period of confusion; (2) a more long-lasting but generally reversible intellectual impairment; ( 3 ) an arousal effect, partly by peripheral ( centripetal) mechanisms, VII. Physiopathology and Neuropathology of ECT

As mentioned earlier, a grand ma1 convulsion is always accompanied by fairly brief and reversible EEG changes. The effects are cumulative when a series of treatments are given. The duration of the EEG changes is variable, but usually does not exceed a month (Hughes et al., 1941). The postseizure pattern is not, or is only to a minor extent, a product of the direct action of the stimulus current (Kirstein and Ottosson, 1960). However, the EEG changes are correlated with the total amount of convulsive activity obtained during a series of treatments (Green, 1960). A positive correlation between the degree of EEG slowing and clinical improvement has been reported by some authors (Fink and Kahn, 1957; Roth et al., 1957; Kirstein and Ottosson, 1960), while no relationship was found by others (Hughes d al., 1941; Johnson et al., 1960). It is most frequently stated that the EEG changes are correlated to the posttreatment memory defects, but sometimes no such correlation is found. The solving of these problems would probably be facilitated if the studies were performed on patients with endogenous depressions and within a certain age group. It would seem, however, that EEG changes are not necessary for obtaining positive results with ECT in depressed patients, and generally the EEG changes show a better correlation with the memory defects than with the antidepressive effect. It is not clear whether the EEG changes are related to the reduction of cerebral excitability found to take place during a series of treatments ( Holmberg, 1954). Changes in the convulsive threshold are not related to the degree of clinical improvement (Brockman et al., 1956). The critical flicker frequency is significantly lowered after one single ECT ( Mowbray, 1961). In animals, electroconvulsive shock produces an increase of the extracellular fluid

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in the brain with reduction of electrolyte concentration, which is related to an increase in the convulsive threshold (Toman and Goodman, 1947). During and after convulsions, a brief capillary leakage takes place in the brain, because of damage to the blood-brain barrier (Bjerner et al., 1944). This may be the cause of the EEG changes (Aird et al., 1956). The increase in permeability of the blood-brain barrier is more long-lasting after a series of electrically induced convulsions (Aird, 1958). There is an increase in permeability to epinephrine and/or serotonin ( Weil-Malherbe, 1955), as well as to norepinephrine (Rosenblatt et al., 1960).Such permeability changes are sometimes claimed to be therapeutically important. However, the influence of ECT on the cerebrovascular permeability shows a striking resemblance to that obtained following inhalation of carbon dioxide mixtures ( CIemedson et aZ., 1958),and it wouId seem probable that an increase in cerebral C 0 2 is responsible for the plasma leakage occurring after the seizures. There is little probability that this phenomenon has any therapeutic effect, since CO, treatment is ineffective in depressed patients. The most common finding at histopathological examination following ECT is edema, commencing with a distension of the perivascular spaces ( Hartelius, 1952). Fairly slight, more or less reversible cellular changes, usually in the form of an increase in the glial elements, and irregular nerve cell degeneration, have been described (Ferraro et al., 1946; Hartelius, 1952). The few human brains examined following death due to ECT usually showed little pathological change, except, of course, in the rare instances in which the cause of death was a cerebral hemorrhage ( Meadow, 1956). Epileptic seizures have been reported as a sequela of ECT, although a critical evaluation of reported cases showed this complication to be very infrequent ( Stensrud, 1958). Although cerebral deaths following ECT are less frequent than cardiac deaths, both are exceedingly rare (Impastato, 1957; Barker and Baker, 1959). VIII. Complications of K T

Fatalities incident to electroshock have been reported to be from 0.0036% (Barker and Baker, 1959) to 0.06%(Kolb and Vogel, 1942). The risks are 10 times greater in patients over 60 than in those under 30 yr of age (Impastato, 1957).

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It has, so far, not been proven that the modification of ECT by barbiturates, muscle relaxants, and oxygenation reduces the mortality risk (Barker and Baker, 1959). It is, however, my conviction that such modification, used by a skilled operator, permits the treatment of many patients who would otherwise be poor risks, without an increase in the total mortality rate. The introduction of technical modifications now makes it possible to apply ECT to many patients who were previously considered untreatable. There is an enormous gain to be obtained from the fact that depressed patients who have received treatment are far less prone to commit suicide than those who have not (Ziskind et al., 1945). Cardiovascular accidents are the most important complication of this therapy (Impastato, 1957), pre-existing pathology being an important c m e (Barker and Baker, 1959; Perrin, 1961). Transient cardiac arrhythmias are common with ECT, but their incidence is greatly reduced by the use of acetylcholine blocking agents, which exert an important protective action against this and other cholinergic effects of ECT (see Section 111). Sympathetic autonomic phenomena, including increased heart rate and blood pressure, would appear to be less dangerous than the parasympathetic, although cerebral hemorrhages have been reported (Perrin, 1961). In hypertensive patients, these risks may possibly be reduced by the use of hypotensive drugs. In my opinion, however, the most important protective factors are atropinization, muscular relaxation, and adequate oxygenation. Barbiturate sedation, which is necessary before the injection of a muscle relaxing agent, is considered by some to add to the risk. This has led to the use of subconvulsive electric stimulation for producing unconsciousness before the treatment proper ( Impastato and Gabriel, 1958). This technique, however, may introduce the risk of severe vagal phenomena, which is known to be undesirable. The careful use of barbiturates does not appear to be contraindicated; on the contrary, it reduces fear as well as postictal excitation and violent autonomic discharge. On the other hand, ataractic drugs, particularly reserpine and phenothiazines, may unduly potentiate both the action of the barbiturate and the autonomic effects of the electroconvulsion, and therefore should be avoided, as far as possible (Perrin, 1961) . Respiratory complications are less frequent. Anoxia and hyper-

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capnia are easy to avoid with muscle relaxing agents and oxygenation. If acetylcholine blocking agents are used to reduce the salivary and mucus secretion, and the airway is kept free by proper technique, the risk of aspiration with subsequent lung abscess should be avoidable. Tuberculosis of the lungs is often considered a contraindication to ECT, but with correct anesthetic techniques, undue strain on the respiratory system and propagation of the tuberculosis process can also be avoided. Fractures, formerly a problem, can be eliminated by proper muscular relaxation, and with well modified ECT no significant mechanical strain is put on any part of the locomotor system. No untoward effects on vision or hearing have been reported. Thus, the only really important risks are cardiovascular accidents. It may not be possible to eliminate these completely, although the author feels that with good technique and, when necessary, with the help of specialists in anesthesiology and cardiology, almost any patient who needs ECT can be treated without any significant risk. Of the several thousand ECT’s, all performed with pentobarbital-succinylcholine relaxation, which the author has given during the past 10 yr or more, none has been attended by any kind of complication. IX. Prognostic Test Procedures and ECT

The method of classifying mental diseases according to the response to autonomic drugs has been in use for over 50 yr (Potzl et al., 1910). Methacholine has been widely used as a test drug ( Myerson et al., 1!337), and epinephrine has been employed for the same purpose (Lindemann and Finesinger, 1938). This type of test has been applied to patients prior to ECT, and a good correlation has been reported between the hypotensive effect of methacholine and the outcome of the ECT ( Funkenstein et al., 1950). A critical evaluation of these test procedures, however, shows that they are of limited prognostic value in electroconvulsivetherapy (Lunde et al., 1958; Sloane et al., 1958). The effect of epinephrine on the blood pressure has not been found to be of any prognostic value (Funkenstein et al., 1952), but the anxietyinducing effect of epinephrine is significantly related to a poor result of ECT (Alexander, 1958). Initially, the responses to autonomic drugs were widely accepted as valuable in predicting the outcome of ECT, but very few reports of the past years have been favorable.

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Atropine is another autonomically active drug that has been employed as a test substance (Hoffer and Callbeck, 1959), although the atropine test is not claimed to be of any clinical importance. Sedative drugs have also been used for testing purposes (Shagass, 1954). The amount of intravenous barbiturate required to sedate a patient (the sedation threshold) is reported to provide an objective index of manifest anxiety in psychoneuroses (Shagass and Naiman, 1956) and to be of value in psychiatric diagnosis (Shagass and Jones, 1958). Owing to the difficulty of determining the “sedation point,” the initial procedure was later modified in the sleep threshold test ( Shagass and Kerenyi, 1958). In more recent studies, however, neither the sedation nor the sleep threshold has shown any significant correlation with either the degree of anxiety or the diagnostic groupings ( Ackner and Pampiglione, 1959; Holmberg and Beck, 1961) , On the basis of neurophysiological experimental results, norepinephrine has been suggested as a more suitable test drug than epinephrine (Gellhorn, 1957) and yohimbine HCl, because of its strong anxiety-inducing and heart-rate increasing effects, may also prove to be a useful test drug (Holmberg and Gershon, 1961) The problem of predictive tests and ECT is far from solved. The role of age must be taken into account (Nelson and Gellhorn, 1957). An appropriate technique, with adjustment of drug dosage to body weight and exact speed of drug infusion, is advocated, and the importance of heart rate as a measure of autonomic and emotional reactivity is stressed (Holmberg and Beck, 1961). I t is possible that autonomic test procedures may be developed to become useful adjuvants in clinical diagnosis and prognosis. So far, however, specific tests would seem to be of limited prognostic value, while. clinical features obtained from the history and examination would seem to be of greater value in the electroshock treatment of depressed patients (Roberts, 1959a,b ) .

.

X. Mode of Action of ECT and Its Relation to Other Therapies

Theories on the mode of action of ECT are numerous (Kalinowsky and Hoch, 1952). It has, however, been possible to eliminate many factors formerly considered to be therapeutically active without reducing the therapeutic effect. This applies to “stress” factors such as anoxia, hypercapnia, muscular exertion, adrenal reactions,

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peripheral excretion of catechol amines, and other biochemical changes detectable in the blood. None of the more peripheral effects reported in the literature can be seriously claimed to be responsible for the therapeutic effect. Induced anoxia and hypercapnia have proven to be of no value in the treatment of depressive states. Concomitant central changes, such as slowing of the EEG and intellectual impairment, have frequently been stated to be related to the therapeutic effect of ECT, but proof has not been provided that these changes are necessary for obtaining a therapeutic effect. It is only to be expected that some correlation would exist between therapeutic effect and side effects, since both result from the action of the therapeutic procedure on the brain. The therapeutic effect of ECT is dependent upon the seizure activity, and not upon the intensity of the electrical stimulus (Cronholm and Ottosson, 1960). Subconvulsive treatment is found to have little or no beneficiary effect (Ottosson, 1962; Ulett, 1962). Nonconvulsive electric stimulation of the diencephalon is of no benefit in depressive states (Breitner, 1958). Softening of the seizures by prernedication with anticonvulsive drugs is reported to reduce the efficacy of ECT (Holt and Borkowski, lQSl), while intensification of the convulsions by the use of muscle relaxants and oxygenation increases the therapeutic effect ( Holmberg et ul., 1955). Metrazol convulsions are often stated to have a better therapeutic effect than electrically induced convulsions, which is probably due to the fact that Metrazol convulsions are more intense (Holmberg et al., 1955). In line with this, it has been found that Metrazol convulsions give rise to a greater increase of the blood sugar (Ewald and Haddenbrock, 1942) and a higher incidence of fractures (Polatin et al., 1939). Because ECT is safer and more practical, this type of convulsive therapy is still preferable in routine treatment. Convulsions induced by inhalation of hexafluorodiethyl ether are claimed to be therapeutically equivalent to ECT (Fink et d.,1961). It is apparent that the intracerebral changes brought about by seizure activity are incompletely understood. The various types of new antidepressive drugs will undoubtedly help to clarify which biochemical mechanisms underlie elevation of mood. Different monamine oxidase (MAO) inhibitors have varied antidepressive effects, independent of their potency as M A 0 inhibitors. Several of the drugs with an antidepressive action, e.g., imipramine

BIOLOGICAL ASPECTS OF Em

405

and amitiptyline, do not inhibit MAO. A release of catechol amines from the brain, e.g., by reserpine medication, may induce depression (Ayd, 1958). It is not known which of the amines are the most important for the action on mood, although so far serotonin has attracted the greatest interest. An interesting finding is that imipramine increases the peripheral sensitivity to norepinephrine both in animals (Sigg, 1959) and in man (Gershon et al., 1962). Electroconvulsive therapy elevates the brain level of serotonin (Poloni, 1956; Breitner, 1961) but has no effect on the amine oxidase concentration (Spilman and Badal, 1960). An interesting observation is that both ECT and certain antidepressive drugs may aggravate anxiety and schizophrenic symptoms. It is my belief that antidepressive therapy produces overstimulation if administered in certain excitatory states. Thus, there is need for valid diagnostic procedures and for restriction of ECT to well selected cases. A considerable amount of work is needed to solve these problems and it would seem worth while to concentrate the studies on ECT, since it is fairly generally agreed that this is an outstanding antidepressant and superior, so far, to any known drug. At least this is the outcome of most comparative studies (e.g., Kristiansen, 196l), even though others may consider drugs such as imipramine equivalent to ECT (Robin and Harris, 1962). Partly because of side effects, such as memory changes, the use of ECT is generally decreasing (Gulevich et al., 1961). However, it must be remembered that the use of chemical antidepressants is connected with considerable risks, greater than those with ECT. Combined electroshock and drug therapy may produce more effective and long-lasting results (Arnold, 1960; Hayes, 1960). Leucotomy is also sometimes advocated in the treatment of chronic depression (Elithorn, 1959; Sargant, 1961). So-called psychosurgery is effective in relieving anxiety and may be of value in chronic depression where anxiety is intolerable. However, it is not indicated in periodic depression, and it is doubtful whether it is of any value in the classical depressive and inhibitory phenomena, The author considers depression and obsessive-compulsive states as fundamentally different, from a physiological point of view. The effects of ECT and leucotomy are likewise, on the whole, entirely different. In opposition to this standpoint, it has been suggested that the

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actions of both treatments are similar, only that ECT gives more temporary interference with the functions of the frontal lobe or thalamofrontal connections ( Roth and Garside, 1962). It seems, however, that this view can be held concerning only the more unspecific sedative action on psychotic disturbance and not if the Specific antidepressive action of ECT is taken into account. Very often psychoanalytically oriented authors express the view that the actions of ECT may be ascribed to fear of death, suggestion, need for punishment, etc. It has also been questioned whether the induction of sleep would be enough to cure depressions. It can easily be understood that such factors would long ago have been utilized if they would work. Most certainly, they have been tried for ages. Statistical studies have shown such factors to play little if any role in ECT ( Ulett et al., 1956). Psychotherapy is of little benefit in endogenous depression. Antidepressive drugs and ECT may, to a large extent, be given in open practice, without no more specific psychotherapy than should be applied always to anxious and depressed patients. However, the differential diagnosis between -pure” endogenous depression, which is accessible to ECT, and neurotic depression and anxiety states is occasionally difkult and, therefore, the therapist must always be ready to regard a case from a different angle than a strictly physiological one. REFERENCES Ackner, B., and Pampiglione, G. (1959).J. Psychosomat. Research 3, 271. Aird, R. B. (1958).A.M.A. Arch. Neurol. Psychlat. 79, 633. Aird, R. B., Strait, L. A., Pace, J. W., Hrenoff, M. K., and Bowditch, S. C.

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Kristiansen, E. S. (1961).Acta Psychiat. Neurol. Scand. 37, Suppl. No. 162, p. 179. Leduc, S. (1902).Compt. rend. acad. sci. 135,199. Lending, M., Slobody, L. B., and Mestern, J. (1959).Neurology 9, 672. Liberson, W.T. (1944).lnst. of Living, Abstr. and Trawl. 12,368. Liberson, W.T. (1945).Yale J. Btol. and Med. 17,571. Liberson, W.T. (1948).Am. 1.Psychiat. 1% 28. Lieser, H. ( 1956).Nervenarzt 27, 69. Lighthart, P. W. K., Johnston, R. P.,and Sussman, E. (1956).Am. J. Psychiat. 112, 619. Lindeman, E., and Finesinger, J. E. (1938).Am. J. Psychiat. 95,353. Lunde, F., Mansfield, E., and Smith, J. A. ( 1958). J. Nervous Mental Disease 127, 430. Lunn, V. (1951).Act0 Psychfat. Neurol. Scand. 26,7. Lunn, V., and Trolle, E. (1949).Acta Psychiat. Neurol. Scand. 24, 33. Masserman, J. H.,and Jacques, M. G. (1947).Am. I. Psychfat. 104, 92. Meadow, L. (1956).Am. J. Psychiat. 113,337. Meduna, L. J. ( 1935).Psychiat. Neurol. Wochschr.37,317. Meduna, L. J. ( 1956). The convulsive treatment. In “The Great Physiodynamic Therapies in Psychiatry, an Historical Reappraisal” ( SackIer, A. M. et at.). Hoeber-Harper, New York. Meyer, J, E. ( 1951 ). Arch. Psychtat. Nmenkrankh. 186,254. Meyer, J, S.,and Gotoh, F. (1960).A.M.A. Arch. Neurol. 3, 539. Michael, S. T. (1956).Psychiat. Quart. 30,63. Mitchell, W.A,, Fox, W., and Funke, H. (1960).1. Clin. Exptl. Psychopathol. 21, 114. MolnBr, L., PBMy, G., and Baldgs, A. (1956).Arch. Psychiat. Nervenkrankh. 195,243. Montagu, J. D. (1953).Acta Psychiat. Neurol. Scand. Suppl. 87. Mowbray, R. M. (1959).Acta Psychiat. Neurol. Scand. 34,330. Mowbray, R. M. (1961).Acta Psychtat. Neurol. Scand. 36,149. Murphree, 0. D.,and Peters, J. E. (1956).J. Nervous Mental Disease 124, 78. Myerson, A., Loman, J., and Dameshek, W. (1937). Am. 1. Med. Sd. 193, 198. Nelson, R.,and Gellhorn, E. (1957).Psychosomat. Med. 19,486. Oliver, W.(1785).London Med. J. 6, 120. Ottosson, J. 0.(1960).Ada Psychfat. Neurol. Scand. 35, Suppl. 145,p . 103. Ottosson, J. 0.(1962).J. Neuropsychiat. 3,216. Perkon, J. ( 1945). A.M.A. Arch. Neurol. Psychiat. 54,409. Perrin, G. M. (1961).Acta Psychlat. Neurol. Scand. 36, Suppl. 152. Pfeiffer, C. C.,Jenney, E. H., and Marshall, W. H. (1956).Electroencephulog. and Clin. Neurophysiol. 8,307. Piette, Y. ( 1958).Acta Neurol. Belg. 58, 219. F’iekenbrock, T. C., Taylor, R. C., and Becka, D. R. (1956).A.M.A. Arch. Neurol. Psychiat. 76, 653. Pikington, T. L. (1957).Practitioner 178,466. Potzl, O., Eppinger, H., and Hess, L. (1910).Wien. klln. Wochschr. 23, 1831.

BIOLOGICAL ASPECIS OF ECT

411

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412

GUNNAR HOLM3ERG

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AUTHOR INDEX Numbers in italic indicate the pages on which the references are listed. Anand, B. K., 254, 292, 305, 306, 309, 332, 342, 345 Abe, N., 136, 176 Anderson, E., 48, 52, 251, 254, 292 Abelson, D., 247, 293 Anderson, E. M., 245, 294 Abood, L. G., 19, 39, 40, SO, 52 Anderson, J. A., 273, 298 Abraham, S., 355, 383 Anderson, R. N., 247, 253, 300 Abrahams, V. C., 326, 342 Andersson, B., 327, 335, 342 Abu Haydar, N., 248, 300 And& M. J., 290, 292 Abul-Haj, S. K., 19, 50 Appel, S. H., 42, SO Ackermann, P. G., 271, 297 Archdeacon, J. W., 321, 343 Ackner, B., 403, 406 Adachi, C., 7, 14, 22, 29, 39, 40, 51 Arduinil A*, 156* 177 Arita, M., 309, 345 Addison, T., 272, 292 Arrnstrong, S. A., 271, 2W Adey, W. R., 62, 91, 110, 113 405* 406 Adrian, E. D., 60, 73, 92, 113, 145, Arvanitaki, A., 76, 113 176 Ashby, W. R., 394, 406 Aiken, J. B., 285, 301 288p 292 Aird, R. B., 92, 115, 273, 292, 400, Asher, R.s 2879 Ashford, C. A., 373, 383 406 Astwood, E. B., 292 Aizawa, T., 168, 179 Ajmone-Marsan, C,, 58, 116, 153, Am% R* w.2 286, 287, 288, 289, 293 Atkinson, R. P., 270, 296 158, 176, 181 Austin, G., 87, 117 Akimoto, H., 156, 157, 176, 177 Ayd, G., 405, 406 Alajovanine, T., 309, 342 Ayer, J. P., 270, 271, 297, 301 Alan, B., 404, 408 Albe-Fessard, D., 58, 113 Albright, F., 267, 292 B Badal, D. W., 394, 395,405, 406,411 Alcaraz, M., 146, 150, 155, 178 Alexander, F., 289, 296 Baehr, G., 270, 271, 301 AIexander, J. B., 392, 409 Bagshaw, M., 312, 344 Alexander, L.,390, 391, 398, 402, 406 Bailey, P., 29, 52 Allen, J. N., 201, 239 Bain, J. A., 31, 50 Bairati, A,, 29, 50 Allen, W. F., 55, 113 Allison, A. C., 55, 73, 102, 113 Bak, R., 391, 409 Bakay, L., 29, 50 Allweis, C. L., 389, 383 Altea, E., 395, 407 Baker, A. A,, 400, 401, 407 Althausen, T. L., 319, 342 Baker, D. H., 374, 387 Baker, R. W. R., 363, 383 AItman, Ia. A,, 57, 113 Altschule, M. D., 263, 292, 392, 394, Baligs, A., 392, 410 Balfour, W. N., 378, 385 406 Amark, C., 392, 411 Ballard, A., 199, 200, 242 Ban, T., 330, 343 Amatniek, E., 230, 231, 242 Ames, A., III, 203, 239 Banga, I., 354, 383 413

A

414

AUTHOR INDEX

Bank, J,, 40, 50 Bankhead, A. J., 392, 407 Bankhead, M. D., 392, 409 Bard, P., 309, 342 Bare, J. K., 338, 344 Barelare, B., Jr., 315, 344 Bargmann, W., 382, 383 Barker, J. C., 400, 401, 407 Barlow, H. B., 166, 167, 177 Barr, M. L., 5, 50 Barrett, A. M., 246, 292 Barrett, R., 350, 386 Bartholomay, A. F., 258, 295 Bartley, H. S., 58, 75, 113 Bartter, F. C., 248, 298 Basowitz, H., 248, 259, 260, 261, 299,300 Bass, A. C., 273, 301 Bates, R., 254, 202 Batt, J. C., 284, 292 Baudouin, R., 316, 323, 345 Bauer, W., 272, 209 Baumgartner, G., 154, 168, 169, 170, 177, 179 Bayliss, M. J., 246, 300 Bayliss, W. M., 347, 383 Beck, L. H., 403, 409 Beck, R. N., 278, 202 Becka, D. R., 273, 294, 392, 410 Beckett, B. G. S., 44, 50 Beek, H., 390, 407 Beer, B., 57, 115 Behar, A., 3, 11, 15, 51 Bell, J., 390, 409 Bell, P. H., 292, 297 Bell, W. R., 245, 253, 294 Bellet, S., 392, 407 Benfey, B. G., 253, 300 Benjamin, R. M., 313, 342 Bennett, A. E., 391, 407 Bennett, A. W., 289, 292 Bennett, L. L., 273, 295, 302 Berberich, J., 276, 297 Bergen, J. R., 44, 52, 273, 292 Berlucchi, C., 55, 113 Berman, M. D., 195, 199, 203, 226, 242

Bernard, L. E., 396, 398, 408 Berry, C. M., 55, 113 Bertman, E. G., 5, 50 Bhargava, S. P., 393, 408 Biasotti, A., 250, 297 Biel, J. A., 39, 50 Biel, J. H., 39, 50 Bini, L., 389, 407 Birke, G., 276, 294 Bishoff, T. L. W., 348, 383 Bishop, G. H., 58, 59, 80, 63, 65, 73, 75, 113, 114, 117, 130, 139, 152, 177 Bishop, P. O., 129, 130, 139, 140, 143, 144, 177 Bjerner, B., 400, 407 Blachly, P. H., 399, 409 Black, E. G., 249, 294 Black, R. L., 248, 299 Blackman, R. B., 81, 91, 96, 113 Bland, J. 0. W., 19, 50 Blau, J. N., 278, 292 Bleuler, M., 244, 267, 272, 274, 276, 287, 289, 291, 292 Bliss, E. L., 257, 258, 259, 260, 261, 263, 264, 265, 292, 203, 394, 407 Blomquist, C., 397, 407 Board, F., 248, 259, 260, 261, 262, 285,293,299 Bogdanove, E, M., 251, 293, 318, 342 Bohr, N., 103, 113 Boling, L., 393, 408 Bollman, J. L., 350, 383 Bondareff, W., 376, 383 Bondy, P. K., 247, 293 Bongiovanni, A. M., 248, 203 Bonhoeffer, K., 274, 293 Borell, U., 324, 342 Borkowski, W., 391, 404, 409 Borman, M. C . , 271, 293 Bornstein, M. B., 7, 42, 50 Borsook, H., 364, 383 Borysko, E., 33, 50 Boudreau, J. C., 75, 81, 82, 92, 109, 113

415

AUTHOR INDEX

Bowditch, S. C., 400, 406 Bowers, C. Y., 276, 293 Bowman, K. M., 282, 283, 285, 293: 323, 343 Boyle, D., 317, 344 Braceland, F. J., 270, 300 Brachet, J., 355, 383 Brack, K. E., 284, 301 Brady, J. S., 79, 113 Brady, J. V., 255, 293, 298, 311, 342 Brady, R. O., 361, 378, 383, 384 Branch, C. H. H., 257, 258, 259, 260, 261, 263, 292, 293, 394, 407 Brannon, E. S., 286, 300 Braunsberg, H., 247, 293 Brazier, M. A. B., 60, 65, 81, 113 Breckenridge, B. M., 377, 383 Breitner, C., 394, 404, 405, 407 Bremer, F., 150, 177 Brenner, C., 311, 344 Bricaire, H., 273, 293 Brindley, G. S., 122, 123, 124, 125, 177

Brink, F., 220, 239 Brinley, F. J., Jr., 186, 195, 205, 209, 212, 213, 215, 217, 224, 231, 233, 235, 238, 240, 241 Brobeck, J. R., 245, 250, 252, 298, 304, 305, 306, 309, 317, 326, 327, 335, 342, 343, 345 Brockman, J. C., 399, 407 Brockman, R. J., 399, 407 Brodish, A,, 246, 293 Brodskaya, N. I., 373, 386 Brody, E. B., 285, 293 Brody, M. B., 398, 407 Brody, S., 270, 271, 293 Bromser, P., 95, 113 Broman, T., 400, 407 Bronovitskaya, Z. G., 374, 383 Brookhart, J. M., 60, 63, 67, 75, 113, 118, 399, 409 Brooks, C. McC., 249, 293 Brown, A. W., 41, 50, 203, 241 Brown, E. A., 272, 293 Brown, G. L., 329, 342

Brown, G. W., 05, 110, 114, 392, 393, 407 Brown, H., 246, 248, 266, 293, 298, 300 Brown, K. T., 123, 124, 125, 137, 177

Brown, M. L., 392, 393, 407 Brown, R. G., 271, 297 Browne, J. S. L., 270, 271, 293 Browning, T. B., 286, 287, 288, 289, 293

Brussel, J. A., 396, 407 Buchanan, J. M., 372, 383 Buchman, C., 390, 409 Buchthal, F., 59, 114 Bucy, P. C., 309, 343 Biichler, P., 276, 293 Bulatao, E., 316, 329, 342 Bulbring, E., 342 Bulbulian, A. H., 266, 301 Buller, A. J., 382, 384 Bureg, J., 212, 213, 215, 217, 218, 219, 220, 221, 222, 223, 224, 240, 241

BureEov5, O., 219, 220, 221, 222, 223, 227, 240 Biissow, H., 278, 293 Burke, W., 144, 177 Bum, G. P., 196, 241 Bum, J. H., 328, 329, 342, 379, 384 Bums, B. D., 65, 75, 79, 114, 383, 384

Bursten, B., 290, 293 Burton, L. E., 394, 405, 407 Burton, R. B., 246, 302 Burton, R. M., 378, 384 Buser, P., 58, 113 Bush, I. E., 246, 247, 293 Byzou, A. L., 124, 177

C Caldwell, P. C., 234, 240 Callan, D. A,, 232, 242 Callbeck, M. J., 403, 409 Cambor, C. G., 289, 292 Campbell, G. A., 89, 114

416

AUTHOR INDEX

Campbell, H. J., 251, 293 Campbell, J., 359, 384 Canti, R. G., 19, 50 Caramanian, M. K., 270, 294 Cardini, C. E., 377, 385 Carlson, A. J., 314, 316, 342 Cannichael, H. T., 289, 296 Carson, R. C., 398, 407 Carter, A. C., 248, 302 Carter, J. D., 270, 272, 298 Casals, J., 380, 384 Caughey, J. E., 276, 293 Cenciotti, L., 394, 411 Cerletti, U., 389, 395, 407 Chaikoff, I. L., 355, 358, 369, 370, 383, 384, 387 Chance, M.R. A., 372,384 Chaney, A. L.,285, 301 Chang, H.-T., 58, 80, 114, 130, 133, 152, 153, 177 Chang, J. J., 2, 28, 50, 51 Chanley, J. D., 394, 400, 411 Chantrenne, H., 350, 384 Cheek, W. R., 253, 296 Chen, P. S., 248, 298, 299, 300 Cheng, C. P., 250, 294 Cherry, R. W., 60, 67, 79, 116 Chevalier, J. A., 260, 261, 299, 300 Chin, L., 394, 405, 407 Chou, S. N., 155, 179 Chow, K. L., 161, 177 Chujyo, N., 329, 342 Clare, M. H., 58, 60, 65, 113, 114, 130, 152, 177 Clark, L. D., 272, 280, 294, 299 Claude, A,, 376, 384 Clayton, G. W., 253, 294 Cleghorn, R. A., 260, 270, 274, 275, 276, 294, 301 Clemedson, C. J., 394, 400, 407 Clemente, C. D., 309, 343 Clouet, D. H., 365, 384 Cobb, S., 267, 289, 271, 272, 294, 299, 302, 311, 312, 342, 345 Cobb, W., 60, 114 Cobey, F., 248, 301 Cohen, H., 247, 294

Cohen, M., 44, 51 Cohen, S., 274, 294 Cohn, P., 325, 342 Cohn, R., 65,114, 160, 177 Cole, K. S., 93, 94, 95, 110, 114 Cole, R. D., 245, 298 Cole, V, V., 301 Collip, J. B., 245, 294 Condon, J. V., 273, 294 Conrad, E., 262, 298 Cooper, R., 60, 65, 79, 91, 92, 114 Cope, C. L., 249, 294 Cop&e, G., 76, 91, 117 Cori, C., 318, 342, 371, 384 Cori, G. T., 371, 384 Costero, I., 3, 50 Coxon, R. V., 355, 358, 364, 368, 374, 375, 384 Cragg, B. G.,55, 91, 92, 114 Craib, W. H., 59, 114 Crain, S. M., 2, 50, 239, 240 Crammer, J. L., 284, 294 Crane, H. D., 95, 114 Crank, J., 197, 226, 240 Cranswick, E. H., 283, 294 Crawford, E. J., 377, 383 Crescitelli, F., 122, 125, 177 Creutzfeldt, O., 157, 176, 177 Crick, F. H. C., 367, 384, 387 Cronholm, B., 397, 398, 404, 407 Crosby, E. C., 55, 114 Cukor, R., 290, 291, 2.97 Cullen, C., 79, 116 Cumings, J. N., 45, 50 Cummins, J. T., 203, 228, 240 Cunningham, A. W.B., 2, *50 Currie, A. R., 266, 294 Curtis, D. R., 231, 232, 23.3, 240 Curtis, G. C., 393, 411 Cushing, H., 267, 294 Czajkowski, N. P., 44, 50

D Dailey, M. E., 282, 283, 285, 293 Dainty, J., 102, 114 Daly, C., 323, 343 Dameshek, W., 402, 410

417

AUTHOR INDEX

D’Amour, M. C,,304, 343 Daniel, P, M . , 251, 294 Daniels, R. S., 405, 408 Darden, E. B., Jr., 187, 240 Darrow, D. C., 203, 242 Darwin, C., 57, 114 Daughaday, W. H., 248, 294 Davenport, V. D., 273, 294 David, G. B., 44, 50 Davies, B. M. A,, 288, 294 Davies, B. N., 329, 342 Davies, E. B., 287, 294 Davies, P. W., 236, 238, 240 Davies, R. K., 352, 386 Davis, A. K., 273, 295 Davis, D. S., 245, 297 Davis, E. W., 392, 407 Davis, R., 143, 144, 177 Davison, A. N., 381, 382, 384 Dawson, G. D., 81, 114 de Ameida, D. F., 41, 50 Deasy, C . L., 364, 383 Debr6, R., 270, 294 DeCaro, D., 395, 407 deCastro, O., 41, 50 Decaulne, P., 84, 103, 109, 115 deGroot, J,, 251, 252, 253, 294, 309, 343 Delafresnaye, J. F., 58, 114 Delay, J., 393, 407 Delfares, A., 352, 386 Delgado, J. M. R., 306, 310, 311, 312, 313, 342, 343 Dell, P., 150, 178 delorenzo, A. J. D., 199,240 Demetrescu, M., 150, 151, 181 den Breeijen, A., 282, 299 De Robertis, E., 7, 28, 29, 50, 199, 211, 240 Desclaux, P., 305, 306, 318, 322, 324, 325, 345 Deuel, H. J., 356, 363, 384 De Valois, R. L., 161, 168, 177, 178 Diana, P . B., 270, 271, 298 Dickens, F., 373, 384 Dickson, J. S., 245, 298 Diczfalusy, E., 276, 294

Didio, J., 62, 110, 113 Di Mascio, A., 393, 408 Dingman, W., 352, 386 Dirac, P. A. M., 102, 114 Dixon, K. C., 373, 383 Dobbing, J., 381, 362, 384 Dobrzecka, C., 308, 346 Dodt, E., 135, 136, 178 Doe, R. P., 259, 266, 294 Dogiel, A. S., 134, 178 Doljanski, L., 48, 50 Dominguez, 0. V., 248, 300 Domino, E. F., 98, 114 Dongier, M., 285, 286, 294 Donner, K. O., 132, 178 Donovan, B. T., 251, 294 Doty, R. W., 151, 178 Dougherty, M., 2, 50 Dougherty, T.F., 245, 294 Douglas, G. W., 359, 386 Douglas, W. W., 328, 342 Dreyfus-Brisac, C., 273, 293 Droogleever Fortuyn, J., 56, I Drujan, B. D., 393, 411 Dua, S., 254, 292, 332, 345 DuBuy, H. G., 40, 52 Duke, H., 333, 342 Dumont, S., 150, 178 Duner, H. A., 323, 342 Dunlap, H. F., 290, 291, 294 Dunlop, C. W., 91, 113 Dunn, A. L., 283, 301 Du Vigneaud, V., 379, 384

E Eagle, H., 4, 50 Ebaugh, F. G., 270, 294 Ebe. M.. 135. 160. 162. 180 Eccles, J. C., 58, 59, 85,’87, 111, 113, 114, 155, 178, 203, 204, 205, 218, 240, 382, 384 Eccles, R. M., 205, 240, 382, 384 Eckert, J. F., 315, 344 Edelman, I. S., 188, 242 Edstrom, J-E., 365, 384 Egdahl, R. H., 253, 258, 294 Eggleston, L. V., 210, 241

418

AUTHOR INDEX

Ehnnan, R. I., 2, 50 Eichelberger, L.,203, 240 Eidelberg, E., 63, 72, 114 Eigher, E. A., 245, 297 Eik-Nes, K., 248, 247, 286, 280, 294, 299, 300 Einarson, L., 33, 50 Eisenberg, J., 266, 267, 301 Ekman, H., 288,295 Eliel, L. P., 270, 299 Elithorn, A., 405, 407 Elliot, K. A. C., 201, 202, 203, 241 Ellis, J. P., 258, 296 Engel, F. L.,245, 302 Engel, G. L., 273, 295 Englert, E., 248, 293 Eppinger, H., 402, 410 Epstein, A. N.,335, 342,343 Epstein, J,, 407 Eranko, O.,325, 343 Erickson, T. C.,392, 393, 409 Erulkar, S. D.,144, 178 Esquibel, A. J., 390, 407 Estes, W. K.,255, 295 Evans, E. A., Jr., 384 Evans, G. H.,245, 295 Evans, H. M.,245, 298 Ewald, G., 274, 295, 404, 407 Ewalds, R. M.,390, 408

Fink, M., 399, 404,408 Fischer, A., 48, 50 Fischer-Williams, M., 146, 180 Fischgold, H.,273, 293 Fishman, J. R., 280, 2@5, 296 FitzHugh, R., 168, 167, 177 Flach, F. F., 393, 407 Flanagen, J. B., 273, 295 Fleckenstein, A., 220, 240 Fleminger, J. J., 272, 295, 297 Flink, E. B.,259, 288, 273, 294, 295,

302 Flint, L. D., 270, 296

Folch, J., 210, 240, 363, 380, 384, 385 Fonberg, E., 310, 343 Forsham, P. H., 245, 273, 295, 302 Forssberg, A., 323, 325, 343 Fortier, C.,250, 251, 253, 295 Foster, R. M.,89, 114 Founnan, P., 278, 295 Fox, C. A., 56, 115 Fox, H. M.,257, 258, 283, 295, 296, 300 Fox, W., 397, 410 Frank, K., 78, 85, 88, 110, 115, 205, 240 Frankenhaeuser, B., 82, 115,220,221, 240 Frankland, M.,317, 344 F Frankl-Hochwart, L., 277, 295 Fabing, H. D., 390, 398, 408 Franksson, C.,257, 295 Fadiga, E., 60,83, 113 Freedberg, A. S., 282, 297 Freeman, H.,283, 295 Farguharson, R. F., 278, 295 Farrell, G.L.,246,251,300 Freeman, W.J., 54, 56, 83, 64, 88, Fatehchand, R., 126, 179, 180, 181 72, 75, 78, 80, 81, 83, 84, 86, 89, 91, 93, 94, 96, 97, 98, 99, Fatt, P.,60, 63, 67, 114, 205, 240 Fazekas, J. F.,323, 343 100, 101, 102, 104, 105, 108, Feldberg, W., 39, 50, 328, 342, 343 107, 108, 109, 113, 115 Femfindez-Guardiola, A,, 146, 150, Frekgang, W. H., 230, 231, 242 155, 178, 180 Freud, S., 243, 295 Fernandez-Morgan, H.,353, 384 Freygang, W.H., Jr., 80, 82, 78, 85, Ferraro, A., 400, 408 88, 115 Fessard, A., 58, 115 Friedman, E., 390, 408 Fielding, U., 250, 299 Friedman, M. M.,404, 411 Fillenz, M.,134, 144, 178 Froman, C. E., 44, 50 Finesinger, J. E., 402, 410 Frosch, J., 391, 395, 408, 409

AUTHOR INDEX

Frostig, J. P., 390, 408 Fry, C. C., 282, 293 Fry, E. G., 245, 250, 298 Fukada, Y., 137, 181 Fulford, B. D., 252, 295 Fuller, J. L., 309, 338, 343 Fulton, J. F., 57, 115, 312, 343 Funke, H.,397, 410 Funkenstein, D.H., 393, 402,408 Fuortes, M. G. F., 205, 240 Furchtgott, E.,337, 343 Furger, R., 288, 295 Furst, W.,392, 407 Furth, J. J., 368, 385 Fuster, J. M., 155, 157, 178

G Gabriel, A. R., 392, 396, 401, 409, 411 Gainer, H.,369, 383 Gaitonde, M. K., 352, 363, 366, 384 Galambos, R.,54, 57, 115, 117 Gale, E. F., 366, 384 Gallinek, A., 397, 408 Ganong, W.F., 251, 252, 254, 295 Garattini, S., 394, 408 Garoutte, B., 92, 115 Gamlove, J. L., 266, 267, 301 Garside, R., 406, 411 Gastaut, H., 55, 115, 390, 408 Gates, G. L., 95, 115 Geiger, A., 31, 50, 352, 368, 370, 378, 384 Geiger, R. S., 3, 4, 5, 7, 11, 13, 14, 15, 19, 22, 29, 30, 31, 33, 37, 38, 39,40, 41, 44, 50, 51 Gellhorn E., 403,408,410 Gemzell, C.A., 257, 295 Gerard, R. W., 40, 50, 73, 116, 381, 384 Gersh, I., 249, 293 Gershenfeld, H. M., 7, 28, 29, 50, 199, 211, 240 Gershon, S., 403, 405, 408, 409 Geschwind, I., 245, 298 Gestring, G. F., 136, 179

419

Gey, G., 2,50 Ghantus, M.,324, 343, 371, 385 Gibbons, J. L.,262, 285, 295 Gibbs, F. A., 273, 294 Gibson, J. G., 285, 295 Gifford, S., 258, 295 Gildea, E. F., 271, 295 Gille, J. C., 84, 103, 109, 115 Gillespie, J. S., 329, 342 Girado, M.,232, 242 Gjessing, R., 282, 295 Glaser, G. H., 267, 271, 295 Glees, P., 134, 138, 139, 144, 178 Glenn, E. M., 248, 296 Gleser, G., 390, 399, 406, 407, 411 Clock, G. E., 361, 384 Glueck, B. C., Jr., 396, 398, 408 Goetz, F. C., 257, 258, 296 Gold, N. I., 252, 295 Goldfarb, W., 323, 343 Goldfien, A., 254, 295, 393, 408 Goldring, S.,232, 240 Goldstein, M. H., Jr., 156, 177 Goldzieher, M. A., 274, 296 Golub, 0.J., 249, 296 Goodhill, V., 337, 343 Goodman, L. S., 250, 273, 294, 302, 391, 393, 400, 411 Goolker, P., 270, 271, 296 Gordan, G. S., 273, 292 Gordon, D. L., 276, 293 Gordon, M. W.,354, 386 Gordon-Smith, E. C.,375, 384 Gore, M.B. R., 238, 241 Gossweiler, N., 221, 240 Gotoh, F., 392, 410 Gottesfeld, B. H., 390, 408 Gottlieb, J. S., 44, 50 Gour, K. N., 393, 408 Gouras, P.,125, 126,178 Grace, W.J., 260, 296 Grad, B., 246, 300 Grady, A. B., 245, 292 Grafstein, B., 223, 224,240 Graham, C.E., 320, 343 Graham, D.,103, 125 Granger, G.W., 122,178

420

AUTHOR INDEX

Hagiwara, S., 115 Haigh, C, P., 284, 296 Hair, G. W., 249, 296 Gray, S. J., 257, 258, 296 Hgkansson, B., 266, 295 Greaves, M. S., 248, 296 Hakas, P., 168, 177 Green, J., 65, 117, 394, 399, 411 Green, J. D., 60, 64, 67, 75, 115, Hale, H. B., 258, 296 Halmi, N. S., 251, 293 249, 250, 296, 309, 343 Halpern, L., 395, 408 Green, M. A,, 399, 404, 408 Halvorson, H. O., 366, 386 Greenblatt, M., 393, 402, 408 Ham, G. C., 289,296 Greene, G., 300 Hamburg, D., 248, 259, 260, 261, Greer, M. A., 251, 296 280, 285, 293, 295, 296, 299, 300 Greipel, M., 317, 344 Hamburger, V., 2, 52 Grenell, R. G., 29, 51 Greville, G. D., 373, 384 Hamilton, C. L., 335, 343 Griffith, J. S., 367, 384 Hamm, F. C.,270, 296 Griffith, W. H., 320, 343 Hamolsky, M. W., 282, 297 Grinker, R. R., 248, 259, 260, 261, Han, P. W., 327, 343 299, 300 Handlon, J. H., 260, 295, 296 Gross, F., 367, 386 Hann, J., 393, 408 Grossi, E., 358, 359, 384, 385, 386 Hara, T., 122, 178 Grossman, M. I., 331, 343, 345 Harada, T., 309, 345 Grosz, H. J., 259, 299 HArd, G., 392, 393, 404, 409 G~~~ssIz,0.-J.,123, 125, 142, 144, Hare, E. H., 284, 296 154, 178 Hare, L.,289, 296 Ha&, W. K., 304, 343 Griisser-Cornehls, U., 142, 178 Griitzner, A., 154, 178 Harris, E. J., 196, 209, 241 Grundfest, H., 58, 115, 230, 231, 232, Harris, G. W., 250, 251, 252, 253, Granit, R,, 135, 136, 137, 158, 161, 178

242

293, 294, 295, 296

Guerra, S. L., 247, 262, 263, 299 Guillemin, R., 247, 253, 294, 296, 300 Guld, C., 59, 114 Gulevich, G., 405, 408 Gullock, A. H., 283, 297 Gurpide, E., 249, 296 Guttman, R., 220, 240 Guze, S. B., 289, 299, 394, 408 Gu&-FIores, C.,146, 150, 155, 178

Harris, J. A., 405, 411 Harris, J. I., 245, 296, 298 Harris, M. M., 404, 411 Harrison, R. G., 2, 51 Hartelius, H., 394, 400, 407, 408 Hartline, H. K., 166, 168, 170, 171, 178, 180

Harwood, C. T., M?,255, 256, 296, 298

Hashimoto, Y., 60, 64,118, 124, 125; 181

H Haagen-Smit, A. J., 364, 383 Haba, D. S., de la, 286, 296 Haddenbrock, S., 404, 407 Haertig, E. N., 331, 343 Hagamen, W. D., 55, 113 Hagbarth, K,-E., 57, 100, 116

Hastings, A. B., 203, 241, 372, 383 Havens, L. L., 393, 408 Hawkes, C. D., 312, 344 Hawkins, R., 398, 408 Hawthorne, E., 254, 292 Hayama, T., 329, 343 Hayes, J. B., 405, 408 Hayhow, W. R., 144, 178

AUTHOR INDEX

Haymaker, W., 254, 292 Heald, P. J., 373, 385 Hearn, W. R., 253, 296 Hearst, E., 57, 115 Heath, H. A., 248, 260, 299 Heath, R. G,, 44, 45, 51, 98, 116 Hebb, C. O., 11, 51, 379, 381, 385 Hebb, D. O,, 57, 115 Hedberg, S. E., 257, 258, 296 Heilbrunn, L. V., 49, 51 Hejmancik, M. R., 392, 409 Helfand, M., 400, 408 Helme, T., 137, 178 Hemphill, R. E., 390, 409 Hench, P. S., 270, 272, 296, 302 Henderson, J, R., 374, 375, 384, 385 Hendrix, C. E., 91, 110, 113 Henry, R. J., 249, 296 Herbert, D., 372, 386 Hemhdez-Pdn, R., 57, 115, 145, 146, 150, 155, 178 Herrick, C. J., 116 Herrmann, E., 277, 296 Herrmann, G. R., 392, 407, 409 Herskowitz, H., 390, 408 Hertz, P. E., 270, 296 Herz, M., 248, 260, 299 Hess, L., 402, 410 Hetherington, A. W., 304, 305, 343 Hetzel, B. S., 260, 288, 296 Hickman, J. W., 270, 296 Hiddema, F., 56, 115 Hild, W., 2, 3, 4, 7, 19, 28, 50, 51, 52, 141, 178, 205, 239, 241 Hill, R. G., 321, 343 Hill, S. R., 257, 258, 288, 296, 302 Hillibp, N.-A., 251, 302 Hills, A. C., 245, 295 Himwich, H. E., 323, 343 Hines, €1. M., 392, 393, 407 Hinkle, L. E., 288, 296 Hinsey, J. C., 55, 113 Hinton, J. M., 278, 292 Hirao, T., 158, 177, 178 Hirsch, S., 278, 297 Hirschfeld, G. R., 390, 409 Hiscoe, H. B., 38, 48, 52, 380, 382

421

Hoagland, H., 44, 52, 263, 273, 292, 298, 299 Hoch, P. H., 391, 393, 394, 395, 396, 398, 409 Hodges, J. R., 246, 258, 266, 292, 297 Hodgkin, A. L., 61, 62, 87, 88, 95, 110, 113, 115, 116, 184, 188, 191, 203, 204, 208, 220, 221, 224, 227, 235, 237, 240, 241 Hoffer, A,, 403, 409 Hoffman, F. H., 398, 409 Hoffman, M. M., 285, 286, 294 Hoffman, R. S., 46, 50 Hoffman, W. C., 273, 297 Hogue, M. J,, 3, 13, 51 Holloszy, J., 248, 294 Holmberg, G., 391, 392, 393, 394, 399, 400, 403, 404, 405, 407, 408, 409 Holmes, E., 372, 386 Holt, L. M., Jr., 315, 344 Holt, W. L., Jr., 391, 404, 409 Hopkins, F. G., 349, 385 Horler, A. R., 278, 295 Horn, G., 57, 116, 146, 156, 178 Horstmann, E., 199, 241 Horwitz, N. H., Q6,117 Horwitz, W. A., 404, 411 Hoskins, R. G., 282, 297 Hotta, S. S., 381, 385 Houdart, R., 309, 342 Housholder, D. E., 253, 296 Houssay, B. A., 250, 297 Hovorka, E. J., 390, 398, 409 Howton, D. R., 383, 386, 387 Howard, K. S., 245, 297 Howard, S. Y., 155, 179 Hrenoff, M. K., 400, 406 Huang, J. H., 232, 240 Hubel, D. H., 79, 116, 142, 144, 154, 166, 167, 178 Huber, G., 288, 297 Hughes, J., 399, 409 Hull, C. L., 97, 116 H u e , D. M., 248, 251, 252, 253, 258, 294, 295, 297, 299

422

AUTHOR INDEX

Humphrey, T., 55, 103, 114 Hunt, C. A., 273, 292 Hunt, H.F., 255, 293 Hurwitz, J., 368, 385

Johnson, L. C., 399, 409 Jobnson, M., 399, 409 Johnson, W. A., 348, 385 Johnston, J. B., 134, 179 Hmthal, L. M.,267, 270, 296, 297 Johnston, M. W.,302 Johnston, R. P., 390, 410 Huston, P. E.,392, 393, 407 Huxley, A. F., 76, 87, 88, 95, 110, Jonas, V., 267, 297 Jones, A. L.,403, 411 113, 116 Hyden, H.,5, 7, 22, 29, 33, 41, 51, Jones, C. H.,399, 409 54, 116, 354, 365, 366, 381, 384, Jones, J. C., 253, 299 385 Jones, M. T.,266, 297 Jones, W.W.,321, 343 I Jonnard, R., 103, 116 Jouvet, M., 57, 115, 116, 145, 178 Iannacone, A., 266, 267, 301 Jung, R., 79, 116, 154, 179, 392, 409 Ikeda, M.,329, 343 Impastato, A., 390, 409 K Impastoto, D.J., 390, 391, 392, 395, Kaada, B., 55,116, 152, 177,254,301 400, 401, 408, 409, 411 Kabara, J. J., 359, 385 Inanaga, K., 216, 242 Kado, R. T.,62, 110, 113 Ingvar, D. H.,162, 179 Kahn, R.,399,404,408 Irons, E. N.,271, 297 Kalinowsky, L. B., 391, 393, 394, Irons, G.,253, 294 395, 396, 398, 403,409 Ison, E. C.,321, 343 Ito, A., 330, 343 Kamp, A., 92, 118 Kandel, E. R.,76, 116, 205,212, 213, Ivy, A. C.,318, 346

J Jackson, B. T.,253, 294 Jackson, J. H.,54, 116 Jacob, F.,367, 386 Jacobsohn, D.,250, 251, 302 Jacobsohn, U.,399, 407 Jacobson, J. H.,136, 179 Jawby, G. A,, Jr., 338,343 Jacques, M. G., 398, 410 Jaeger, J. C.,195, 241 Jakob, A., 276, 297 James, V. H.T., 247,293 Jamieson, B., 285, 301 Janowitz, H.D.,331,332,343 Jardon, F.,399, 409 Jasper, H.,58, 79, 116, 392, 393, 409 Jenkins, D.,248, 300 Jenney, E. H., 390, 410 Jennison, R, C.,81, 94, 102, 116 Jeremy, D.,129, 130, 177 John, E. R., 57, 58, 116

215, 217, 224, 231,233,240, 241 Karnovsky, M. L., 359, 385 Karoly, A. J., 161, 177 Karp, E., 404, 408 Karrer, A., 247, 262, 263, 299 Katayama, S., 168, 179 Kato, R., 394, 408 Katz, B.,61,116, 191,204, 241 Katzen, H. M.,375, 376, 387 Katzenelbogen, S., 290, 297 Katzman, R., 209, 211, 241 Kaufman, M. R., 394, 400, 411 Kawata, N.,220, 242 Kay, D. W.,394, 399, 411 Kay, W. W.,284, 292 Keigbley, G., 364, 383 Keller, A. D.,304, 343 Kellogg, 0.D.,60, 116 Kelsey, F. E., 283, 297 Kelsey, F. O.,283, 297 Kendall, E. G., 270, 272, 296 Kennedy, E. P., 362, 385

423

AUTHOR INDEX

Kennedy, G. C., 317, 343 Kent, H. S., 266, 299 Kerenyi, A., 403, 411 Kerly, M., 377, 385 Kerman, E. G., 391, 409 Kerr, D. I . B., 57, 100, 116 Kerr, S., 371, 385 Kerr, S. E., 324, 343 Kershhaum, A., 392, 407 Kersting, G., 46, 51 Kety, S. S., 241 Keutmann, H . H., 246, 302 Keynes, R. D., 186, 187, 188, 191, 196, 197, 208, 224, 227, 235, 237, 241 Kheim, T., 271, 297 Killam, K. F., 57, 116 Kind, H., 276, 277, 288, 297 Kinnen, E., 61, 116 Kipfer, K., 221, 240 Kirstein, L., 399, 409 Kitai, S . T., 161, 177, 178 Kleinherg, W., 247, 294 Kleinschmidt, H. J., 290, 29, 297 Kleinzeller, A., 372, 385 Kleitman, N., 337, 343 Klenk, E., 359, 385 Kling, I., 285, 301 Klippel, M., 274, 297 Kliiver, H., 309, 343 Kniper, J., 92, 118 Knowlton, K., 254, 292 Koch, A., 199, 200, 242 Koelle, G. B., 7, 11, 41, 51, 52, 326, 333, 342, 343, 381, 385 Koenig, E., 381, 385 Kohata, T., 136, 180 Kok, D. J., 390, 412 Koketsu, K., 205, 240 Koletsky, S., 251, 300 Kollros, J. J., 203, 240 Komora, E. J., 390, 409 Korchin, S. J., 248, 259, 260, 261, 299, 300 Korey, S. R., 353, 385 Kosman, A. J., 309, 310, 344 Koster, M., 268, 297

Komtz, W. B., 271, 297 Kraepelin, E.,243, 274, 297 Krakauer, L. J,, 257, 258, 296 Krantz, J. C.,Jr., 390, 407 Krasne, F., 308, 343 Kratochuil, C. H., 258, 296 Krehs, H. A,, 210, 241, 348, 355, 370, 385 Krell, A,, 390, 409 Krieg, W. J. S., 304, 343 Krieger, D. T., 280, 297 Kristiansen, E. S., 405, 410 Kfidnek, J., 212, 213, 217, 218, 221, 222, 223, 240, 241, 373, 385 Kmjevic, K.: 203, 241 Kruger, L., 56, 117, 152, 153, 179 Kuhicek, W. G., 61, 116 Kuffler, S. W., 166, 167, 177, 179 Kulb, L. C., 400, 409 Kumamoto, T., 325, 345, 375, 386 Kurland, A. A., 390, 407 Kurland, G. S., 282, 297 Kurotsu, T., 330, 343 Kuypers, H. G.,254, 299

1 LaBarre, J., 323, 346 Labesse, J., 270, 294 Laidlaw, J. C., 248, 263, 300 LaigneI-Lavastine, M., 244, 297 Lajtha, A,, 364, 366, 387 Lamesta, L., 394, 408 Lance, J. W., 129, 130, 177 Landau, W. M., 62, 115 Laqueur, G. L., 251, 297 Larrahee, M. G., 186, 195, 209, 235, 238, 240 Larsson, S., 306, 323, 325, 326, 327, 335, 342, 343 Laszt, L., 319, 346 Latham, L. K., 44, 50 Laufer, M., 126, 179, 180, 181 Lamas, K. R., 249, 297 Lazarow, A,, 376, 385 Leach, B. E., 44, 51 Leiid, A. A. P., 126, 179, 217, 225, 226, 227, 241

424

AUTHOR INDEX

Leaf, A., 81, 117 Le Baron, F. N., 363, 372, 380, 384, 385 Leduc, S., 390, 410 Lees, M., 210, 240, 380, 384 Lefkowits, H. J., 404, 408 Le Gros Clark, W. E., 138, 180, 179 Lehmann, J., 268, 295 Lehninger, A. L., 355, 385 Leiderman, P. H., 209, 241 Leloir, L. F., 377, 385 Lending, M., 394, 410 Lennox-Buchthal, M. A., 168, 179 Lennox, M. A., 130, 133, 153, 180, 179 Leone, L., 248, 301 Lesse, H., 98, 116 Lesse, S. M., 390, 408 Lettvin, J. Y., 174, 175, 179 Levi, G., 2, 7, 52 Levi-Montalcini, R., 2, 52 Levin, M. E., 282, 297, 394, 408 Levitt, M. F., 270, 271, 301 Levy, A, L., 245, 298 Lewis, A. J., 272, 297 Lewis, B. I., 270, 272, 298 Lewis, D. J,, 402, 411 Lewis, P. R., 188, 187, 208, 241 Lewis, R. A., 258, 273, 297 Li, C., 79, 116 Li, C. H., 245, 296, 297, 298 Li, C.-L., 155, 179 Liang, E., 393, 407 Liberman, E. A., 133, 179 Liberson, W. T., 390, 398, 410 Libet, B., 73, 116 Liddle, G. W., 288, 298 Lidz, T., 270, 272, 289, 290, 298 Lieberman, S., 249, 296 Lieser, H., 390, 410 Lighthart, P. W . K., 390, 410 Ligon, E. W., 321,344 Lilly, J . C., 60, 87, 79, 116 Lin, R. C. Y., 328, 343 Lindeman, E., 402, 410 Lindsay, A. E., 288, 298 Ling, A. S. C., 390, 407

Lingjaerde, O., 282, 285, 298 Lingjaerde, P., 285, 298 Lipmann, F., 348, 385 Lipner, H. J., 318, 342 Lipscomb, H. S., 247, 253, 300 Livingston, R. B., 57, 116 Lloyd, D. P. C., 80, 116 Lluch, M., 319, 344 Locke, W., 278, 293 Lolley, R. N., 378, 385 Loman, J., 402, 410 Long, C. N . H., 245, 248, 250, 293, 298, 300, 302, 304, 342 Long, J. M., 247, 253, 300 Long, R. G., 150, 151, 179 Loraine, J., 249, 298 Lorente de N6, R., 58, 59, 80, 81, 63, 65, 87, 88, 75, 79, 88, 116 Lowenstein, J. M., 348, 355, 370, 385 Lowry, A. H., 203, 241 Lowry, 0. H., 385, 385 Lowy, P. H., 364, 383 Lumsden, C. E., 3, 15, 19, 48, 52 Lunde, F., 402, 410 Lundsgaard, E., 319, 343 Lunn, V.,393, 397, 410 Luse, S. A., 7, 52 Lusk, G., 351, 385 Luton, F. H., 290, 297 Lynden, F., 381, 385

M McAlister, A. J., 78, 118 McCann, S. M., 251, 252, 295, 297, 298 McCarthy, D. A., 57, 117 McCarthy, J. D., 286, 295 McCleery, D. K., 84, 85, 102, 117 McConnell, K. P., 351, 386 McCoy, C. M., 203, 241 McCulloch, W. S., 58, 79, 117, 174, 175, 179, 392, 407 McDermott, W. V., 245, 250, 298 McDonald, I. R., 251, 295 McDougall, E. J., 348 Mach, R. S., 271, 298

AUTHOR INDEX

McHugh, P., 262, 295 McIlwain, H., 203, 228, 238, 240, 241, 355, 379, 386 McIntyre, A. K., 60, 116 McKeon, C., 276, 278, 302 McKinley, W.A., 56, 115 McLaughlin, J. T., 270, 271, 298 McLean, P., 361, 384 MacLean, P. D., 95, 117 MacLeary, R. A., 337, 343 McLennan, H.,209, 241 McLeod, J. G., 139, 140, 177 McMillan, P. J., 359, 386 MacMillan, W.D., 117 McMurtry, M., 259, 299 MacNichol, E. F., Jr., 125, 159, 166, 179, 181 McPhail, A., 390, 411 Macpherson, L., 125, 179 McQuame, I., 273, 298 McRuer, D.,103, 115 Madsen, A., 160, 179 Maffly, R. H., 61, 117 Magath, T.B., 350, 383 Magoon, H. W., 56, 97, 115, 117 Majno, G., 359, 385 Malamud, W.,271, 295 Malis, L. I., 152, 153, 179 Malone, E. F., 324, 344 Man, E. B., 285, 293 Mandelbrote, B. M., 289,290, 298 Manery, J. F., 199, 241 Mann, F. C., 350,383 Mann, J., 249, 296 Mansfield, E.,402, 410 Marchbanks, V. H., 258, 298 Marg, E., 135, 136, 178, 179 Margolin, S. G., 273, 295 Margolis, P. M., 405, 408 Mariz, I. K., 248, 294 Marks, I., 274, 294 Marmorston, J., 252, 300 Maroc, J., 262, 299 Marsh, J. T., 57, 117 Marshall, A., 405, 408 Marshall, N. B., 324, 344 Marshall, W.H., 139, 151, 179, 181,

425

211, 212, 213, 215, 217, 224, 225, 227, 231, 233, 240, 241, 390, 410 Martins-Ferreira, H., 225, 241 Martynink, E., 331, 345 Mason, H. L., 246, 298 Mason, J. W., 247, 254, 255, 256, 257, 258, 259, 260, 295, 296, 298, 299 Masseman, J. H., 331, 343, 398, 410 Matthes, K. J., 355, 383 Mattsson, E., 405, 408 Mattsson, N., 405, 408 Maturana, H. R., 134, 174, 175, 179 Maxwell, A. E., 285, 295 Maxwell, D. S., 64, 75, 115 Mayer, B. F., 282, 283, 285, 293 Mayer, J., 306, 316, 319, 322, 323, 324, 331, 337, 344, 345 Mead, J. F., 363, 386, 387 Meadow, L., 400, 410 Means, J. H., 289, 298 Meath, J. A., 380, 384 Meduna, L. J., 389, 390, 410 Mellinkoff, S. M., 317, 344 Mercer, D.M. A,, 81, 117 Merkin, M., 391, 393, 411 Meschan, I., 321, 344 Mestern, J., 394, 410 Metuzals, J., 249, 298 Meves, H.,199, 241 Meyer, D. R., 344 Meyer, H., 2, 7, 52 Meyer, H . E., 2, 52 Meyer, J. E., 398, 410 Meyer, J. S., 392, 410 Michael, S. T., 394, 410 Michel, F., 57, 116 Mickle, W.A., 96, 116 Migeon, C. J., 257, 258, 259, 260, 261, 263, 264, 265, 266, 292, 293, 298, 394, 407 Milici, P., 390, 409 Miller, E. R., 282, 283, 285, 293 Miller, R., 258, 298 Miller, W.H., 96, 116 Mills, I. H., 248, 298

426

AUTHOR INDEX

Minkowski, M., 144, 179 Mirsky, I. A., 256, 298 Mises, R., 273, 293 Mitarai, G., 125, 126, 179, 180, 181 Mitchell, W . A., 397, 410 Mittelman, A., 263, 298 Moersch, F. B., 290, 291, 294 Molander, L., 397, 407 Molnir, L., 392, 410 Monnier, A. M., 76, 91, 117 Monnier, M., 135, 180 Monod, J., 386, 367, 386 Monroe, R. R., 96, 116 Montagu, J. D., 391, 410 Montemurro, D. G., 344 Montgomery, D. A. D., 278, 292 Moore, F. D., 248, 256, 298, 301 Morgan, L. O., 324, 344 Morgan, R. S., 361, 362, 384 Morgane, P. J., 306, 307, 309, 310, 313, 344 Morillo, A., 158, 176 Morita, H., 115 Morrell, F., 58, 117 Morris, A. W., 286, 302 Morris, R. S., 270, 271, 301 Morrison, R. S., 225, 241 Morrison, S. D., 306, 344 Mortensen, R. A., 359, 386 Moruzzi, G.,60, 75, 113 Moser, H., 359, 385 Motokawa, K., 123, 125, 126, 127, 128, 131, 132, 133, 135, 136, 158, 159, 180, 161, 162, 163, 184, 185, 166, 167, 168, 170, 171, 172, 173, 180, 181 Mountcastle, V. B., 79, 117, 309, 342 Mowbray, R. M., 397, 399, 410 Moxham, A., 266, 297 Mozziconacci, P., 270, 294 Miiller, J., 57, 62, 76, 95, 117 Muller-Limmroth, H. W., 180 Muller-Limmroth, W., 122, 135, 180 Muller, P., 117 Mullins, L. J,, 76, 87, 95, 118

Mundy-Castle, A. C., 60, 65, 79, 91, 92, 114 Munson, P. L., 246, 299 Murakami, M., 80, 84, 118, 124, 125, 181 Murawski, B. J., 257, 258, 295, 296 Murison, P. J., 276, 293 Murphree, 0.D., 398, 410 Murray, M. R., 2, 3, 7, 38, 52 Myerson, A,, 402, 410

N Nabarro, J. D. N., 266, 299 Nadas, E., 270, 296 Naiman, J., 403, 411 Najjar, V. A,, 350, 386 Nakagawa, D., 136, 180 Nakai, J., 4, 19, 52 Nakamura, Y., 156, 176 Nakasawa, F., 319, 344 Naquet, R., 55, 115, 146, 180 Narkentin, L., 318, 346 Nauta, W. J. H.,254, 299, 311, 342 Nehlil, J., 309, 342 Nelson, D. H., 246, 247, 248, 252, 284, 266, 292, 294, 296, 299, 302, 394, 407 Nelson, P. G., 240 Nelson, R., 403, 410 Nesbett, F. B., 372, 383 Nesbett, F. J., 203, 239 Nims, L. F., 186, 241 Nissl, F., 385, 386 Noailles, J., 270, 294 Noell, W. K., 137, 180 Nokes, G., 248, 299 Norton, A. C., 137, 181 Norton, J. A., 259, 299 Norymberski, J. K., 249, 299 Nugent, C. A., 266, 298, 299 Nurnberger, J. I., 354, 386

0 Ochoa, S., 354, 383 Ochs, S., 62, 118, 227, 242 Ogawa, T., 123, 125, 127, 128, 158,

AUTHOR INDEX

427

159, 166, 170, 171, 172, 173, Pavlov, I. P., 57, 95, 117 P a y b g Wright, G., 361, 362, 384 Payne, R. W., 245, 292 Pearson, 0. H., 270, 299 Pblegrin, M. J., 84, 103, 109, 115 Pennell, R. B., 44, 52 Perkon, J., 398, 410 Perrin, G.M., 392,401,410 Persky, H.,248, 259, 260, 261, 262, 285, 293, 299, 300 Peters, G., 287, 299 Peters, J, E., 398, 410 Peters, R. A., 354, 383 Peterson, E. R., 2, 52 Peterson, R. E., 247, 248, 262, 263,

180 Oikawa, T.,123, 125, 126, 127, 131, 132, 133, 136, 170, 180 Okamoto, M.,3, 52 Okita, G. T., 359, 385 Okuda, J., 147, 149, 150, 155, 157, 162, 163, 164, 180, 181 O’Leary, J, L., 55, 63, 73, 75, 113, 117, 139, 144, 177, 180, 232, 240 Oliver, W., 389, 410 Olsen, C. W., 391, 411 Orchen, M., 353, 385 Ore, G. D., 309, 345 Orgel, L. E., 367, 384 Orlov, 0.Yu., 133, 180 Orr, A,, 256, 302 Orrego, F., 139, 140, 154, 155, 181 Orstrom, A., 324, 342 Ortiz-Calvin, 155, 179 Ostern, P., 372, 386 Ostfeld, A,, 39,50 O’Sullivan, J. B., 267, 297 Ottosson, J. O., 398, 399, 404, 407, 409, 410 Ottson, D., 124, 180 Overman, R. R., 273, 295, 301

299 Petersson, H., 392, 393, 409 Petit, D.W., 285, 301 Petsche, H.,60, 67, 115 Pfdmann, C.,313, 338, 342, 343 Pfeiffer, C. C., 390, 410 Phillips, C. C., 205, 241 Phillis, J. W., 231, 232, 240 Picchioni, A.,394, 405,407 Pickford, M.,333,342, 344 Piekenbrock, T. C., 392, 410 Piette, Y., 392, 410 Pigou, A., 54, 118, 354, 381, 385 Pilkington, T. L., 397, 410 P Pincus, C., 263, 298, 299 Pinsley, I., 390, 409 Pace, J, W., 400, 406 Pitts, F. N., 289, 299 Padula, L.,390, 409 Pitts, W., 58, 79, 117, 174, 175, 179 PQlffy, G.,392, 410 Planas, J., 319, 344 Paintal, A. S.,315,330, 344 Plantin, L. O., 276, 294 Palade, G.E., 37, 52 Platt, B. S., 350, 386 Palay, S. L., 250, 299 Patzl, O.,402,410 Palladine, A. V.,373, 386 Polatin, P., 404, 411 Pampiglione, G.,403, 406 Paoletti, P., 358, 359, 384, 385, 386 Pollack, M.,404, 408 Paoletti, R., 358, 359, 384, 385, 386 Polley, E. H., 139, 140,154, 155, 181 Polley, H.F., 270,271,272, 296, 302 Papez, J . W., 55, 117,341, 344 Poloni, A., 405, 411 Pappius, H.M.,201, 202, 203, 241 Patlak, C. S., 61, 62, 117, 200, 242 Polyak, S., 134, 180 Pomerat, C. M., 3, 4, 19, 45, 50, 52 Paton, W . D. M., 328, 342 Pon, N. G., 245, 298 Pattee, C. J., 275, 294 Ponz, F.,319, 344 Patton, H. D., 305, 345

AUTHOR INDEX

Popa, G. T., 250, 299 Pope, A., 41, 52, 380, 384 Porath, J, O., 245, 298 Poretti, G., 221, 240 Porter, C. C., 247, 299, 301 Porter, J. G., 253, 299 Porter, R. W., 252, 254, 299 Pover, W. F. R., 284,294 Powell, T. P. S., 79, 117 Press, G. D., 200, 242 Pribram, K. H., 58, 117, 309, 312, 343, 344 Price, D. B., 257, 299 Priestley, J. T.,266, 301 hitchard, M. M. L., 251, 294 Prokhorova, M. I., 373, 374, 386 Prunty, F. T. G., 245, 295 Purpura, D. P., 57, 58, 59, 73, 117, 232, 242

Reger, J. F., 48, 52 Regis, H., 146, 180 Reifenstein, R. W., 257, 258, 296 Reiss, H., 398, 398, 408 Reiss, M., 283, 284, 292, 300 Reiss, R. S., 273, 295, 302 Reitan, R. M.,287, 300 Renold, A. E., 248, 300 Reyes, E.,335, 342 Reznik, S., 390, 408 Rice, S. O., 81, 117 Richard, J. B., 256, 294 Richard, M., 287, 300 Richter, C. P., 312, 315, 318, 336, 344 Richter, D., 325, 342, 352, 363, 305, 366, 384 Riley, E., 256, 302 Rioch, D. M., 251, 254, 292, 297, 311, 344 Q Ritchey, J. O., 289, 298 Quarton, G. C., 272, 299 Ritchie, E. A,, 271, 300 Quick, A., 350, 386 Rizzo, N. D., 283, 300 Quigley, J. P., 315, 321, 344 Robbins, L. R., 289, 300 Quinn, B., 270, 271, 298 Roberts, J. M., 397, 403, 411 Roberts, S., 252, 253, 301 R Robertson, J. S., 187, 193, 200, 242 Raacke, I. D., 245, 298 Robin, A. A., 405, 411 Raben, Y. S., 245, 292 Robinson, B. W., 64, 117 Rabin, A. I., 398, 411 Robinson, C. V., 180, 242 Racamier, P. C., 289, 299 Robinson, F., 96, 117 Radzow, K. H., 321, 344 Robinson, R. J., 368, 384 Rall, D. P., 61, 62, 117, 200, 242 Roche, M., 273, 295, 302 Rall, W.,61, 85, 117 Roger, A., 55, 115 Ram6n y Cajal, S., 2, 48, 52, 55, 117, Rohdewald, M., 377, 387 134, 180 Rohmer, F., 273, 302 Ramqvist, N., 392, 393, 404, 409 Robin, L., 400, 408 Randall, R. V.,266, 301 Rollman, H., 285, 301 Ranson, J. W., 304, 305, 343 Roma, M.,203, 240 Rasmussen, A. T., 249, 299, 300 Romanoff, L. P., 283, 298 Ratliff, F., 168, 170, 171, 178, 180 Rome, H. P., 270, 300 Rauschkolb, E. W., 246, 251, 300 Ronzoni, E., 271, 295 Read, M. R., 321, 344 Rosanoff, W. R., 391, 411 Redd, D., 300 Rose, A. S., 19, 52 Reddy, W. J., 248, 257, 258, 208, Roseman, E., 392, 407 298, 300, 302 Rosemberg, E., 251, 297 Redlich, E., 276, 300 Rosenberg, B., 253, 296

AUTHOR INDEX

Rosenblatt, S., 394, 400, 411 Rosenfalck, P., 59, 78, 114, 117 Rosenthal, N., 255, 298 Rosnagle, R. S., 246, 300 Ross, D. A., 288, 301) Ross, E. J., 288, 300 Rossiter, R. J., 362, 386 Rosvold, H. E., 309, 343 Roth, M., 60, 65, 117, 118, 394, 399, 408, 411 Rothenberg, M. A,, 187, 242 Rothschild, D., 396, 411 Roux, J., 314, 345 Ruch, T. C., 305, 345 Rummel, W., 221, 240 Rumsfeld, J. W . , 253, 299 Rushton, W. A. H., 81, 116 Russell, D. S., 19, 50 Russell, G. F., 394, 411 Rybova, R., 372, 385 Rylander, B., 2, 50

S Sabshin, M., 248, 259, 260, 281, 299, 300 Sachar, E., 260, 295, 296 Sachs, H., 379, 386 Sachs, W., 369, 386 Salde, H., 394, 411 Saffran, J., 246, 300 Saffran, M., 246, 253, 260, 300, 301 Sainton, P., 274, 300 Saito, Y., 158, 176 Salassa, R. M.,266, 301 Salisbury, R., 336, 345 Sammantine, R., 250, 297 Samson, F. E., 378, 385 Samuels, L. T., 246, 247, 257, 258, 259, 260, 281, 263, 284, 288, 292, 293, 294, 299, 300, 394, 407 Sanguinetti, A., 394, 41 1 Sandberg, A. A,, 248, 293 Sanders-Woudstra, J. A. R., 58, 114 Sands, D. E., 284, 292 Saravis, C. A., 44, 52

429

Sargant, W., 395, 396, 405, 411 Sasaki, Y., 124, 181 Sato, M., 87, 117 Sato, Y., 125, 181 Saur, G.,144, 178 Sawa, M., 309, 345 Sayers, G., 245, 248, 250, 292, 294, 300, 301 Sayers, M. A., 245, 263, 300 Schachter, M., 328, 342 Schad.6, F. P., 219, 242 SchadB, J. P., 58, 82, 118 Schally, A. V., 246, 253, 300 Schaltenbrand, G., 29, 52, 311, 345 Schapiro, S., 252, 300 Schedl, H. P., 247, 248, 298, 300 Scheibel, A. B., 5, 52 Scheibel, M. E., 5, 52 Schein, J,, 270, 271, 296 Scheinberg, P., 286, 300 Scherrer, H., 57, 115, 145, 178 Scheuer, J., 247, 293 Schimizu, N., 375, 386 Schindler, W. J., 64, 75, 115, 253, 300 Schmallenberg, H. C., 271, 293 Schmidt, C. F., 241 Schmitt, 0.H., 85, 117 Schneider, J., 398, 407 Schneider, J. H., 248, 300 Schoenheimer, R., 358, 386 Scholl, D. A,, 218, 229, 242 Scholz, D. A., 288, 301 Schottstaedt, W. W., 260, 296 Schreiner, L. H., 251, 297 Schrodinger, E., 103, 117, 348, 386 Schumsky, D. A., 390, 409 Schwab, R. S., 286, 300 Sears, T. A., 60, 114 Segre, G., 102, 117 Selye, H., 256, 300 Setekleiv, J., 254, 301 Shagass, C., 403, 411 Shanes, A. M., 184, 195, 199, 203, 208, 220, 224, 226, 227, 230, 231, 235, 236, 237, 242 Shapovalova, N. S., 374, 383

430

AUTHOR INDW

Share, I., 331, 345 Sharma, K. N., 332, 345 Sharp, L. I., 396, 411 Shaw, J., 65, 117, 394, 411 Shaw, J. C., 60, 118 Sheatz, G., 57, 115 Sheehan, H. L., 275, 276, 278, 301 Shepperd, R. G., 245, 297 Sherrington, C. S., 57, 118 Sherwood, S. L., 39, 50 Shimizu, N., 325, 345 Sholl, D. A., 55, 58, 102, 103, 118 Showacre, J. L., 40, 52 Shuster, S., 265, 301 Sicheneder, T., 390, 411 Sidman, M., 255, 298 Siebert, W . M., 81, 118 Sigg, E . B., 405, 411 Sikkema, S. H., 283, 301 Silber, R. H., 247, 299, 301 Silfverskiold, B. P., 392, 411 Silva, P. S., 144, 181 Silver, S., 276, 301 Simmonds, M.,275, 301 Simon, A,, 282, 283, 285, 293 Simons, E. L., 248, 293 Simpson, M. E., U5, 298 Sinclair, R. G., 351, 386 Sines, J. O., 399, 409 Singh, B., 332, 345 Sjodin, R. A., 76, 87, 95, 118 Sjogren, B., 268, 295 Skaug, D. E., 254, 285, 298, 301 Skinner, B. F., 118, 255, 295, 301 Slater, E., 395, 411 Slater, P., 402, 411 Slaton, W. H., Jr., 363, 387 Sleeper, F. H., 282, 297 Slessor, A,, 273, 295, 302 Sloane, R. B., 260, 301, 402, 411 Sloane-Stanley, G. H., 210, 240 Slobody, L. B., 394, 410 Slocumb, C. H., 270, 271, 272, 296, 302 Slotin, L., 384 Slusher, M. A., 251, 252, 253, 301 Smart, P., 326, 342

Smith, C. J., 161, 177, 178 Smith, J. A., 402, 410 Smith, K., 390, 399, 406, 409, 411 Smith, L. L., 248, 301 Smith, M. H., 336, 345 Smith, P. E., 245, 301 Snider, R. S., 80,75, 113 Snyder, R., 289, 301 Sobel, C., 249, 296 Sobel, H., 252, 300 Sobotka, H., 394, 400, 411 Sodd, M . A,, 378, 384 Soffer, L. J., 286, 267, 270, 271, 301 Soklova, G. P., 373, 386 Solomon, A. K., 187, 195, 200, 242 Solomon, H . C., 393, 402, 408 Solomon, H. D., 402, 408 Somerfeld-Ziskind, E., 401, 412 S o m e d e , W., 273, 302 Sonnenschien, R. R., 227, 242 Soulairac, A., 305, 306, 312, 313, 315, 316, 318, 319, 32Q, 321, 322, 324, 325, 328, 329, 333, 334, 335, 336, 339, 345 Soulairac, M-L., 312, 313, 333, 334, 335, 339, 345 Sourkes, T. L., 393, 411 Spence, K. W., 96, 118 Spence, W . T., 254, 292 Spencer, W . A., 67, 75, 76, 116, 118, 205, 241 Sperry, W. M., 356, 357, 361, 382, 386, 387 Spiegelman, S., 220, 242, 386, 386 Spillane, J. D., 269, 301 Spilman, E. L., 394, 395, 405, 406,

411 Spirtos, B. N., 251, 293 Sporn, M . B., 352, 386 Sprague, R. G., 268, 301 Stadie, W . C., 372, 386 Staehelin, B., 276, 301 Stampfli, R., 382, 387 Stamm, J. S., 225, 227, 242 Stanley-Jones, D., 110, 118 Starling, E. H.,347, 387 Starr, A. M., 267, 301

431

AUTHOR INDEX

P., 285, 301 Stead, E. A., 286, 300 Steenburg, R. W., 248, 301 Steenkiste, J. N., 335, 345 Stein, M., 256, 298 Steinbach, H. B., 220, 242 Steinberg, G., 363, 387 Stellar, A., 306, 345 Stensrud, P. A., 400, 411 Stephens-Newsham, L., 285, 286, 294 Steriade, M., 150, 151, 181 Stem, T. L., 273, 301 Stetten, D., Jr., 375, 376, 378, 387 Stetten, M. R., 375, 376, 378, 387 Stevens, J. D., 283, 301 Stevenson, J. A. F., 306, 344 Stewart, M., 7, 14, 22, 29, 39, 40, 51 Stitch, S. R., 263, 300 St. Marc, J. R., 257, 258, 296 Stockham, M. A., 266, 297 Stockholm, M., 319, 342 Stokes, P. E, 393, 407 Stoll, W. A., 274, 284, 301 Stone, W. G., 3, 11, 19, 29, 30, 31, 33, 37, 39, 40, 41, 51 Stoupel, N., 150, 177 Stout, A. P., 3, 52 StoYanoff, V, A,, 356, 357, 361, 362, 386, 387 Strait, L. A., 400, 406 Straub, W., 122, 181 Strauss, E. B., 390, 411 Streicher, E., 200, 242 Strickland, K. P., 362, 386 Strisower, E. H., 370, 387 Strominger, J. L., 317, 345 Stuart, C., 390, 407 Stubbs, R. O., 249, 299 Stumpf, C.,64, 75, I15 Sudsaneh, J., 345 Sudsaneh, S., 331, 344 Sugi, Y., 59, 118 Sulzback, W. M., 392, 406 Summers, V. K., 275, 276, 278, 301 Surratt, C., 270, 272, 298 Sussman, E., 390, 410 Sutton, D., 96, 118 Starr,

Suzuki, H., 147, 148, 150, 161, 166, 167, 181 Svaetichin, G., 76, 118, 124, 125, 126, 179, 180, 181 Sweat, M. L., 247, 301 Swensson, A., 400, 407 Swinyard, C . A., 250, 294 Swinyard, E. A,, 391, 393, 411 Sydnor, K. L., 246, 301 Symington, T., 266,294

T Taira, N., 147, 148, 149, 150, 155, 161, 162, 163, 164, 165, 166, 167,180,181 Tait, J. F., 249, 297 Tait, S . A. S., 249, 297 Talbot, S. A., 139, 151, 179, 181 Taliaferro, I., 248, 301 Tane, T., 330, 343 Tasaki, I., 139, 140, 141, 154, 155, 178, 181, 186, 205, 239, 241,242 Tasaki, J., 2, 51, 59, 67, 118 Tasaki, K., 122, 123, 124, 125, 126, 127, 131, 132, 133, 136, 137, 170, 177, 180 Tasaki, N., 59, 67, 118 Taylor, J. L., 216, 242 Taylor, R. C., 392, 410 Taylor, R. E., 230, 242 Taylor, S. G., 270, 271, 301 Teitelbaum, P., 306, 338, 345 Temperley, H. N. V., 91, 114 Tepperman, J., 245, 302, 304, 342 Teorell, T., 95, 103, 118 Temer, C., 210, 241 Terzian, H., 309, 345 Teysseyre, J., 305, 306, 345 Thaler, M., 257, 299 Thesleff, S., 391, 392, 393, 409 Thiebaut, F., 273, 302 Thiel, J. H., 268, 297 Thompson, W., 362, 386 Thomson, D. L., 245, 294 Thomson, J. M . , 151, 181 Thorn, G. W., 245, 248, 257, 258,

432

AUTHOR INDEX

Vallecalle, E., 126, 179, 180, 181 Valsecchi, A., 393, 412 297, 300, 302 ValzeUi, L., 393, 394, 408, 411 Tietz, E. B., 391, 411 Van Arsdel, P. P., 276, 302 Tillotson, K. J., 392, 408 Timiras, P., 199, 200, 242, 273, 302, Van Bremen, V. L., 48, 52 Vance, V. K., 266, 302 374, 387 Vandewiele, R. L., 249, 296 Tingley, T. O., 286, 302 Vane, J. R., 328, 342 Titeca, J., 290, 292 Van Gordon, D. J., 396, 411 Toepel, W., 246, 299 Toman, J. E. P., 391, 393, 400, 411 Van Haareveld, A., 219, 221, 222, 223, 225, 227, 242 Tomita, T., 60, 64, 76, 118, 124, Van Harreveld, A., 62, 118, 390, 408, 125, 128, 137, 181 412 Tonnies, J. F., 76, 118 Van Itallie, T. B., 316, 323, 345 Torrens, J. K., 392, 407 Van Steenkiste, J. N., 345 Tosaka, T., 126, 181 van Storm Leeuwan, W., 92, 118 Towbin, E. J., 331, 345 Varjabedian, A., 396, 411 Tower, D. B., 58, 118 Vasquez-Lopez, E., 249, 302 Trethowan, W. H., 267, 269, 302 Vastola, E. F., 141, 143, 144, 145, Trolle, E., 397, 410 181 Truelove, S. C., 271, 302 Vennes, J. A., 259, 266, 294 Trufant, S. A., 271, 295 Vernikos, J., 246, 297 Truit, E. B., Jr., 390, 407 Tschirgi, R. D., 29, 40, 50, 52, 61, Verstraeten, P., 395, 412 Verzar, F., 319, 321, 348 118, 216, 242 Villegas, G. M., 62, 118 Tseu, T. K., 247, 293 Villegas, J., 126, 179, 180, 181 Tucker, B. R., 274, 302 Villegas, R., 62, 118 Tui, C. E., 256, 302 Villey, R., 309, 342 Tukahara, S., 162, 180 Tukey, J. W., 81, 91, 96, 113, 118 Vinson, D. B., 289, 300 Vogel, V. H., 400, 409 Tunturi, A. R., 73, 118 Vogt, M., 246, 302 Turchetto, E., 394, 411 Voit, C., 348, 383 Tusques, J., 291, 302 Volpe, R., 302 Tyler, D. B., 337, 343, 390, 408 von Baumgarten, R., 154, 179 Tyler, F. H., 246, 266, 299, 300 von Brauchitsch, H., 284, 302 Tyrode, M. V., 4, 52 von Braunmiihl, A., 390, 412 U von Dardel, O., 392, 393, 409 Vondehrae, A. R., 324, 344 Ucki, Y., 309, 345 von Monakow, C., 134, 181 Ueki, S., 96, 114 Ulett, G. A., 390, 399, 404, 4.08, 407, W 409, 411 Wackenheim, A,, 273, 302 Ulstrom, R. A,, 266, 294 Wadeson, R., 260, 262, 285, 293, 296 Upton, V., 247, 293 Wadsworth, R. C., 276, 278, 302 V Waelsch, H., 356, 357, 381, 362, 364, 365, 366, 381, 388, 387 Vale, J., 302 263, 286, 273, 275, 295, 298,

AUTHOR INDEX

Wagner, H. G.,159, 166, 170, 171, 178, 181 Walker, A. E., 203, 240 Walker, G., 299 Walker, R. M., 216, 227, 242 Wall, P. D., 60, 118 Wallace, E. Z., 248, 302 Wallach, S., 248, 293 Walshe, F. M. R., 57, 118 Walter, D. O., 113 Walter, V. J., 60, 118 Walter, W. G., 60, 64, 65, 79, 92, 118, 390, 409 Walters, W., 266, 301 Wang, H., 2, 7, 52 Wang, J. H., 186, 242 Ward, J. W., 54, 118 Ward, L. E., 271, 302 Warkentin, J., 318, 346 Warren, J. V . , 286, 300 Watanabe, K., 123, 126, 177, 181 Watkins, J. C., 232, 233, 240 Watson, J. D., 367, 387 Waxenberg, S. E., 290, 291, 297 Weaver, G. M., 398, 408 Webster, A. G., 60, 64, 119 Webster, G . R., 362, 387 Weidmann, S . , 231, 242 Weil-Malherbe, H., 393, 394, 400, 412 Weinberg, H., 336, 345 Weiner, H., 286, 287, 288, 289, 293 Weinman, E. 0.. 370, 387 Weiss. P., 2, 7, 38, 48, 52, 380, 382, 387 Werle, J. M., 321, 344 West, H. F., 248, 249, 296, 299 Westman, A., 250, 251, 302 White, A,, 245, 294, 300 Whitehead, T. P., 278, 279, 302 Whitehorn, J., 290, 298 Whittaker, S. R . F., 278, 279, 302 WidBn, L., 153, 158, 181 Wiener, N., 81, 91, 119 Wiersma, C. A. G., 390, 408

433

Wiesel, T. N., 79, 116, 123, 124, 125, 137, 144, 166, 167, 177, 179 Wigton, R., 399, 409 Wilcox, K. W., 397, 412 Willcox, D. R. C., 285, 295 Williams, E. W., 258, 296 Williams, R. H., 276, 302 Willingham, W. W., 337, 343 Willstatter, R., 377, 387 Wilpizeski, C., 335, 343 Wilson, H., 254, 292 Wingstrand, K. G., 249, 302 Winkler, C., 102, 119 Winokur, G., 394, 408 Wipf, H., 278, 302 Wirth, A., 161, 178 Wislocki, G. B., 250, 302 Wittenstein, G . J., 253, 297 Wittkower, E. D., 285, 286, 289, 290, 294, 298 Witts, L. J., 271, 302 Wojtowski, H., 270, 296 Wolbarsht, M. L., 159, 166, 181 Wolff, H. G., 260, 296 Wolfram, F., 19, 52 Wood, C . D., 309, 346 Woodbury, D. M., 199, 200, 242, 272, 273, 302, 374, 387 Woodbury, L. A., 245, 300 Woolsey, C. N., 151, 181 Works, A. S., 390, 409 Worthington. W. C., 250, 302 Wortis, J., 323, 343 Wortis, S . B., 391, 396, 408, 409 Wunsch, L. A., 398, 409 Wynvicka, W., 308, 346

Y Yagasaki, Y., 125, 180 Yamasaki, S., 378, 384 Yamashita, E., 127, 128, 137, 158, 159, 168, 171, 173, 180, 181 Yannet, H., 203, 242 Yanonni, C. Z., 319, 344 Yaxley, D. C., 372, 384 Yeandle, S., 186, 242

434

AUTHOR INDEX

Yokota, T., 126, 181 Young, I. J., 19, 52

Z Zabarenko, R. N., 270, 271, 298 Zaffaroni, A., 240, 302 Zamecnik, P. C., 366, 387 Zapp, J. A., 372, 386

Zeifert, M., 390, 412 Zierler, K. L., 195, 242 Zileli, M. S., 393, 408 Zingler, M. R., 273, 298 Zinneman, H.H., 286,294 Ziskind, E., 401, 412 Ziskind, L., 401, 412 Zunz, E.,323, 346

SUBJECT INDEX Anesthetics, effect on nerve membrane A permeability, 230-232 Acetylcholine, effect on brain cell culture, 39-40, Anorexia nervosa, 341 endocrine role in, 285 47 Anterior pituitary gland, effect on intestine of, 328 central nervous system and, 249Acetylcholinesterase, in brain cell cul256 ture, 4 1 , 4 9 5 0 control by hypothalamic Acetylthiolcholine, effect on brain cell neruohumoral agents, 253 culture, 7 Anxiety, endocrine role in, 261-262 A m , see Adrenocorticotropic Appetite, hormone control, neurological factors in, Addison’s disease, psychiatric aspects 30%346 of, 27%275 glucostatic theory of, 316, 322-326 Adenohypophysis, emotion and, 245hypothalamus effect on, 304308 282 intestinal control of, 318-322, 327Adenosinetriphosphatase, effect on 332 brain cell culture, 41 lipid deposits and, 317 Adrenal cortex, nervous mechanisms controlling, disorders of, psychiatric aspects of, 314-341 265-279 rhinencephalon and, 308-31 1 psychological states and, 25&282 sensory role in control of, 336-339 Adrenochrome, effect on brain cell thennoregulation of, 326327 culture, 39 Asparagine, role in cortical depression, Adrenocorticotropic hormone, 222 characterization of, 245 determination of activity of, 245- Aspartic acid, role in cortical depres247 sion, 222 emotion and, 245-282 Astrocytes, psychological effects of, 270-272 in brain cell culture, 14, 48 Alkaline phosphatase, in intestinal abmovement of, 19 sorption, 321 Atropine, Allergic encephalitis, sera, effect on effect on appetite, 334 normal brain cells, 42 use in mental disease tests, 403 y-Aminobutyric acid, Axons, potassium in, 208 effect on EEG waves, 72-73 effect on nerve membrane permeability, 232 6 2-Aminotricyanopropene, effect on Barbital, see Sodium barbital brain cell culture, 5, 29-30 Amitriptyline, as antidepressive drug, Barbiturates* effect on optic nerve, 136 405 use with ECT,401 Amphetamine, effect on food intake, 334 Bemegride, use in psychotherapy, 390

435

SuBJEcr INDEX

438

Brain, extracellular space of, 61, 198402 fatty acid synthesis in, 356.363 functional subdivision of, 54-59 glycogen turnover in, 371-379 ionic composition of, 202-207 potassium in, 209-211 protein synthesis in, 363-371 (See also Central nervous system) Brain cell culture, 1-52 brain extract effect on, 40 capillaries in, 29 enzyme activity of, 40-41 narcotics effect on, 30-31 neuron interaction in, 14 in neuropathology, 41-46 stimulant effect on, 31 Butyrylthiocholine, effect on brain cell culture, 7, 11

C Calcium, in cortical depression, 22% 221 Camphor, use in psychotherapy, 389 Cat, possible color vision in, 162 Central nervous tissue, biosynthetic activities of, 347487 control of anterior pituitary by, 249-256 drug effects on membrane permeability of, 229-238 exchangeable and nonexchangeable ions in, 207-211 ion flux in, 183-242 tracer experiments on, 185-198 Chlorpromazine, effect on plasma corticosteroids, 256 Choline acetylose, occurrence, 379 Color vision, 158-166 amtopic blue shift in, 161 Cortex, prepyriform, anatomy of, 5 5 5 6 electrical activity of, 53-119 behavior and, 96-102 distribution of, 66 evoked and spontaneous potentials of, 79-98

input-output relationships of, 102-111 signal of, 76-79 Cortical depression, 211-229 compounds effecting, 223 postmortem potassium fluxes in, 217 potassium released in, 216-217, 224, 228 prevention by divalent ions, 219221 recovery processes in, 225-229 sodium and chloride movements in, 217-2 19 “trigger substance” in, 221-224 Corticosteroids, plasma, environmental stress and, 255-256 Cortisone, effect on brain cell culture, 5, 11 psychological effects of, 270-272 CRF, characterization of, 253 Curare, use in ECT, 391 Cushing’s syndrome, psychiatric aspects of, 265-270 Cytochrome oxidase, in brain cell culture, 4 0 4 1

D Dementia praecox, as endocrine disorder, 243 Depression, endocrine role in, 262 ECT of, 395 2,4-Dinitrophenol, effect on spreading cortical depression, 224, 234-238 DOCA, in therapy of Addison’s disease, 273

E ECT, see Electroconvulsive therapy EEG waves, alpha, 65 analysis of, 53-119 cortex, behavior and, 97-102 origin of, 59 slow, in sleep, 65

437

SUBJECT INDEX

Electroconvulsive therapy, biochemical changes in, 393395 biological aspects of, 389-412 clinical effects of, 395-397 effect on brain serotonin, 405 endocrine changes in, 393-395 fatalities from, 400-402 mode of action, 403406 neuropathology of, 399-400 physiological effects of, 391-393 physiopathology of, 39-00 prognostic tests and, 402403 psychological effects of, 397399 Emotion, endocrine glands and, 245282 Endocrine system, neuropsychiatry in, 243-302 Enzymes, in brain cell cultures, 40-41, 49 Epinephrine, as appetite inhibitor, 331 effect on brain cell culture, 7, 3839, 40, 47 mental disease test using, 401-403 Eserine, effect on brain cell culture, 39--10,47 Extracellular space, of brain, 198-202 definition of, 199 determination of, 199-201

F “Feeding center,” 305-306, 307, 328, 333 Fructose, me in appetite studies, 337

G Gaucher’s disease sera, effect on brain cell culture, 4-8 Gemistocytes, in brain cell culture, 14 Gitter cells, in brain cell culture, 14 Glial cells, in brain cell culture, 1415,49 Glucagon, as appetite inhibitor, 332 Glucose, use in appetite studies, 337 Glutamic acid, role in cortical depression, 222

Glutamine, role in cortical depression, 222 a-Glycerophosphate dehydrogenase, effect on brain cell culture, 41

H Hallucinogens, effect on brain cell culture, 39 Hexafluorodiethyl ether, therapeutic convusions by, 390, 404 Hunger, see Appetite Hydrazides, use in psychotherapy, 390 Hyperthyroidism, psychiatric changes in, 289-291 Hypothalamic hyperphagia, 304 Hypothalamus, effects on feeding behavior, 304-308 Hypothyroidism, psychiatric changes in, 275-279, 286-289 I Imipramine, as antidepressive drug, 404 Insulin, effect on food intake, 334 Intestine, appetite control by, 3 1 s 321 Ion fluxes, in the central nervous system, 183242 permeability coefficient and, 190191 theoretical aspects in study of, 187198 Iproniazid, effect on food intake, 335 K Kluver syndrome, 309

1 Lactoflavin phosphate, use in appetite study, 319-320 Lateral geniculate body, 138-145 anatomy of, 138-139 binocular interaction in, 144-145 electrical activity of, 139-142 neurons, excitability of, 142-143

438

SUBJECT INDFX

Leumtomy, use in depression therapy, 405 Limulw mmutidkr eye, border contrast effect in, 168-171

LSD-25, effect on brain cell culture, 28,3940 effect on food intake, 335 Lysergic acid, use in study of stress, 259 Lysergic acid diethylamide, see LSD25

M Malic dehydrogenase, in brain cell culture, 41 Mania, endocrine role in, 26%263 Megaphen, use in psychotherapy, 390 Mental disorder, endocrine role in, 261-265 Methacholine, classification of mental diseases by, 402 Methionine, use in study of protein synthesis, 363364 N-MethyI-3-piperidylbendate, effect on brain cell culture, 39 Metrazol, effect on optic nerve, 136 use in psychotherapy, 389, 404 Monamine oxidase inhibitors, as antidepressant, 404 Mucopolysaccharides, in oligodendrocytes, 14, 19 Myanesin, in optic nerve study, 136

N Nerve tissue, ionic composition of, 203 potassium in, 208-211 Nerve tumors, diagnosis by tissue culture, 46 Nervous system, electrical fields of, 69 Neurons, number of in cerebral cortex, 383 reaction with glial cells, 22 sodium in, 206

Neuropsychiatry, endocline system and, 243-302 Nissl granules, stains for, 7 Norepinephrine, as appetite inhibitor, 331 effect on brain cell culture, 40 in mental disease tests, 403

0 Optic nerve, antidromic acition potentials of, 131-133 centrifugal fibers of, 134 fibers, 130-131 types of, 174 impulsive conduction in, 129-134 neurograms of, 133-134

P Pattern vision, 166-175 Pentobarbital, effect on EEG waves,

73 Pentylenetetrazole, effect on brain cell culture, 31, 37, 40 Phloridzine, use in appetite study, 319, 320 Pituitary disease, psychiatric aspects of, 265-279 Potassium, in nervous tissue, 208-211 transport in nerve, 227-228 Procaine, effect on nerve membrane permeability, 230-232 Psychiatry, thyroid and, 282-292

R Reserpine, effect on food intake, 335 effect on plasma corticosteroids, 256 Retina, information transfer in, 122-134 lateral conduction in, 126-129 neuronal layer of, 124-126 receptor potential of, 122-124 role in pattern vision, 174-175 Rhinencephalon, feeding behavior in, 308-311

439

SUBJECT INDEX

Rhodopsin, use in retina study, 122 Ribonucleic acid, “messenger,” 367 Ringer’s solution, ionic composition of, 203

Succinoxidase, effect on brain cell culture, 4 1

S

Taraxein, effect on brain cell culture, 45 Taste, appetite and, 337 Thyroid gland, psychiatry and, 282292 Transaminase, serum, in ECT, 394 Trypsin, use in brain cell culture, 4

Saccharose, effect on feeding, 337 “Satiation center,” 333 Schizophrenia, electroconvulsive therapy of, 396 endocrine role in, 263-265 radioiodine uptake in, 283-285 sera, effect on normal brain cells, 42-45 Seawater, ionic composition of, 203 Serotonin, effect on brain cell culture, 28, 37-

T

U Uridine, role in glycogen synthesis, 378

38,40 V in electroconvulsive therapy, 394 Vasopressin, biosynthesis of, 379 effect on food intake, 335 Vision, information transfer in, 121Sodium transport in, nerve, 227-228 181 Sodium azide, effect on spreading Visual cortex, 151-155 cortical depression, 224 role in color vision, 162, 166 Sodium barbital, effect on brain cell Visual pathways, culture, 30 corticifugal influence on, 158 Sodium cyanide, effect on spreading corticipetal influence on, 155-157 cortical depression, 224 Visual transmission, Sodium 1,3-&methylbutylethyl barbinonspecific afferents and, 145-151 turate, effect on brain cell culture, 31 Y Strychnine, in optic nerve study, 136 Succinic dehydrogenase, effect on Yohimbine, in mental disease tests, 403 brain cell culture, 44

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