International Review of Neurobiology Volume 3

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INTERNATIONAL REVIEW OF

Neurobiology VOLUME 3

This Page Intentionally Left Blank

INTERNATIONAL REVIEW OF

Neurobioloav -

-

-

" I

€dited by CARL C. PFEIFFER N e w Jersey Neuropsychiatric lnstitufe Princeton, N e w Jersey

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

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

VOLUME

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

C. A. K. S.

Hebb Hoffer Killam M8rtens

3 1961

ACADEMIC PRESS

New York and London

Copyright 0, 1961, by Academic Press Inc. ALL RIGHTS RESERVED

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

ACADEMIC PRESS INC. 111 FIFTHAVENUE NEWYORK 3, N. Y . United Kingdom Edition Published by ACADEMIC PRESS INC. (LONDON) Lm. 17 OLD QUEEN STREET,LONDONS.\V. 1

Library of Congress Catalog Curd Number 59-1 3822

PRINTED IN THE UNITED STATES O F AMERICA

CONTRIBUTORS VAHE E. AMASSIAN,Depurtnrcnt 01 Physiology, Albert Einstein College of Medicine, Urons, N e w York A. BAIN, Departnicnl of Phnrnmcology, Emory University, Atlanta, Georgia

JAMES

EDUARDO DE ROBERTIS,Inslitiitc, of Gciierul Anatomy, Facultad cle Merlicina, Buenos Aires, Argentina H. h4. GERSCIIENFELD, Instilzitc of General Anatomy, Facultad de hledicinu, Bucnos Aircs, Argeiitina HIROSHINAKAIIAMA,Department of Physiology, School of Medicine, Keio University, Shinjukri-kii, Tokyo R. RODNIGHT,Department of 13iodieinistry, Institute of Psychiati y, Mazidsley Hospitul, Lontlon, England D. R. VOWLES,Institute of Experimental Psychology, University of Oxford, Oxford, E ngltr n tl ARmun A. WARD,Jn., Difiisioii of Neurositrgery, University of Washington School of Mctlicine, Seattle, Washington G. R. WEBSTEII, Deparlment of Cheinical Pathology, Guy’s Hospital Meclical Scliool, London, Englund

HARRYL. WILLIAMS, Depurinicnt of Pharmacology, Emory u n i versity, Atlanta, Georgia

V

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PREFACE The present era is one of the explosive growth of science in terms of its scope, range, and the volume of work done and number of publications. Each scientific discipline and sub-discipline is the responsibility of the appropriate university department and the research organizations attached to hospitals, industry, etc. The important task of liaison between these various fields is, however, no one’s particular responsibility, in spite of the fact that many important new hypotheses can arise only from areas where different disciplines overlap. Something is being effected by the construction of the multidisciplinary research teams but much more still remains to be done. It is therefore a major aim of this Review to provide a forum where the many major and different sciences that make up neurobiology can present the latest progress in these fields for the edification, not only of scientists working in the same science, but also of those working in other disciplines.

CARL C. PFELFFER JOHN R. SMYTHIES

September, 1961

vii

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CONTENTS CONTRIBUTOR> ................................................

v

PREFACE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

vii

Submicroscopic Morphology and Function of Glial Cells EllU4RDO DE ROIILRT15 1x0 H . xf . GI R>CHENFEI D I. 11. 111. 1 v. V.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Submicroscopic Morphology of Gliul Cells . . . . . . . . . . . . . . . . . . Fuiictional Significance of Astroglia . . . . . . . . . . . . . . . . . . . . . . . . I.”incticinal Significance of Oligodendroglia . . . . . . . . . . . . . . . . . . General Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 3 28 52 59

61

Microelectrode Studies of the Cerebral Cortex E.

\’.AlIl?

AhLISSIAN

I.

Technical Proccdnrcs in Obtaining. Localizing. and Handling of Unit Recordings . . . . . . . . . . . . . . . . . . . . .. I1. Classification and Intcrprctation of Single Cortical Neuronal Spikes ancl 1ntracellul;ir \Vaves . . . . . . . . . . . . . . . . . . . . . . . . . . . I11. Microelcctrode Analysis of Brain Waves and the Relationship of Slow Waves to Unit Activity ............................. IV . Patterns of Unit Kespnnsc to Spccific Thalamocortical Affcrcnt Volleys . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V . Patterns of Unit Response to Direct Cortical Stimulation . . . . . . V I . Patterns of Unit Respoiisc to Corticocortical Afferent Volleys . . VII . Integrative Responses l o hlincd Corticipetal Volleys . . . . . . . . . . ....... VIII . Discussion and Summary References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

68 76 89 109 123 124 125 126 131

Epilepsy AHTHUR

A.

WARD. JR .

................... I . introduction . . . . . . . . . . . . . . . . . . . . The Epileptic Focus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I1. I11. 1V V VI

. . .

The Seizure . . .... Propagation of the Seizure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Precipitation of Seizures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding Remarks ........... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

137 140 169

177 181 181 183

CONTENTS

X

Functional Organization of Somatic Areas of the Cerebral Cortex HmosHr NAKAHAMA I . Introduction ........................................... Afferent Projections ..................................... I11. Efferent Projections ..................................... IV. Regulatory Mechanisms .................................. V Corticocortical Projections ................................ VI. Behavioral Conditioning Studies .......................... VII Conclusions ............................................ References .............................................

.

I1

. .

187 191 202 215 225 236 240 242

Body Fluid Indoles in Mental Illness R. RODNIGHT I. Introduction ............................................ Normal Body Fluid Indoles .............................. Body Fluid Indoles in Mental Illness ...................... Concluding Discussion ................................... Summary .............................................. References .............................................

I1. I11. IV. V.

251 254 265 284 287 288

Some Aspects of Lipid Metabolism in Nervous Tissue G. R. WEFISTER I. I1. I11. IV V.

.

Introduction ............................................ Lipid Turnover in Nervous Tissue ......................... The Breakdown of Lipids ................................ The Action of Lysolecithin on Brain ....................... Summary and Conclusions ............................... References .............................................

293 294 303 308 313 314

Convulsive Effect of Hydrazides: Relationship to Pyridoxine HARRY L . WILLIAMS AND JAMES A. BAIN

.

I 11. 111. IV. V. VI . VII .

Introduction ........................................... 319 General Pharmacological Responses to the Hydrazides ........ 321 Neurophysiological Aspects of Hydrazide Action ............ 325 Biochemistry of Hydrazide Action ........................ 329 Neuropathology and Clinical Aspects ...................... 339 Miscellaneous .......................................... 341 Summary .............................................. 343 References ............................................. 344

CONTENTS

xi

The Physiology of the Insect Nervous System D. M. VOWLES

..................... I. Introduction ..................... I1. Sensory Physiology ................. ..................... I11. The Insect Central Nervous System ........................ References ..............................................

351 364 371

AUTHORINDEX................................................

375

SUBJECTINDEX ................................................

392

349

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SUBMICROSCOPIC MORPHOLOGY AND FUNCTION OF GLIAL CELLS’ By

Eduardo De Robertis and

H. M. Gerschenfeld

lnstituto de Anatomia General y Ernbriologia, Facultad de Ciencias M.+dicas, Buenos Aires, Argentina

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Submicroscopic Morplrolo~yof Glid Cclls . . . . . . . . . . . . . . . . . . A. Astrocytcs . . . . _. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Oligodcnclrocytes ................................. C. Intermediary Typc f Glial Cclls . . . . . . . . . . . . . , , , . . . . . . D. Special Types of Glial Elcmcnts . . . . . . . . .......... E. hlicroglial Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. Functional Significance of Astroglia A. Astroglia and the Extracellular B. Astroglia and the Watcr-Elrctrol C. Astroglia and Cerebrospinal Flui D. Extracellular Spacc and tlie Sothe CNS . . . . . . . . . . . . . . . . . . . . . . . . . E. -4stroglia and “Edema” of thc CNS . . . . . . . . . . . . . . . . . . . . F. Astroglia and the Physiological Barriers of the CNS . . . . . . G. Astroglia and the Pcrmclnliilit the CNS . . . . . . . . . . . . . . . . . . . . . . . . . H. Neuroglin and Ncaronnl Activity . . . . . . . . . . . . . . . . , . . . . . IV. Functional Significance of Oligodc A. Oligodendroglia and Ccllular the CNS . . . . . . . . . . . . . . . . . . . . _ .. .... ..... B. Oligodendroglia and tlie Di\posaI of hl! clin in Brain Lcsions V. General Conclusions . . . , . . , , . , . . . . . . . . . . .. . . . . References . . . . . . . . . . . . . . . . . , , . , ,

I.

1 3 6

18 19 19 28 28 28 33 34

35 37 39 44 47 52 52 58 59 61

Introduction

Although the different types of glial cell constitute quantitatively an important part of the central nervous system (CNS), 1 The original work presented in this paper has been supported by a grant of the National Multiple Sclerosis Society of New York.

1

2

EDWARD0 DE ROBERTIS AND H. M. GERSCHENFELD

until recent years their special functions and relationships with the neurons and vascular elements were practically unknown. The classic work of the Spanish school (Cajal, Rio Hortega) defined the different types of glial elements-astroglia, oligodendroglia, and microglia-and their histogenetical relationships but were unable to throw definite light on their physiological significance within the realm of the CNS. These classic studies, well summarized by Penfield (1932), Rio Hortega (1942), and Glees (1955), marked the end of the purely morphological and descriptive analysis of glia and after them the interest of investigators waned. Only in recent years with the development of new cytological and cytochemical techniques-e. g., the culture of the different strains of glial cells, the methods for mass isolation, enumeration and biochemical study of glial elements, and submicroscopic analysis with the electron microscope-has the interest for these cells been revived and new data been accumulated about their possible role in myelination, nerve conduction, and brain permeability. Most of these modern approaches are considered in the recent symposium on the “Biology of Neuroglia” edited by Windle (1958). The present work is not intended to duplicate or bring up to date any of the above mentioned reviews of the field. Emphasis will be essentially concentrated on the submicroscopic analysis of the CNS and on the physiological implications of this study. The conclusions reached will be mainly based on the results obtained in our laboratory. The analysis of the CNS with the electron microscope is of paramount importance since large territories of it entirely escape study with optical instruments. In the gray matter more than half of the volume is composed of elements of submicroscopic dimensions that cannot be seen by the light microscope. This lack of information has given rise to many faulty interpretations and theories that are still in vogue. It is only with the high resolution provided by the electron microscope that it is possible to demonstrate that these vast territories are filled with tightly packed neuronal and glial processes and nerve endings leaving no real extracellular space for interstitial fluid or for a fundamental intercellular substance. This type of analysis permits a direct observation of the relationship of the

MORPHOLOGY AND FUNCTION OF GLIAL CELLS

3

astroglial cells to the capillary wall and to the active surface membrane of the neurons and the synaptic endings. It also illustrates the fine intracellular changes that occur in the oligodendrocytes during myelination. The material of this essay will be divided into four main parts. The first part (Section 11) will be dedicated to the submicroscopic analysis or ultrastructure of the two more important types of glial cells of the CNS: astroglia and oligodendroglia. The special types of glial cells and microglia will not be considered. In Section I11 the morphological data on astroglia will be discussed and interpreted and several experimental approaches to the study of the water and electrolyte metabolism of these cells will be mentioned. Furthermore the functional significance of astroglia in relation to water and ion exchange, capillary permeability, and the physiological barriers of the brain and their possible role in excitation and synaptic transmission will be discussed in a critical and speculative way. The importance of an integrated correlation with physiology and pharmacology at a subcellular level will be emphasized. In Section IV the ultrastructure of the interfascicular oligodendrocyte of the white matter will be considered in relation to the cellular mechanism of lipoprotein synthesis in myelination and to the destruction and disposal of myelin in some experimental lesions of the brain. In the last section some of the general conclusions about the submicroscopic morphology and function of glial cells will be presented.

II.

Submicroscopic Morphology of Glial Cells

Structural analysis of the central nervous system with the socalled selective histological techniques is hindered by the fact that only partial views of the total organization are obtained. Thus the methods for the demonstration of astroglia, oligodendroglia, microglia, myelin sheaths, Nissl substance, and so forth emphasize only one component at a time and do not permit a spatial integration of all structural elements. On the other hand, with the electron microscope, all these and other components can be visualized simultaneously in thin sections fixed in osmium textroxide and can

4

EDUARDO DE ROBERTIS A N D H. hl. GERSCHENFELD

FIG. 1. Astrocpte ( a s t r ) , two oligodendrocytes ( olig), and onc microglial cell (micro) in the white matter of an adult rabbit near a stab wound; nu: nucleolus. Magnification: x 8000.

6

EDLCTA\IIDO DE ROBEHTIS .\ND H . hi. CERSCHESFELD

be followed in their topograpliicd relationships down to diliiensions beyond the rcwlving p v e r of the optical microscope. \\;it11 this technique, the different tyiies of glial cells tliat mist in the central nervous system may be recognized, mid their inorpliology, their relationship to ncr~rons, nerve f i h c ~ s and cwclings, antt so fort 11 can be tle t c w n i n e :l. The identification of tlie differcwt tvpes of glial cc~lls\\.it11 tlie electron microscope is not, liowe\~cr,without tlifiiciiltics iuitl this has led to conflicting views ;iiiiong tlie \vorkers pionc.c~riiigin this field ( De Kohertis, 1955; Lrise, 1956; Farqiiliar and Hartinmn, 1957; see \Vintlle, 1958). In oiir description \ve shall f o l h t - the itlentification c.ritcLriii set by Furqiihar and Hartinaim ( 1957 ) ( SCY also IIartmann, 1938; Scliultz cf (11. 1957; Dc. Holwrtis cf ul., l958a) tor t l i c ~r c u p i t i o i i of tlie tlirw tvpes of glial cc~lls.

Astrocytes c i ~ i1)e recognizcd I)y scvcriil niorphological C l i i l r xtcristics pertaining to tlic nricleiis, c!~toplasm, ant1 tlic, ccll pro cr sscs . Th 11iiclci is is irrcg ii 1;ir I ? ovoi tl an tl frcqii c ~t 1Ji s 11o11-s a wcl1- t l c h c d 11 ucl ear m cml)ranc~ and a 11i d e()I I IS wit 11 I I s 11[ )iig!’likc structure (1 Fig, 1) . Cliromatin is contlenscd n n c l c ~tlic nuclear !iieinl)riinc~,antl tlierc. irre tlcfinitc. s p c m oecirpietl by niiclwr sap. 1h r cytoplasm is cliaractc~rizcdl y tlitx IOW clectron tlcnsity of the inatris whicli gives to it ;in c ~ i i p t yor watery aslwct. This aspect has hcwi obscwc>tl hy i i s in astrowtes of tlie r;it ( Fig. -3) and cat ( l k I
S).

Sevcr‘d cell organoid\ C ~ I I >be recognizrd in the ‘tstroc) tc. \Iitocliondria are ratlier 1.1rg~ ‘15 coinl~,ired\vith similar clemeiits within the neiironnl element\ or t h e oligode17tlrocytc.s. L O I Imito~ chondri,i can he o b s t ~ v e t l\vitlrin tlrc cell proccssc\ a n d i n tlre

I ~ I c . 2. Astroglia frotii a .5-cIay-111~1rat sliowing tlw chiractcwstic ck>;ir cytoplasm and n large pro In1 < t i i d 1 astrogli,il pro t h r vicinity of tlw cell ( u p ); A’: I I I I ( , ~ C Y I S ,t i i i : ~iiitochontlria,G : Chlgi siilxtancc~, PI’: cndopl:ismic rcticuliin. hlagtiification: >< 30,000.

vascular foot. A few scattcrcd lirofil(,s sirrroundetl l y ri1,osomc.x are fomid around t h c nucleris a n t 1 in t h e cell processes. Thcy liecome more conspiciious at the vascular feet (see Section 111, F ) . Also small tlictyosome-like eleinriits o f the Golgi complc>w with

s

EDUhHDO DE ROBERTIS AND 11. 11. GERSCHENFELI)

FIG.3. Astrocytc of thc Iruinan cerelxd cortex showing the clwr cytciplasm and astroglial 1)roccsws ( ( i p ) : Ax: axon, A’: niicl(ws, f t t i : mitoclrontlria. hIagnification: x 12+500.

10

EDUARDO DE ROBERTIS A N D H. M. GERSCHENFELD

FIG.4. Greatly swollen astrocyte of the rat brain incubated for GO minutes in isotonic fluid without glutamate. The nucleus ( N ) , mitochondria ( mi),and the astroglial processes ( u p ), are also swollen; er: endoplasmic reticulum. Magnification: x 14,000.

12

EDUARDO DE ROBERTIS AND H. M. GERSCHENFELD

FIG.5. Nervous tissue incubated for 60 minutes according to Pappiim and Elliot. All astroglial processes ( u p ) are notably swollen and the dendrites ( d ) are well preserved. There is no extracellular space; er: endoplasinic reticulum, Ax: axon, mi: mitochondria. Magnification: x 14,000.

14

EDUARDO DE ROBERTIS AND H. M. GERSCHENFELD

their parallel membranes and small vesicles can be observed near the nucleus. Farquhar and Hartmann (1957‘) have described dense “grapelike structures” in the cytoplasm of astroglia. These cor-

F I G . 6. Astrocyte of the huinan brain cortex near a tumor. The cytoplasm is swollen and contains lipochroirie inclusions ( l i ) ; N : nucleus, nu: nucleolus, Ax: axon, er: endoplasniic reticulum. Magnification: x 16,5GO.

respond to lipochromes and are found mainly in adult and old animals. In Fig. 6 some of these dense inclusions in a human astrocyte are shown.

MORPHOLOGY AND FUNCTION OF CLIAL CELLS

15

The astrocyte processes can be recognized by their clear cytoplasm and well defined limiting membrane. In the cross section they are round or oval and may show mitochondria and a few vacuoles. These processes are of different width depending on the distance from the cytoplasm. The electron microscope studies do not permit us so far to establish a clear difference between the two classic types of astroglia. Astrocytes of the white and gray matter (protoplasmic and fibrous astrocytes ) have essentially the same submicroscopic organization. It is possible that the two types of astrocytes seen with light microscopy may depend only on the size and shape of the cell processes. In what may be interpreted as protoplasmatic astrocytes the processes are larger, shorter, and more irregular. In the so-called fibrous astrocytes the cell processes are thinner and longer constituting the “glial fibers” seen with light microscopy. The electron microscope clearly demonstrates that the “glial fibers” are not extracellular, as was supposed by Weigert at the end of the last century, but cytoplasmic expansions that extend from the cell body and are probably endowed with flowing movements as those revealed in tissue culture (Pomerat, 1958 ) . The problem of glial fibers is also related to that of the g l i d fibrils. Although most of the astrocytes of the gray and white matter do not show intracellular fibrils, these may be found in certain regions of the CNS within the cytoplasm and in the astrocytic process. Observations of this type were made by us in regions of the brain stem near the fourth ventricle. Recently Gray (1959) has observed bundles of fibrils 100 A or less in diameter within astrocytic processes in the occipital cortex of the rat. Similar fibrils were previously observed by Luse (1956) in normal and pathological glia and by Fleischauer (1958) in reptilian glia. In unpublished observations we have found that in the astrocytes surrounding a stab wound there are fine intracellular fibrils that increase in number with the time after the oper at’ion. From the morphological viewpoint it seems evident that these glial fibrils are similar to those found in different epithelial cells. The early claim that gliofibrils were of collagenous nature should be dismissed For more details on the glial fibrils see Bairati ( 1958). By their long and numerous processes astrocytes establish close connections with the other components of nerve tissue and

16

EDUARDO DE ROBERTIS AND H. M. GERSCHENFELD

particularly with the blood capillaries and pial membrane (Figs. 7 and 8 ) . The relationship to the capillaries by means of the so-called vascular feet is of particular interest in connection with their probable role in the barrier mechanisms of the CNS and will be described in the corresponding Section 111, F. Glial

FIG.7. Astroglial process ( u p ) surrounding a capillary ( C a p ) vessel (vascular foot) ; bm: basal membrane, mi: mitochondria, er: endoplasmic reticulum, d p : dense particles. Magnificati'on: x 24,000. See description in the text.

processes may also have close relationships with the nerve cells and oligodendrocytes of the gray matter. The soma and processes of neurons are not only related to perineuronal oligodendroglia satellite cells and nerve endings but a larger or smaller portion of their surface (depending of the neuronal type) is in direct

FIG. 8. Diagram showing the different topographical relationships of an astrocyte of the gray matter. Processes adjacent to a capillary ( C u p ) or vascular foot, to the pioglial membrane, and surrounding a nerve cell are shown. The position of the so-called blood-brain barrier ( BBB ), liquor-brain barrier ( L B B ) and synaptic barrier ( S B ) are indicated. The thick arrows represent the possible movement of fluids and solutes within the astrocytic cytoplasm. A perineuronal oligodendrocyte is also indicated. The drawing emphasizes the clear cytoplasm of the astrocyte and astrocytic processes and the lack of tnie extracellular space. An extraneuronal recording microelectrode with its implantation into the glia is shown (end., endotheliwn, has. m., hasement membrane).

18

EDUhHDO DE ROBERTIS .\ND 11. XI. (XRSCIIESFELD

relationship with the clear processes of the astrocytc, ( Figs. S and 9 ) . In the neiiropil regions of the gray matter, the astrocyte procmses fill all intervening specs in betwccm the tlendrons, iixons, and mye1iiintc.d nrrve fibers. In the adult animal all cells, cclliilar procvsses, antl v a s c ~ ~ l u r tleinents of the CNS are tightly packcd among tl~cnisclves,antl there is an exact corrcsponclcncc o f the imm1ir;ines h i v i n g 110 extracellular sixices i n bctwccvi thein. A distance of only 120-2.50 A is foinid betwew tlie adjacent mernliranes ;ind this is filled wit11 a material of definitc electron clcmsity ( lle Rolwrtis ct ol., 1938a, 13). The lack of real estracellnlar spice: in the adult animal is o f great physiological interest m c l nil1 be discussed at lcngtli in Scction 111. It is important to point out liere that in tlie developing CNS true spices may Iw foiind in Iwtweon tlie celldar clrnients (see Fig. lo), h i t t h e disappcxr s o o n aftcr birth lw tlic~ h i d i n g togcther of all cell meinliranes.

H. OLIGODEXD~KX :Yms Following tlie criteria laid down liy Rio Hortega (192S), oligodendrocytes can tmily 1)c recognized untlcr the clectron microscope in two distinct locations: ( N ) iicar the lxrikiir!.on of rieurons \diere they constitute t.he perinciironal satellites nntl ( 11 ) in 1,etwecn tlie I~undlcsof nerve fibers of white matter \VI~CYC they constitute ro\vs of intrrfascicular cclls. Perincirroncil oli~ot/[.nt/rocyfesarc srnall cells with scanty cytoplasm and short thin processes. The nucleus is roiintl or o v d and lias blocks of condensed chromatin, separated by nuclear sap. Sometimes a sinall dense nucleolus can lie observed ( Fig. 11) . The cytoplasm sliows a m i d i higher electron density that the s mitochondria, antl is crowded astrocyte, contains i i i i ~ n e r o ~small with dense ribosomes. As shown in Fig. 12 two oligodendrocytes m a y sometimes be seen side by side ( t\vin-satellites) located in a depression of the ncuronal cytoplasm. The density of both types of cells is somewhat similar and contrasts with the clex cytopliisni of the surrounding astrocytic processes. These c,lectron microscope findings are in agreement with the recent microchemical study of Hytlcn and Pigon ( 1960) demonstrating, in isolated perineiironal oligocytes, that there is high dry matter, protein, and RN.4 content, and a liigli energ!. incta1)olisni.

Thc oli~or:oirll.oc,!/Ccsof /lie tcliite multcr have been descri1)cd at length hy IIe h b e r t i s [,/ t r l . ( 19.58a). In the \diitc matter of \miiig animals during m ~ ~ l i n a t i o the n oligodcndrocytes arc in intimate relationsliip with tlie axons ( Fi g. 13. ) T he cytoplasm is inota1)ly dense ant1 slion~s1)rofilw of tlie cmdoplasinic reticulum, iiiiiiicroiis RNA particles, iii(>iiil)rmcs of the Golgi coinplex, and mitochondria. T h e iiucltws is roriiitl and sliows a doiible nuclear cmvelope and condenset1 c:ironiatin constituted by fine filaments. In longitutlinal sections tlic oligocytes arc' in ro\vs l)et\t7een the nerve fibers. In transversi. swtions the intirnatr relationship v i t h the myelinating iisoiis is l)c.ttc,r ol)servecl ( Fig. 13). T h e iiwns are iir;ictically embcddetl \vitliin tuinncls that cross through the oligotlcmtlrocyte cytoplasin. The nicinbrane of tlic axon is in close contact wit11 the siirf'ace n r c . i i i l ~ r i i i i ( of ~ tlic cell constituting \\.hat we have called t he axon-oligoc!+c ineiinl~raiie ( AO!vl ) complex (Fig. 13) ( De Robertis (11 ol., 1938a). T11e myelin sheath, tliat is being laid clo~vn,is coirtaiiictl cintirely within the cytoplasm of the oligoclentlrocyte. As we slid1 see 1atc.r in Section IV, the oligoclcndrocyte of \vhite matter seems to be prii~cipillyrelated to the syntlie ~naintenanceof tlie inyeliii slic~itli in the normal anirnnl antl to the disposal of it in some pt l i ol ogi cd conditions.

TYPESoi+' (:LIAI. C ~ L L S C. INTERLIEDIARY Farqiihar and Hartnianii ( 1%7) have described cells that may have morphological cliar:ictc.ristics ( 110th of the nucleus antl cytoplasm ) in l~et\i,eentlw t!Tpiciil astrocytes ant1 oligodendrocytes. Although trai~sformations 1)ctnww the t\vo ct,ll types m a y b e p(;ssil)le (Rio Hortega, 193-7), VY: think that at prcsent it is still premature to speak of tIic.sr traiisitional forms. hluch work is iieeded on tlie iiornial atliilt glial cvlls, and tlie entire field of glial tlevelopment and liistogcvic~sis still recjiiires flirther study at the siibinicroscopic level. D.

SPECIALTYPESOF G L I A I1.':l>Khll.:P;'l'S

O t i ~ e rspecial types of glial cclls siicli ;IS tlic epentlyma cclls ( Palay, 1958) the pituicytc%s ( l'aliiy, 1958; Gerschenfeld ef al., 1960) of the iieuroliypopliysis, tlic. hliiller cells o f the retina lvill not lie consitlcred in this rcxviciv.

Fic:. 9. To tlic lo\\w right a large cleiisc ncurori ( N ) of the vc.ntr;il acoiistic iinca pig. 'rile edge of tlw I I ~ . L I ~ O I Iis surrorintlctl by c l r x ' ( o p ) and two synaptic endings (.y)fillctl with synaptic vesicles ( S D ) . ' l l c liiiiit of tlic cnding is intlicatctl with arrows. The disposition of the glia siiggcst its prolxilhle action iis syniiptic 1)arric.r; tni: niitoc~lroiitlri;~. \lagnification: x :36,000.

22

EDUARDO DE ROBERTIS AND H. M. GERSCHENFELD

FIG.10. Brain cortex of a rat embryo of 20 days of age. Note the existence of true extracellular spaces (es) in between the different cell components: d : dendrite, nc: nerve cell, Ax: axon. Magnification: x 36,000.

Fit:. 11. l’criiieiiroiid olifii)dciidrocytc, adjaccnt to a ncrve cell. W i c ~contact is intlicated with arrows. Note tlic tliflcrerice in elcsctron tlcnsity \\.it]) the tistrocytic processes ( o p ) ; mi: riiitoclrontlria, i t i i : nucleolus, A’: nuclvris. \ 1 q nification: x 15,000.

FIG. 12. Twin perjiiwroii;il oli~otlt.ndroc]vtesin contact with :I i i c n ~cr.11 ., Ax: iix011. hlagiiification: x 11,000. (arrows); u p : astroglial proc

26

28

EDUARDO DE ROBERTIS AND H. M. GERSCHENFELD

E. MICROGLIAL CELLS ihlicroglial cells have been described by Farquhar and Hartmann (1957) and Schultz et a,?. (1957). These cells have an elongated and very dense nucleus. The cytoplasm is also notably dense showing mitochondria and numerous small dense particles, and sometimes irregular inclusions of dense material presumably phagocytosed debris. The microglial cells are more numerous near lesions in the brain as shown in Fig. 14.

FIG. 14. hlicroglial cells (micro) in the brain of an adult rabbit near a stnb wound (ncte the high density of the cytoplasm); Ax: axon, N : nucleus. Magnification: x 11,000.

Ill.

Functional Significance of Astroglia

AND THE EXTRACELLULAR SPACE A. ASTROGLIA

As was mentioned previously, the first electron microscope observations on sections of the central nervous system ( Wyckoff and Young, 1954; De Robertis, 1955, Dempsey and Wislock, 1935)

MORPHOLOGY AKD FUXCTION OF GLIAL CELLS

29

that have been confirmed later by many other workers (Luse, 1956; Horstmann, 1957; Niessing and Vogel, 1957) showed that the glial elements fill all intervening spaces between the neurons and the vascular elements and that no real extracellular spaces are present within the central nervous system in the adult animal. The plasma membranes of all cellular components of the nervous tissue are in intimate contact with themselves and with the basal membranes of the capillaries. A distance of only 120 to 250 A can be observed between the adjacent membranes, and this is filled by a material of definite electron density. These results cannot be considered artifactual, as van Harreveld (1957, van Harreveld and Schadk, 1959) has proposed, based on a supposed increase in size of dendrites with consequent diminution of the extracellular space after fixation. The conditions of fixation for the electron microscopy have been sufficiently analyzed by several authors ( Palade, 1952; Rhodin, 1954; Fernhndez-Mor6n and Finean, 1957) and there is sufficient evidence to establish the good preservation of the tissue. In our studies of the CNS careful control was applied to pH, osmotic concentration, ionic composition, and also to the oncotic pressure of the fixative by the addition of polyvinylpyrrolidone. Furthermore on pure morphological grounds it would be difficult to explain the fact that the distance between the cellular components of the adult CNS is always 120-250 A on the basis of artifacts. The good preservation by osmium fixation has also been demonstrated in certain experimental conditions. Elliot (1959) has observed that osmium tetroxide preserves the swelling of the material incubated in vitro whereas formaldehyde does not. Recently Horstmann and Meves (1959) have made calculations of the “extracellular space” on electron micrographs of the CNS of Scylliorliinus. Rased on measurements of the mean diameter of the nerve fibers and other cell processes and on geometric consideration they concluded that this space cannot exceed 5% of the total volume of the CNS. This calculation must be even less in the CNS of mammals which contain more myelin and probably a higher percentage of cellular volume. Determination of the extracellular space by physiological methocls. The total volume of the extracellular space may be obtained by calculating the volume of the distribution of a substance in-

30

EDUARDO DE ROBERTIS AND IT. hl. GERSCHENFELD

capable of penetration through cell membranes ( Robinson, 1960). Thus one of the methods to calculate the extracellular space is by determining the distribution of Naf and C1- which are considered to be mainly extracellular ions. The ionic space is the per cent relationship between the C1- concentration in 100 gm of brain tissue and the concentration of the same ion in 100 gm of plasma dialyzate ( Davson, 1958). Using radioactive ions an extracellular cliloride space of 31.4 to 50yb was calculated while the sodium space was of 26.6 to 40% (Table I ) . These results were conTABLE I EXTRACELLULAR S P A C E ( ECS ) IN PERCENTAGE OF BRAINVOLUME AS DETERMINED HY PHYSIOLOGICAL OR BIOCHEMICAL METHOIB ECS 26.640 30 34.6 50 33.2-37.6 50 40 24.7 31.4 42 4-5 17 m1/100 gm 14.5 m1/100 gin 4-5 14-15

Method Na+ Na + Na t N a + ( i n uitro) c1c1c1c1C!c1-- (in uitro)

so,-

Ferrocyanide (in Gitro) Iniilin (in vitro) Inulin (in Gitro) Sucrose

Author Mannery and Hastings (1939) Mannery and Bale ( 1941) Davson (1958) Davson and Spaziani (1959) Mannery and Hastings ( 1939) Mannery and Haege (1941) Elliot and Jasper (1949) Woodbury et al. (1956) Davson (1958) Davson and Spaziani (1959) Woodhury et al. (1956) Allen ( 1955) Allen (1955) Woodbury et al. (1956) Davson and Spaziani (1959)

sidered too large and it was thought that these electrolytes might in part be intracellular. So far there are no data on the possible content of N a + and C1- in neurons and glial cells. Because of the special permeability properties of the CNS (see below ), the substances generally used to measure the extracellular space (inulin, ferrocyanide, etc. ) do not penetrate the so-called blood-brain barrier (BBB) and thus cannot be used in the CNS of the living animal. This difficulty can be obviated by incubating sIices of tissue, a technique frequently used in neurochemistry (see McIlwain, 1955). Allen (1955) incubated brain slices in isotonic Ringer with glucose and glutamate for 30-60 minutes in a special chamber under continuous flux of oxygen. By adding

inulin or ferrocyanide thc, innplitiitle of the cstracellulnr spice was calculated. Since iintlcr t l i c w x circiiinstances t h e is also swelling of tlie tissue, thc (l(~t~~riiiiii~itioii can be onlv rnadc after a theoretical extrapolation to zoro time. Il’ith this tcclinicjiie valiies of for the iiiuliii space a i i t l of 17r!i for ferrocyanide wcre obtained (Table I ) . Pappiiis mid Elliot ( 1956) incubated brain slices in Ringer phosphate witlioiit glutaniate for 60 minutes in oxygen and found a swelling of thv slicc, of 30‘ which w a s considered to b e extracelldar by the addition of sncrose antl thiocyanate. In connection with these esperiincmts it is intcresting to point oiit that lx-ain slices swell both i i i mi acro1,ic or in an anacrohic medium (Elliot, 1946, 1955) but tlir prcsence of glntamate in the aerobic esperiincnts of Pappiiis a i d l of ( I t

32

EDUARDO DE ROBERTIS AND H. M. GERSCHENFELD

electron density of the cytoplasmic matrix (Figs. 4 and 5 ) . Mitochondria also showed considerable changes with fragmentation and vesiculation of the crests and diminution in electron density. The cell body and dendrites of neurons showed, to some extent, a normal structure without swelling. On the contrary, the edges appeared compressed by the adjacent swollen glial processes. With incubation in hypotonic solution after 30 minutes the weight increased 25.9%. Electron micrographs showed even more conspicuous changes, while the perikaryon and dendrites of neurons showed an increased electron density, probably due to retraction

FIG. 15. Swelling of the brain slices incubated in different solutions, expressed in percentage of the wet weight gain.

or compression. The astrocytic cytoplasm and all astrocytic processes showed considerable swelling with watery aspect of the matrix and mitochondria1 alterations. The synaptic endings also showed some swelling. In this case, as in all other in vitro incubations, there was no apparent extracellular space and all cell membranes were well preserved and in contact. Slices treated for one hour according to the technique of Pappius and Elliot increased 28.1% in weight. The electron microscope observations showed no extracellular space but an enormous swelling of the astrocytes. With this treatment, mitochondria and synaptic endings were better preserved. Neurons showed normal structure, but their edges were sometimes compressed by the swollen glial processes.

TIIE

BRA~X

The abovc-mentioned o1)scw:itions on t h e in uifro experiments slio\v the strikingly dii+ercwt bcthavior of tlie various cell types found in the central nervoIis system in relation to the surcharge of fluid. While the neuronal clemciits (probably with the e s c c p tion o f the synaptic endings ) , thc oligodendroglia, and thc cntlothelial cells do not show tnarkcd changes, the astrocytes are consitlerably s\voIlcn and ha\.(, a morc watery aspect tlian in the normal tissue. From the comparison 1)c~tweentlie biocliemicid niid submicroscopic obscrvutions it seems possible that a large part of thc C1k a i d Nil--- ions esperiincntally introtliiced into tlie brain is contained i n the astroglia cc~lls. I n tho experiments of Pappius and Elliot ( 19%) thiocyanate and siicrosr might also have penetrated into the glia cells iis a result of damage to cc.11 processes. Elliot ( 1958) h a s discnsscd the importnnce that the section of nerve fibers and glial processc’s tnay have in thcw e\-periinents. This pro1)lein is also cliscussrtl 1)y h l c I h a i n ( 1955) i n connection witli the metabolism of hriiin sliccx Because both nerve and glial cells have processc’s runiiing in all directions, tlw proportion of cells damaged in the slicing procedure is certainly Iiighcr than i n other tissues. Howcvc~r tlic thy \wight loss in the slice is lcss i n tlie brain that in liver I~c~caiisc~ t h r structures are long and vcry narrow and can probably l w s c ~ i l ( doff rapidly. hlcIlwain ( 1 h a s calculate-tl that the cut siirfacx~in the h a i n slice m a y r c p e sent only about 0.2~;4of tlw ci.11 surfacc. Davson and Spaziani ( 1. ) tliminishetl the risk of sectioning cell processes liy tising . s 2-#3 inin thick. 111 spite of tliis care thc>yh n n d the valucs of vxtriicc~llrilar shown in Talllc I. In our esperiincnts ( Gersch(mfe1d cJt (iZ., ) siinilar c;xe \vas taken. For these reasons it scvtiis to 11s itnprolxible that tlie pcnc’trairose, ant1 tliiocyiiniite i n the tion of water, electrolytcxs, sii astroglia cells is due to tlw svction of thc cell processes. In the slice the neiironul proct~sst~siirv l ~ r ~ l ) ; ~ ciit l ~ l yin tlie samv proportion as in glia but tlicrc. is no swelling o f the nerve cells. T h i s may hc indicative of it difl’crcmcc, i n pernieahility lwt\vceii tlic

glia an d the neuroiis with a correspontliiig tlifl’crence in elwtrolyte content. Tlic results slipport tlie concept that astroglin constitiitrhs a water-ion compartment interposrd between tlic 1)lood capjlliiries and the cerchrospinal fluid on one side and tlic, cc~lhilarcotnixwtinent rcprcscmtccl by the neiiroiis and all nerve exp;itisiotis and Iiossildy oligotlcntlroglia ( Fig. 16) on tlir other. This \\-iitcxr-ion compartment w o d t l be involvctl in thc: sel c~t i on and trailsport of inctabolitc~s and fluid eschatiges l ~ ~ \ v r ethe n blood a n t 1 the ccrehrospind finitl \vitli the ccllrilar compartment.

c:.

AS-I-RO(:LIAAND

~EHI~:l3I~OSPINA F LLU I D

Davson ( 19%) has recently stirdicd tlw tlifferenccs in c011cc11tration of ions, a i d otlicr substances osmotically importaiit. I)etween the blood plasma and the ccrclwospinal fluid ( CSF ) . It w a s f o i i i i d that thc osinol~irityof thc CSF is 2;; liiglicr tlran that of thc blood plasnia. This corrcyionds to aboiit 180 inm of incrciiry in osmotic pressure. Furtliermore the intimate I-elationahips existing bctwcen tlw CSF and the n c ~ v ctissiic ant1 tlic liigli rate of ionic and 1iietal)olic iiitercliangc between botli coinpartmcwts (Hakay, 1957; Dnvson, 1959) arc’ \vc>ll known. On the h i s of these premises Davson postulntes that t he t~stracclltil~irfluid o f the brain and the CSF should have ;I similar ioitic composition wliiclr is different from tliat of the I)lood plasma. €It. flirther postulates that, in opposition to other tissiics, the CNS lias an rstracelliilar flriid that is Iiypertonic and is pro1)ably sc.cwtcd by tlic cc.lls of the capillary wall o r thc pioglial nicm1)raiie.

The electron microscope. ohcwations leave no dou1)t about tlie lack of such “intercelli~larfiuitl.” Furtlirrinorc there is no evidence of secretory activity by tlrc glia or tlie entlothelial cells of the capillary ~ l l The . rcsiilts of our in o espcriments in hypertonic Hinger sliowing ;I swelliiig of ustroglia ( Fig. 15) suggest that it may have ;i tlilfcwnt osmolarity tlian tlir other cell components of the 11rain. It s w i m also possilile that astroglin, which arc: in tlircct contact \vitli tlic CFS 1)y means of the I ~ I I I I I C Y ous proccwes ending on tlw pia in;itc,r, may inHnence its osniolar composition. This relationship may also osplniii the esistmce of some barricr mechanism for radioactivc ortlio~~liospliatewhose passage from tlie CSF to the nervous tissric, invol\~esan nctivc transport tliat may be inhibited with -SH antagonists (Ilcrrlin, 1Y56, 1958). Siiinmarizing these concvpts tlicl following \vorking hypothesis will be advanced with rcgaid to thc function of astroglia: ( ( I ) astroglia i n spite of being ;I cc,lhilar coinpartincnt lias fnnctions ~ ~other that are to some extent similiir to tlrc extracellular s p ; of tissues ( b ) astroglia is in osiiiotic eqiiilibrium with CSF and has a hypertonic content with reslwct to blood plasma, ( c ) astroglia represents n pool of watcLr a i i t l clectrolytcs l)et\\7een the blootl plasma and tlie nciiroiis in atltlition t o having importnnt inc.clianic~11 a n d lioincwstatic fmctions. I i r Soction III, F, the relationship of astroglia \\7ith tlw so-called 1)Ioo~lLl)r~iiii barrier will be considered.

D. EX~I.IL\CELLITI.AH S I ~ A C\xi) I ~ : mI:SO-CALLEI) GKOLJSII SUBST.\ 01; ‘1‘11E CNS Tlie problems \ ~ h i c h\ve have, discussed in this section are also related to the so-called groiiid or frintlarncntal substance of tlie CNS, which h a s been supp:svc.‘l to constitutc~an jinportant coinponent of the I I C ~ V O L I Stissricb. l ’ l i ( ~ olmrvation under the light microscope of the p a y m a t t r r lctl histologists to lielic,ve in tlic csisteiicc of a large extracc~lliilars ~ x i c e This . is not siirprising since in a recciit culculation of Da\itl and lhown ( 1959) in \vliicli the relative surface occupied 1 ) ~ . the nc’rvous, vascular, and glial elements \vas deter~ninedciriantitativc:ly, it w a s found that 59.35; of the area of the cortex coiwsponds to nonidentified elcinents. At thc beginning of tliv centiiry Nissl ( 1903) postulated the

36

EDUARDO DE ROBERTIS AND H. M. GERSCHENFELD

existence of a plasmatic substance-the so-called interzelluldres Grau-which was independent of the nervous components and which was interposed among all cellular elements. Nissl, relying on some phylogenetic arguments, attributed a great importance to this substance, since it was found to increase in quantity in the gray matter of higher animals. For many years, until the advent of electron microscopy, the names interzelluliires Grau, neuropil, plexiform substance, and fundamental reticulum served to cover our ignorance about the precise relationships existing between the different cell types and their processes in the nervous tissue. In 1953, with the use of the periodic acid-Schiff (PAS)reaction on brain sections, Hess revived the concept of fundamental substance. He concluded that while the glia and nerve cells gave a negative reaction this was positive in the “ground substance” interposed between the cellular elements. It was further postulated that because of some special hydrophilic properties this substance could be related to the water metabolism of the brain and to the blood-brain barrier ( Hess, 1955a, b, c ) . Leaving out the discussion of the cytochemical aspects it is now evident that there is no extracellular space in the brain to account for this ground substance (Wyckoff and Young, 1956; Niessing and Vogel, 1957). Hess (1958) has recently postulated that the labyrinthine space of 120-250 A shown by the electron microscope in between the cellular components could in thicker sections give rise to the PAS reaction attributed to the ground substance. This is a concept that has to be proven at the submicroscopic level since no extrapolation can be made from the observations of the light microscope to those of the electron microscope. Furthermore, if the PAS substance were confined in between the cellular components, it would represent only a small proportion of the total volume. In addition, we have seen that this material is not influenced by changes in the water content of the tissue. There is no doubt that the concept of a ground or fundamental substance has now only a historical interest and that it corresponds to regions of the CNS composed of a multitude of completely packed submicroscopic components of nerve and glial cells.

LtORPHOLOGP

z\

UI) IXJNCI‘IOS OF GLI.4L CELLS

37

Another problem that should be reinterpreted on the basis of the ncw submicroscopic data is that of so-called brain edema. By definition edema is an increase of extracellular fluid antl is thns related to the problem of estracellulnr space in the CNS. Elliot and Jasper ( 1949 ) tricd to evaluate cpntitatively the factors involved in the “s\\vlling” antl “shrinkage” of the CNS bv determining the relatioir ship hcat\vcen the wet and dry weight of the brain. The authors tl(wonstratec1 that most cases generally consiclcred a s brain edema correspontled to situations in which thcre was congestion of hlootl vesscls or dilatation of the ventricles or cisterna. These were wcognizetl by tlie name of “inflation” by Elliot and Jasper ( 1949). This mc~chaiiism w a s also involved in thc cxperiments of Weed iind hlcKibben ( 1919) in \vhich by the injection of hypotonic antl li!pcrtonic solutions considerable changes of brain volume \very prodiiccd. This is at variance with what Iiappens in traumatic Ivsions i n \vhich there is an increase in the bloocl and water contcmt at the site of the lesion. The near1)y region is frequently coiisitlewtl to represent a real edema and is not influenced by tlw injcction of hypertonic solutions as in the case of the “inflation” ( C1asc.n ct u / . , 1953, 1957). Since tlie work of Penfieltl ant1 C o n c x ( 1926 ) the “acute” s\velling of tlic oligodentlroglial cc.11~ wiis considered the most common finding in traumatic and toxic Ic3sioiis of the CNS and the most sensitive indicator of c h a n g c ~ in the nervous tissw. Howcvcr Gersclienfeld et a!. (1959) i n stab wounds of the brain cortex observed that in the region o f tlica so-called trauinatic edema the astrocytes appeared consitl(wil)ly s\vollen. I n addition there w i s 110 extracellular space around tlie Irsion. Some discoiitinnitics due to necrosis of the tissiiv o n l ~ ,in tlie wound itself could be observed. Similar findings I i : i v c ~ 1)rcn intlepend(~ntlyreported by Klatzko et (11. ( 1958) using silver staining and histochemical rcactions in their study of local brain injury by cold. Enlargeincnt and intense PAS-positive stiiining of astrocytcs coincided with development of traumatic c d ( m i a . In hoth works oligotlendroglia \vas aplxirently not involvctl in the sndliiig of tlic 1)rain tissue. In some unpiiblished obscwatioiis of thc> so-called peritumorul cdcma in liuman brain W E coriltl also observe ii marked swelling of t h r astrocytcs without airy iipp(’;ir;ince o f cxtracellular space

35:

EI)UARDO DE ROBERTIS ,\A]) 11. 31. GERSCHEAFELD

(Fig. 6 ) . Fwthcrmore Torack et al. (1959) in electron microscope observations of secondary swclling after cold injury f o m d no extracellular s p c ~hut~ great en1:irgcment of what they called glia cells with clear cytoplasm. Another experimental approach to the problem of brain cdeina in relation to generalized edeina was undertaken by Gerschc.nfcld et nl. ( 1959). In neplirectomized rats injected with isotonic solution to prodiicc ;I 401; increase of the total estracellular space of the 1)ody there w a s no change in tlie water content of the Nephrex + Isot. P>O.l controls

Cronex +nephrex+ isot.

P > 0.1

cranex controls

Pitressin +water P> 0.1 controls

Cronex t pitressin +water

P > 0.1

cranex controls

FIG.17. LVutcr coiitcnt in milligraiiis per 100 g m 1)odp \vci,glit of tlic I m i n in the tliffcrciit grotips of ciperiiiic~iital ant1 control aiiiiiials. Tlw shatlecl portions corrcvpoiid to c>xperimental aniiii;ils a n d tlic. oprn pi)rtions to controls. Diffcrcwcc>sbctwccw all paired groiips of aniinals arc not significant.

brain, and the image under the electron microscope w a s iiormal. Similarly nrgativcx findings wcrc~ o1)tnined with the s ; m c trcatInent plus cstcnsive craniectomy and also in the expc.riincirts of water intoxicatioii according to Ronmtrec (19%) (Fig. 17). These last resiilts tlo not support the intcrprctation of Rowntree that tlie convulsions prodiicctl by this troatincwt were due to cerebral e dcm a. All tliesc c~spc~rimental data lead 11s to concliidc~that the e s t a b lishment of a rcal cdemu in the C N S is lxmtically iinpossililc~. Even under the inost drastic conditions, in \vhich therc) is coilsiderable e s t r x c l l u l a r edema in all other tissues, tlw cc~llular

39

MORPHOLOGY A N D FUKCTION OF GLIAL CELLS

elements in the brain maintain their compact structure without real intercellular discontinuities.

F.

A s - m o c L r A AND THE PHYS1OLOGK:AL BARRIERS OF THE

CNS

The discovery of Ehrlicli ( 1885) that intravenously injected coerulein A does not stain the h i i n , while it does stain other tissues, was the first indication that the CNS differs from other tissues in relation to the liptake of substances from the blood stream. Since then this property was recognized as a protective mechanism and several attcmipts have been made to interpret it and to determine the anatomical locus of the physiological barriers of the CNS and especially of this so-called blood-brain barrier (RBB). W c , do not intend to inake here a complete survey of this problem and the reader is refcrretl for this to the reviews of Friedemann ( 1942 ) , Tschirgi ( 1953) , Mannery ( 1954 ) , Uakay ( 1956, 1957), Dobbing (1956), a i d Davson (1957). We would like to consider only those points in wliicli modern studies of ultrastructure of the CNS may I)c, pertineiit to clarify the relationship between glia and the barrier mechunisms. Throughout the years tliifcreiit matomica1 loci have becn postulated a s the site of tlw 13BB. The blood capillaries were considered b y S p t z ( 1 3 ) on the h i s o f a diflerential reaction toward vital staining. This thcorv lias been more recently reafhnecl by Aird and Strait ( 1944)-, Bronian ( 1955), van Breenien aiid Clcmente ( 19-55),and 1Xotlriguc.z ( 1955). Schatelbrand and Bailcy ( ICE8 ) postulatcd the pioglial inenihrane that snrrounds the l)lood-l)riiiii vessels as the possible site of the BBR. 3Iore rccently Tschirgi (19S2) backed this theory with new physiological facts altlioiigh later he considered the possibility that two levels ( c~itlothrliiimand astrocytic membrane ) could be involved in the rcpil;ition of the transport of water and solutes between the blood plnsma and the nervous tissue ( see also Mayer aiid Bain, 1956). According to Lumsden ( 1957), wlio also postulated the participatioii of astroglia in the BBB, the locus is not in the cell memlxane h i t i n tlic gellcd matrix of tlic cytoplasm. King ( 1939) suggested tlrikt, l)c.cause of the lack of connective

40

EDUARDO DE ROBERTIS A N D H. X I . GERSCHEXFELD

tissue in the CNS, the fixation of vital stiiins in it w o d d he different than in tlie rest of the organism. Hess (1955a, 11) 1)c~lieved that tlie so-called ground substance ( vide supra ), was the matomical substratum of the HBR. In supliort of this tlieory it has lieen argued that the PAS reaction disappears in expe~imcntal I(~sions of tlie brain wherc tlie BBB is drstroyed. In all these papers, with the esccytion of Lumstlt~n ( 1957), the BRB is suplmsed to be located between the blood pl;!siixi and the “extracelliilar compartment” of tlw ner\70iis tissucx. Rcccntlv Edstriiin ( 1958), postulated a thcwry I)asctl 011 the data ol)tiiined by electron microscopists on tlie sul~n~icroscopic strtictm‘cx of the CNS and which were described in tlie first section of this rcview, in which he docs not find it necessary to asstime the cxistcncca of a I3BB. He c~xpliiins thcl restrictions csisting for the traiisport of substances fro111 tlrc blood to the nerve tissue, on the basis o f the lack of extrwellular space iincl gronntl suhstmce. Accortling to him the fact that all cellular components iire tightly 11;iclicd in the CNS is (moiigh to c~xplainthe BEE-effect antl the tlircct p;issage of sut)stancc,s into tlie cells. oscopic mw/!y.sis of cqdlnrics in f h c C N S . Bcforc, eonsidering further these different tlwories of the BBB it is pc~tinent to describe tlic most recent results of the submicroscopic analysis of the brain capillaries. This is of consitlerable iinportancc~ in order to interpret their permeability properties in difl’erc~ntregions of the CNS antl to coinpare them wit11 that o f other rc~gions of the body. Bennett vt NI. ( 1959) have recently proposccd a ~norpholi)gieiil classification of 1)lood capillnric~i Ixiscd ( (1 ) on tlw prwcwcc’ or Ixisement meml)rane, ( 11 ) o n the 1)r(~sence absence of a contii~i~ons or absence of intracellulur or intcwcllular fcncstrations in the endotlielium, antl ( c ) on the 1~rcwiiceor absence of a coniplete lwricapillary investment of cellular elcments. It n . a s sriggestetl that thesc wrying capillary strnctural feattires may 1)c. rc)l(vant to prol~lems rcllatcd to tlie escliangc. of materials l i e t \ i ~ c n tlie blood plasrna and the parenchymal cells. li simple tlirc~~-tligit notation system w i s used b y tlie a~itliors to designatc~tlrc type of capillary in relation to the above incmtioned variables. According to this classification most capillaries of the C N S lx~long to the A-1-13 type ~vhichcorrcspontlents to capillarics having ;I con-

tinuous bnscmcnt meml)r:inc~, a nonfenestrated endotlielial wall, and a continuous cellular prricapillary investment (Deinpsey and IVislocki, 1955; Maynard c l ol., 1957; De Robertis et al., 195821). These three morphological cliaracteristics of most brain capillaries are readily o1)serwd in Figs. 7, 8, antl 18. The endothelium may show complex digitations untl microvillous projections into tlic, lunic~nh i t there is no evidence of true fenestrations within or l)ct\vewi tlicx endothelial cells. Tlie density of these cells is very high. Tlic>!, have nunieroiis clense particles in the hyaloplasm, a n d this contrasts with the low density of the lumen antl the vasciilar f c y t of tlic astrocytes. These capillaries are surroiindetl l y a continiioiis amorphous basement membranc which is clearly limited by t!ic adjacrnt cellular niemliranes of tho endotlieliiim and tlie pericapillar!, cxlcinents (Figs. 7 aiid 1 8 ) . At the level of the brain capillaries thcrc are no retic~ilar-collageii fibers a s c;in be observcd i n larger vessels, and the h s e m c n t membrane is amorphous, lacking filirillar elements. The dcscription of the third component of tlie capillary \ w l l is related to the old 1irol)lrni of thc existence of a pericapillary or His-Held space. Several olwmxtioiis with the light microscope had alrcatly indicated thc artificial character of this space ( Patck, 1944; Woollam and Millen, 1954, 1955), h i t the electron microscope has definitely slio\v~i tlic, nonezistcmce of sricli perivascular spaces at tlie capillary 1evc.l ( llcwipwy and IVislocki, 1955; Luse, 1956; hfaynard ct nl., 1957; Ilc, Rolic,rtis ct d . ,1958a). I t is now clear that all contours of the basement rncmbrane are swroiintled by tightly packcd cc~llnlarelements that leave no rval space in 1)etween their siiri:ice mcm1,ranes. The nature of these cellular elements may vary, lint according to Maynard et trl. ( 1957 ) and our own olxc~rvationsmost of them are represented by the vascular feet of tliv astrocytcs. The former authors calculate that about YO:!; of tlic: capillary surface is covered by the astrocytes which make vcr>r special contacts. Oligoclendroglia cells and even neurons may lie i n partial contact with the capillary wall h i t in both cases there arc 1 1 0 spccializetl processes involved. T h e rclationsliip o f astroglial processes to the capillary wall may iissiirne different configurations. In some cases almost the entire cross section of thc capillary is surrounded by a single huge vascular foot ~ h i c hcontiiins few niitochondria, large vacuoles of

48

EDUA€U)O I)E HOBEHTIS A S D €1. h f . GI..HSC€IENFELD

FIG, 18. C a p i l l q ( C u p ) of tlir rat brain showing ii dense cntlotlielium, a continuous I)asal itieinl)rane ( bm ), and several astrocytic processes ( u p ) (\,ascular leet ) . Notice that tlie tlriisity in thc glia is dmost simi1;ir to that of blood plasma; C T : cdoplasmic reticulum, mi: initochoi1dri;i. hlagiiific~ation: x 36,000.

44

EIIUARDO D E HOBEHTIS ;\AD 11. h l . GERSCHESFELD

the entloplasmic rc,ticnlrim with RNA graniiles, antl ;I clear ( witcry) matrix containing a f a v scattered groups of free ri1)osoinc~ ( Fig. 7 ) . I n others (Fig. 18) a few enlarged processes ( : 3 or 4 ) with a watery aspect are :ipplied 011 the lxiseincmt mein1~r:ine and cover it entirely. In this case the pericapillary coinponent appears to be tlivided into waterlike compartinents by the contacting inembranc~sof the adjacent vascular feet. In other cases thcl-c are a few large vascular feet h i t most of the capillary surfacc is coverctl by a feltwork of thinner astroglial processes ( glial fillers ). I t is obvious that at the 1o\v resolution of the optical microscope this pericapillary glial envelopment may give the iinl”cssion of a pericapillary spice. In addition it may lit: a Iociis minoris resistenfirre to the action of teclinical handling and artifacts may be easily produced, resulting i n tlie so-called His-Ileltl spice. It is interesting in this connection that there ;ire a fcw sinnll areas of the C N S where the BBR meclianisn~is not presciit niid where vital dyes or colloidal substances may easily pass. These are, for ewaniple, the nreri postrcnzci and some iiticlci of the chiasmntic infiindibular region of the liypothulamiis, where the cupil1aric.s are surroiinded by collagen fibers ( van Iireemen and Clementc, 1955; Dempsey antl Wislocki, 1955 ) , ;und the r~curoliypopliysis and pineal hody, her^ vital stains and colloidal silvcr are kuo\vn to penetrate ( Wislocki and Letluc, 1952 ) . ‘ k s e regions ;ire also pcrmeable to large protein molecules and to PC2 and otlicr ions. We have recently stndied the ultrastructure of the 11c~iiroIiypophysis of the toad ( Gerschenfeld c f ( / I . , 1960) in which the wide capillaries are siirroundetl by a continuous l~asement nic:mbrane and t1ic.n b y nuinm)iis tightly picked nervc cndings. It is interesting that these capillaries that liuvc no BBB incclianisms also lack typical glial processcs or vascular feet 011 the capillary surface.

G. ASTROGLIAA N D I N THE CNS

TI113 PEl73lEAHILITY ~’HOPER.I.IES O F C A P i L L A n I E S

The intimate rclationship of astroglia to most brain capillaries is for 11s an important reason to relate the UBB inechanisni to tlie membrane of the astrocyte ( De Robertis ef a!., 19581,; Gerschenfeld cf (/I., 1959).

I’cwncdility in brain capil1:uic.s is very difl‘crcmt from that in otlier territories of the vasciilar s),stcm. Tliiis tlic intcrcliange of ratlioactive ions bet\vecn hlootl plasma and the CSS is c,straordinarily slow as compared with most other tissiivs ( c g . miiscle) (sce Hakay, 1956, 1957). For t ~ : i i i i p I c ~ratlioactivc , sodium ecliiilibrates its conwiitration between tlic hlootl plasma aiid iiiusclc in 20 minutcs (Rfanncry and Bale, 1941) \ i , l i i l c A it takes 21 hoiirs to do the same with the CNS (Grcxmberg ct ( / I . , 194:3). Tlie same slo\v penetraK’?, C1 mid otlicr ions (see tion h a s bwn observed w i t h F, I3akay, 1957). O n l y a f ( w sii1)stances such a s ancdietics, barbitiiratcls, and some liposoliil~lecomponents may easily pass the RBB. Osygeii and other g ; i s ~ s penetrate freely ( Hering, 1952). Also tleuterium oxide m a y p i s s w r y rapidly, and even heavy watcr may go ~icrossthe 1313 t3 inorc~rapidly than in muscle ( cited hy Coulter, 1958). Scveral authors since Krogli ( 1946) hu\,c compared the HRB with tlie mechanism of activc transport occiirring across cell mcnilmnes. This ~nechanismmny also involve clc,ctrical plienoincna resulting from ion gradients. Recently Tscliirgi and Ta~rlor ( 195s) liave foiintl a potential diffcrcnce ( e m f ) hetween tlrc blood stream antl many points within t!ic CSS. Tlie authors tliscrisscd the possiljlc anatomical locus of tlic o t t i f and the HHH. T\\w strrictiires \ \ w e consitlcred: tlie capillary c~ndotlieli~im, wliicli is ~\ccptioiialIyi m ~ ~ e r i n c ; i l ~tol c plasma proteins, antl the atljaccv it mcml,rane, which is thought to he engagcd in “secretory” activity, transporting inorganic electrolytes I)ctwccm the plasma a n t 1 tlw “interstitial” flriid. Since no rcal ertraccll~ilarfluid is prt’stliit in tlw C N S it c m I)c assumed that this emf, between the blood and thc nervoiis tissiic, could be the result of a differential ionic pcrnieability and tlie active transport of some ions. Coiilter ( 1958) dc~ternriucd tlic filtration cocfficient o f brain capillaries with a inethotl Ixiscd on the llonro-Kcllic doctrine wliicli states that the CNS a n t 1 associatcd fluid compartments arc enclosctl in a rigid container antl t h i s pliysiologically tlw total voliime of the system is practically coiistmit. He foruitl that tlie coefficient of filtration of brain capilliirics is Iiiglicr than that foiind by Pappeiihcinier ( 195:3) in niiisclc~,indicating that lipoinsolluble moleciiles conld pass as rapidly as i i i iniiscle. Discussing the fact that the

ions and gliicosc concentration equilibrates more slowly ill the CNS than in otlwr tissues, Coiilter postulated thc, Iiypotlic,sis of an active transport mechanism-that the water and ions oiic(’ they have crossed the cclpillary wall coiild be piimped Ixwk 1)y ii srcretory imchanism. According to this concept thc. BE13 slioiiltl not be considercd ;i l’hysical obstaclc due to the virtual iilwnce of an extracellnlar spice its in Etlstriini’s ( 1958) concept, h i t ;is an active I)arric,r mechanism for diffusion from 1)lootl to nervous tissue (see also Dnvson and Spuiani, 1959). It is cvident that such pliysiological p r o ses need a metabolic sul~stratiim that cannot lie prescnt in ;L Ixisement mcmlirane or i n an irit may be located in the cell mernbranc of tlic p~ricapillary glinl processes. In our prcvious work we have postulated that the pliysiological intercliange represtwted by the so-called RED is 1~rodiicctlI)etween the blood plasma ilnd tlic astroglia across the siirface mcmilwane of the astrocytes ( I l e Robertis et nl., 195821, b; Gersclienfcltl ct al., 1959). In this concept astroglia is thought to I)cl a special coinpiirtment intcrposetl lxttwecn Mood and neiirons. This cc.llular conilxirtment of tlic CNS h a s properties that to some r’utent are analogous to the cstracellular space found in other tissws ( Fig. 16). The prescncc of a ERR mcclianism with active pumping hack of ions and other substances may explain the irnpossibilitv of producing a real brain edeina in the in oioo espcrimcnts in which there is an incrcase of 40‘ of cstracelldar space in the body (Fig. 1 4 ) (Gerschenfeltl et NI., 1959). That this mechanism is not clependent on mechanical factors, siicli as thc presence of a solid bony casc’, was proven by the experimt~nts in wliicli an extensivc: craniotoiny ~ 7 ; t s performed. It is evidcwt that thc HBB rcyresents a homcostatic protcctivc meclinnism that is al)le to avoid aciite changes in brain volume that could bc noxioiis to its structurc ( FVecd and Flexncr, 1932). The incrc1;tse in volume, producecl by swelling of the astrocytcs, is only possilile \vlien tlie HHH across tlie ~tstrocytic-vasc~il~ir rnembrane is not operating, a s in tlie case of o w in oitlo cxlieriments, in which other processes a n d tlie soma of the astrocytes may be exposed to tlic incubating medium. The fact that the amount of fliiid taken is not only dependent on the osmotic 1)res-

lLfORPHOLOGY .iND F U N C T I O N OF CLIz\L CELLS

47

sure but also on the prtwnce o r lack of inetalx)lites such as

glutamate (Fig. 15) indicates that the swelling of glia is also ;in active phenomenon. Ho\vc~vc~, in this casc one h a s to suppose tliat the s\velliiig is caused bv thc failure of metabolic phenomena related to permeability. So far there is no clcwr eviclcwce that tlie astrocytic-vascular nieinbranc is diiferent from the other membranes of the astrocyte. This tlemonstration may prolxibly need tlre m e of some special histochcmicd techniques at tlrc electron microscope level. However, it is evident that n w r thc vascular membrane of the astrocytcs the mitochondria and most of the elements of tlie endoplasniic reticulum are concc~ntratetl, indicating that this surface may be metabolically more active. Other experimental cvitlcnce supports the role of astrocytes in the BBB mechanism. Since the work of Macklin and Macklin ( 1920) it was known tliat ;I “stab \ ~ o i i n d ”caiises the disappearance of the BBB around the lesion. Other drastic treatments such as heat necrosis, air embolisin, passive congestion, electroshock, aiid ultrasound radiation, also r c d t e t l i n alterations of the BBB ( Bakay, 1955; Bakay ct ol., 1956; Rozdilsly and Olszewski, 1957; Lee and Olszewski, 1959). In all those cases, however, the action is too destructive to determine exactly tlre hasis of the physiological changes. A bctter method is the irradiation of the CNS with Xrays. Clemente and Holst (1954) ollservetl, in the monkey, that with 1500 r there is no altcration of the BBB. With irradiation of 4500 to 6000 r there wvre intense changes in the BBB, with degeneration of the astrocytes and neurons. It was found that there was a correlation Iwt uwn BBR and the astrocytes and not with the neurons. Thesc cxperiincmtal results also indicate that the astrocyte has the main rwponsibility for the BBR mechanism.

H. NEUI~OGLIA AND NEUIWNAL ACTIVITY In the previous sections we Iiave disciissed h o w the recent histophysiological knowlctlgc of glia and tlie cxtracellular space has changed our classic conception of brain structure a i d has led to a better understantling of the physiological barriers and the water and electrolyte meta1)olisni in the CNS. Now we would like to discuss the impact these new concepts may have on some of

the modern aspects of neiirophysiology, s r d i ;IS the 1,ioc~lwtric activity of iie~iroiis iiiclutling the ionic imec1i;inisms in 11crvc conduction antl the transmission of nerve i m p d . For thc moment we can 01-ily examine t l m e pro1,leins in a speculative way Iroping that new experimental approaclios may 17c developed in tlic future to solve them. The celliilar Ireterogeneity of the CNS and the complex topogruphy of the different coniponc~ntsexplains tlie grcat tlifFicultics that aiialysis of glial elemrnts. To learn exist for a rnicrophysiolo~ic~il ahout the participation of iicuroglia in the gctieral 1,ioclectrical activity o f the iicrve tissue it \vould be extremely important to known the clxact ionic composition of this tissue and how it (lifters from the otlicr cells. The blind penetration of a microelectrodc that permits the detection of neuronal activity is not sensitive or csact enongh to analyse the bioelcctric 1)ropertie.s of glia. IEild ef ril. (1958) have tried to overcome this difficulty by studying the electrical tictivity of astrocytcbs antl iie~iroiisseparately cultured ill c i f r o ( Hild, 1957). In both cell types ii mcmbrane potential of 50 in\’ was found. In neurons, stimulation by a11 estracelliilar electrotlc produced an action potential of 1 msec a n d 30 to 70 iiiv amplit~itle. Astrocytes hat1 a response that consisted in a quick depolarization of 40 mv followcd by a very slow (about 1 scc) recovery of tlie resting potential. \\Tit11 two or m o r c stimuli rcpeatcd at sliort intervals thcre w i i s a summation effect. Recently Cliaiig antl Hiltl ( 1959) havo tlcmonstr~ited that this reaction is accompanicd hy ii contraction of the astrocytcs \vhicli may follow, to ;I certain limit, tlic intensity of stiiniilatioii. After this rc!action tlic cell expands and regains the protoplasmic and inembranoils movcmeiit. Siriiilar contractioiis Iiavc heeii o1,sc~ved to occiir sl~oiitanrously in cultiirctl astrocytcs ( Hiltl, 1954 1 and oligodendrocytcs ( I’omcrat, 1951) . Tasaki ant1 Chmg ( 1959) with microelectrotles insert(d in the cerebral cortex liavc foiiiid electrical responses that \verc’ coinpletely diffrrent from those of the neurons antl morc similar to the “glial potentials" found in uitro.

The new structiiral concepts tliat have enit~gcclfrom the sulimicroscopic analysis pose ;I crucial qucstion for tlie nciiropli!.siolo-

hfORPIIOLOGY . \ h D E’USCTIOS OF G I . I A 1 ~CELLS

49

gists. In view of the organization of the CNS it seems evident that by using micropipettcy 5000-10,000 A in diameter one cannot obtain information about :in intercelliilar cleft of 120-200 A . The so-called “extracellular potential” ( Eccles, 1957; Freygang and Frank, 1959 ) should be lwttcr named “extraneuronal potential” because the tip of the micropiptitte is probably implanted in the cytoplasm of another cell wliicli in most cases may be of glial nature (Fig. 8 ) . The probablc infiuence that the glial membrane may have on the extraneuronal potential is open to investigation. Tlie interpretation of 1ncni1~r;11ic potentials in the C N S presupposes-as in tlie case of pcriplierial nerves ( Hodgkin, 1957 )the existence of an ionic g r d i c n t with an extracellrilar fluicl similar in composition to plasnia ultrafiltrate. Ecclcs ( 1957 ) postulates that the 70-mv qiiilihriiini potential for sodium implies foiirteen times less sodiiirn ions within the motoneurons than outside. The following pro1)lem is immediately raised: is tlie intercellular cleft of 120-200 A foiind between all cellular elements of the CNS large enough? C h i it hold a sufficient amount of ultrafiltrate to explain the ionic. intc~rcliange and thc maintenance of some of tlie fundamental cllcctrical properties of nervoiis tissue? Horstmann antl Meves ( 1059) have answered these questions positively indicating that thc. 5‘ c.alculated space agrecs with the specific resistance of the h a i n . W e have already discrt tl orir ohjections to this point of view (see above). The clcft is not it true intercellular space and we have found that even with a 30: water increase it remains the same. Horstmann antl R Icw.s’ ( 1959 ) calculations are made on several indirect data and tlicwrc,tical basis without experimental proofs. Thus tlie specific resistance of the glial membrane and the differences in ionic composition 1)ctwccn the different cell types are c o m p l c t c l ~unknown. ~ ‘I’lic~ spwific resistance may be the result of more complex factors tl tan those postulated hy Horstmann and Meves (1959). We have alreatly tnentionetl that in onr experiments there is a great difl’cwnce in permeability between astroglia antl neurons. Furthermore the, permeability of the glia surface membrane may also differ in the various parts of the cell (Fig. 8 ) . Van Harreveld ( 1957) pointetl out that it is difficult to correlate the data of electron microscopy wit11 the relatively low specific resistance of tlie cortex ( Frcl!piig a i d Lantlaii, 1955). This c m

be easily esplaiiictl by postulating for astroglia ;in ionic coinpsition more or less similar to that of tlie estracellular fluid o f otlier tissues. This last possiljility was mentionr:d in the p 1 1 1 c ~ of van ~:irrevclda n d Schacli: ( 1959) . If astroglia functions a s a special water electrolyte compartment interposed bet\verii the hlootl and the neuron, it secnis possiblc that it iniiy act I S iin ionic pool for the excitable ~nembraiiesof the CKS. Sclimitt and Ccwhwind ( 1957 ) have discussed the site of interaction Iietwcen nriironal and estraiieiiroiial phases in poriplieriil nerves d e r e Sclnva~incells rcplucc glia. Bccaiisc, of tlic lack of aii estracellulnr spice thcy find it clifFicnlt to explain tlie iniinctliate source of sodillin ions for nerve escitation. This would not IIC, tlie case if Sch\r,ann cell ( atid astroglia ) h a s ;I high co1iccmtr:itioii of this ion. Ionic excliaiigrs l)et\veen netirons and astroglial proccsscs \\mild permit tlie muiiitcwance of ionic gradients across the niein1,raiies m d their replaceinelit during excitation. This action may also avoid accmnulation o f potassium ions in the interce1liil:ii- clefts during physiological excitation ( Horstmann and :\levcs, 1959 ) . Another iiitercsting prolilein is tliat of tlie rclatioiiship that glid elements might have with synaptic transmission. Confirming thc classic findings of Ranioii y Cajal ( 191;3) and Rio Hortcga ( 1928 ) , tlrc! electron microscope observations tlcmonstrate the direct relationship that perineural oligotlendrocytes antl h a v e with the surface mcm1)rane of iiciirons and ncrvc endings. I11 t h r past this close 1-elationship \viis interpreted a s iiiclicatiiig that tliclse perineuronal elements play ;I role in nerve escitatioii and synaptic traiis~nission. Thus Rainon y Cajal ( 1923) madc the Iijpthesis that the distaiice ljet\z7eeii iierve endings and iieurc)nal surface could be altered by tlw swelling of gliul processtJs tliiis providing a way to intcwiipt nerve transmission. Accortling to Dc Castro ( 1951) glia is iiiteriiosed a s a curtail1 in betwccw endings a n d neuroiis antl thus is crtcnsively cngagcd in synaptic activity. Tlie electron rnicroseope studies of De Robcrtis aiitl 13cniirtt ( 1954, 19S5), Palav arid Palade ( 19#54),and many otlicrs, Iiave conclusively demonstrated that glial processes iirc not iiitcrpose'd in the synaptic junction aiid that at this level ;i tlircct contact with special submicroscopic cliariictrJristics esists.

T h e synaptic cleft 1)etwc~c.ntlic prc- and sri1)synaptic mcmlxines is also 120-250 A in size, ivitli iiiore enlargcd regions at tlie level of tlic “active points” of tliv synapse (sccx De Robcrtis, 1959). During synaptic transmission tlie “clearing” of this spice by diffusion of the chemical transinittc,r inay take, some time. Eccles aiitl Jaeger (19.58) have calciilatctl that, if the cleft wcre free of other diffusion harriers, ;I fraction of ;I millisc~contl W O U I ~ be enoiigli for the clearing of the syiiaptic cleft for acetylclioline. However, in different synapses an(1 especially i n the Renslian7 cclls (Curtis and Eccles, 19,58a,11 ) ii 1110re prolonged or resit1ri:tl action of the transmitter hiis b c ~ w Ioiiiicl indicating a barrier effect on the diil‘usioii (Curtis and Ecclcs, 1959). Since the i i c ~ v e cndings Iwyond the svnaptic cleft are entirely surrounded b y astroglial proce:sses ( Figs. 8 and 9 ) it sceins to 11s possilile that these may Iiave ;I barrier activity that prevents tlie t l i h s i o n of tlie mediator sul)stiince rclwsed at tlie junction. This m a y explain the long tlriration of the r.xcitatory postsynaptic potentials in some neuroiis, t h i s giving a rcpetitive discharge in respoiisc to a single presynaptic stiniiiliis. This kind of “synaptic glial Ixirrier” woiiltl also protect the underlying postsynaptic rc.ccy>tor froiii ccrtuin drugs applicd in tlic vicinity of tlie s y n a l i w , ant1 tliis may csplain the slo~vnessin tlie action of soine tlrugs \vliicli ;ire’ ineffective i11ioii the first two or tlircc responses. The topography of the astroglia a t the synapse and the lack of cstracellnlar s p i c e niay thus act as a “protective device” for the secretory and ionic mechanisms iiivol\wl in syniip tic transmission (Fig. 8 ) . Reccmt stiidic>s with niic.rot,lr.ctrodcs lw Curtis m c l Eccles ( 1958a, 1) ) on tlie pliariii~~colog!,o f Re11sliiiw cclls indicate the> existence of such ii synaptic 1)arrir.r to tlrugs injected in sift,, in addition to the BUI3 which is acti\rc on sulxtanccs iiijccted into tlie vascular systcrn. A4ccorc!ing to the authors both diEnsiona1 barriers have different peritwi1)iIit~~properties ant1 certain s u b stances that (lo not cross tlic 13HH may c w i l y p a s s the synaptic 1iiiiiier . : hen injected locally. Tliris prostigmilie and etlroplionii~m have practically no action upon Iiensliaw cells \vl;en xlministerecl intra\wiously but arc efft.cti\,c. ;is a11ticliolirie~stc~rases whcn applied 11e;1r the cell. T h e physiological data a r c ;tlso indicative of a regional

specialization n7itI1 portions of different permcability propc,rties of the plasma ineinbrai~e of the astrocyte, a s was tlisciisscd in Section 111, G. The role that perineiironal oligodendrocytes may play is p r x tically unknown. According to HydCtn and Pigon ( 1960) these cells corild l x mctabolically very active and might also be in ii dyiiamic interchange with neixons.

IV.

Functional Significance of Oligodendroglia

Myelinogcnc,sis of the CNS has I)cx31 attributed, for inany \’ears, either to axons, oligodendroglin, or astroglia. Rio H o r t c p ( 1928, 1942) ~ ~ o s t u l a t ethe d role of oligodendrocytes in myclinatioii b u t there was no direct proof of this i n his well-clociiiiiente(1 histological observations. The role of the axon, long ago supported by Kiilliker ( 1904), h a s recently been reaffirmed b y Hild (1957) in nerve’ fibers grown in tissuc culture. Finnlly Alpers and Haymaker ( 1934) suggested that not only oligodeiidroglia but astroglia i s impliratctl in myc>lination. In ;i first attempt to analyze this process with tlic e l c ~ ~ r o n microscope Luse ( 1956), hesicks interpreting incorrectly the type of glia cell, did not reach definite conclusions regarding the, site and mechanism of myelin formation. Her interprctations were very much infliienced by Gercn’s ( 1954 ) widcly accepted “jelly roll” theory of the. wrapping of tlic incsaxon around thc a x o n in peripheral myelination. Also in Lux’s work emphasis w a s placed on the siirface incmlmine of the glial cells neglecting any possibblc contribution o f tho cell cytoplasm i n the process of myelin synthesis.

A.

OIXODENUROGLIA AND CELLULAH h JECHAKISM IN TIW c:NS

OF ~ l ~ e i , i , u ~ ~ i o s

De Robcrtis et d.( 1958a) in their study of myelination in >.ouiig cats and rats tlcmonstratcd the fiindainental role played in mvelination bv the intcrfascicular oligotleiidrocytes of the white matter. This process seeins to be essentially a syntlwsis of menilxinous structures within thc cytoplasm of the oligodentlrocytc depending on the rneloplasniie wticdtim. The oligodendrocytc cytoplasin siir-

FIG.19. General diagram showing the process of myelination within the cytoplasm of the oligodendrocyte. A. The axon ( A x ) is surrounded by the axon-oligocytic membrane ( AOM ) . The first myelin membranes (Mym) are deposited, at a distance, by a process of confluence of membrancs ( m ) and fusion of vesicles ( V ) . B. Several myelin membranes are deposited in irregularly folded and discontinuous layers. The laying down of new membranes is indicated. C and D. The process of myelination progresses with the deposition of new layers. The myelin lamellae ( M y ) become continuous, smoother, and more compact. The AOM of lower electron density can be recognized (mi:mitochondria).

54

EDUARDO DE ROBERTIS AND H. M. GERSCHENFELD

FIG. 20. To the left, a myelinating axon with myelin layers. Notice the folding and discontinuities of the layers, and the irregular distance between them. See also the lower electron density of the AOA4 and the continuity of the outer myelin layers with the membranes ( m ) and vesicles ( z j ) present in the oligodendrocyte (some of these continuities are marked with an arrow 1. To the upper right a myelin sheath ( M y ) with 9 perfectly smooth, continuous, and packed layers. The period is of the order of 80 A ( e r : endoplasmic reticulum, mi: mitochondria). Magnification: x 102,000.

56

EDWARD0 DE ROBERTIS AND H. M. GERSCHENFELD

FIG. 21. White matter of the rabbit 7 days after a stab wound showing degenerating axons ( d A x ) and myeloid bodies ( M y b ) within the cytoplasm of an oligodendrocyte. Magnification: x 36,009. FIG.2.2. The same as Fig. 21. The oligodendrocytic cytoplasm is filled with numerous myeloid bodies ( M y b ) . Magnification: x 23,500.

58

EDUARDO IIE ROBERTIS AND H. h f . CERSCHESVELI)

rounding the axons ( Fig. 1.3) becomes filled with mcrnlnmous material in the form of vesicles and tubules that fuse together and become deposited aroiund the iixon at a distance from the axonoligocytic inenihranc complex ( AOAI, Fig. 19). \Vhile analyzing the myelination of a single axon, tn7o consecutive periods m;iy be described. One involves the laying clown of the first fiw to six myelin lamellae i n which the structure is still not well integrated antl shows discontinuities, folds, trapped vesicles, antl irregnlar repeating periods. The second period, pro1)al)ly starting aftcsr the formation of tlie eighth to tenth myeliii lamellac, involves tlir cornplete coalcwence and alignment of the membranes iintil they acquire the regiilarly ordered nnd continnous miiltilamellar structure that is cliaractcristic of adult myelin (Fig. 20). These observations can be correlated with rrcent biochemical work relating the biosynthesis of lipoproteins antl of thc, niyelin sheath to intracellular enzymatic processes ( Korey, 1960). This synthesis has been associated with the microsomal fraction ~ h i c h derives froni the endoplasmic reticulum and with tlie aqiieous cellular phase ( Brady et nl., 1958 ). Thc individnal lipids are apparently synthesized in a definite sequence on the meinl)rane, which indicates that they “remain united to the endoplasmic reticulum constituting lipoproteins \vhich move into place alongside the axon as a niyelin membrane subunit. By this process a bimolecular lipid ant1 protein layer consisting of segments of the endoplasmic reticnluni with attached lipids in a definite order, align themselves concentrically about the axon, which proba1)ly serves largely as an oricnting structure” ( Korey, 1960). In unpuhlishcd olxervations on periphcral myclinat ion at this laboratory a syiitliesis of membranes by way of the endoplasmic reticulum of the Schwann cell w a s also found. ;\Icmbranes are being formed in the cytoplasm and addcd to the mesaxon in myelination. This iiiay explain the growth of thc. incsaxon and tlie so-called wrapping of the myelin lainellae.

R.

OLIGODENDIIOGLIA AND THE DISPOSAL OF A ~ Y E L I N LESIONS

IN

BRAIX

Oligoclendrocytes are not only engaged in the formation of myelin but also in its clisposa1 in some pathological contlitions that lead to degeneration of central nerve fibers. In regions surround-

XIORPHOLOCY .\hU k’lJ,\rCTION OF GLIAL CELLS

59

ing stab wounds of the braiii, d i e r e section of nerve fibers has occurred, we liave observcd all stages of nerve degeneration and disintegration of the myelin slicath taking place within the cytoplasm of the oligodendroglial elements. For esainple, in Fig. 21 where several degenerating filwrs arc’ observed, the axoplasm has disappemed and the niyclin slicatli still remains normal or has started to collapse. In Fig. 22 ;I flirther stage of tlie fragmentation of inyeliii into round-shapcd concentric myeloid bodies can be observed within the oligotlc,ntlrocyte. Only at the site of the wound, where free debris is prcscvit, is myelin actively pliagocytosed by microglial cells. From all these results it ci111 be concluded that throughout the life of the individual tlie synthesis, maintenance, and disposal of myelin is a fiinction of oligodcntlroglia.

V.

General Conclusions

1. Sirbniicroscopic Morphology of Glial CelLs With the electron microscopr all components of the CNS may be siinultuneously analyzecl and thc, different types of glial elements identified. Ill the adult all cell11lar ~”o‘c’ss”‘s and vascular eleme11ts are tightly packed leaving no r e d c~strncellular space. Discontinuities between tlie cellular elements may 1~ fouiid i n brain before birth but the binding of the cell nic~in1)ranessoon causes these to disappear. A. The ustrocgtes are charactc,rizcd by tlie low electron density of the cytoplasm and tlwir lirocwses and by tlie rclationsliip that they bear to nerve cells, pial nic~nibrane,and the blood capillaries (vascular feet ) . Certain striwtural cliffercnces were detected among different species of ~nammals. No spccial distinction can be m x l e between tlie so-callctl “protoplasmic” and “filiroiis” astrocytes. This Iiistological iclentification may depend on tlie length and thickness of the cell prowsscx There are no extracellular “glial fibers.” Glial filaments iirc. oliscrved within certain astrocytes particularly in experimental gliosis. B. Two types of oligotlcrttlrocytc~s are recognized: ( N ) perineuronnl located near the ncviroiis and ( h ) interfascicul(ir in be-

t w m i the iicrvc fibers of the wliitr inattvr. Tlie c).topliisiiiic tlensity of thesc cc,lls is 1iighc.r than iii tlicl astrocyte ailcl the cytoplasm is rich in orgiuioicls antl ri1)osoinc.s. In thc, \vliitv matter tlic, asons are cxnbcdtletl \vitliin tiinnels that cross tliroiigh thc oligo(1c~iidrocytic cytoplasm.

2. Fiinctioricil Significcuicc of Asfroglici

,4. The concept of cvhacclliilar s p c v in tlic CNS is tliscrissed taking into consitlcration the confiictiiig results l)c)t\vcwi cletcwninations Ily electron microscopy :tiit1 by physiologicd nicthotls. I n incnlxitetl Ixaiii slicc>sin \vliicli tlicre may 1)cl an incrvase in water contcnt of 25-30: ; , \ve f o i i i i d no c~stracc~llnlar spicc> \isilile with the electron microscope. Kc.urons sliow no s\vc~lliiig wliik astrocytes antl their proccsses arc. greatly s\\wllcii. H. Thesc rrwilts are intcrprctetl a s indicating that \vatc,r ant1 ii large amount of C1 and Nla . is containccl in tlic astroglia which coiist i t tcs ;L special coin part incn t or pool i n t e r p o s d 1 t w c ~ i it 11e blood antl the iicwons (Fig. 16). C. Tlic fact that the cerelirospinal fluid has a liighrr osinolarity than 1,100~1 p l a s m a is discusscd in view of tlic close> r(3l:itionsliip of glial proccwes to tlie pioglial mc~niliriiiit:. 11. The old conccpt of ;in rstracclliilar grouiitl sul~stancein the CNS is tlisciissetl nnd disniissecl in view of t!i(, lack of a true extracellular spec in thc CNS. E. Tlic conccpts of cdcmia, s n ~ ~ l l i i i gand , “infhitioii” of tlw CNS lire annlyzd. Esprriinc~nts leading to thc incrcasc of the gc~iic~r;iI c~strucrllrilarsliace antl of Irydric intosicatioii did not change tlic water contcvit of tlie h a i l l :und did not altrr thc striictrire of thc C X S . 17. Tlie diflercmt anatomical loci postulated ;is sit[. of the 1)lootl-brain 1)arric.r ( R R B ) are tliscussctl. l h c submicroscopic analysis of tlic 1)rain ciipillarics slio\vs the prvsencci of :I coritiniious bascincnt incmil)rme ant1 a tiglit celliilar iii\wtnicnt fornied iiiainl!. by the vuscular k e t o f nstrocytcis. Tlicrc, is no pcricapillar~.or HisHeld spac~’. There ;ircx a few csccptions to this ride in rcyions of thc CNS lacking I3BR. G. I t is postdated that thc HER mechanism is relatc(l to the incLrn1,rune of the astrocyte. An active Iiomeostatic inc~chanism,

protecting the brain from acrite changes in volume, operating continuously across the astroc.\.tic-\:nsciilnr membrane is siiggestetl. Swelling of the astrocytes oc‘c~irsw h c ~ nthis inembrane is altered or othcr parts of the ccll surfacc, i ~ r eesposcd to the fluid medium. K. Recent data of thc. 1itc~r:iturc~related to the bioelectrical activity of astrocytes are mcwtionetl. The impact that tlie sill)microscopic analysis of thc C N S inay have on some neuropliysiological pro1)lems is discussed. Among these is the possil%lity that ionic eschanges may occur l)ct\vccn astroglia a n d neiirons to muintain ionic gradients cross tlir. niein1)ranes and their replacement ~ of extraneuronal rccordduring excitation. Thc p r o l ~ a h l rposition ing microelectrodes is also consitlercd. Glial processes, by their intimate relationship to n e r \ ~ lcclls a n d ncrve enclings, may act a s a synaptic barrier whicli prc.vc.nts tliiimion of mediator substances a t the junction or m a y slo\v thc. action of drngs app!ied near the synapse

(Fig. 1s).

3. Functioiicrl Sipificcince of O l i ~ o ~ l ~ i ~ ~ l r o c ! / t ~ s

A. The relationship lwtwcvn oligodendroglia of the white matter and tlie ineclianisin of iny,lination is analyzed. Thc formation of membranes within tliv oligotlcndrocytc cytoplasm is 01)served and the different steps of tliis ccllular niechanism of myelination i r e tlescrihed. These olwrvations are correlated with reccmt biocliemical work relating tlw 1,ios)mthesis of lipoproteins of the myelin sheath to intracellular c~izymaticprocesses. H. Oligodendrocytrs arc’ also cmgaged in the disposal of myelin after clcgeneration of the ne filxrs. All these results indicate that the synthesis, niainteniuncc~,antl disposal of myelin is 21 function of oligodendroglia. 1

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62

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MICROELECTRODE STUDIES OF THE CEREBRAL CORTEX By Vahe E. Arnassian Department of Physiology, Albert Einstein College of Medicine of Yeshiva University, N e w York, N e w York

I. Tt&nical Procrdurcr in Olitaitiing, Imcalizing, and Handling of Unit Recordings . . . . , . . . . . ,

, ,

. .., ... , .., .. , , . ..... ., . .. .

11. Classification and 1ntc~rprc~t;itionof Single Cortical Nciironal Spikes a n d 1ntracellul;ir \f’;ives , , . . . . . . . . , . . . . . . . , . . . . . , . . 111. hlicroelectrodc Analysis ot Brain \\’aves and the Relationship of Slow \V\;avcs to Unit Activit!. . . . . , ..................

Spontaneous Activit!. . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . n. Recruiting Wavc>s , . . . . , . . . . . , , , . . . , . . . . , . . , . . . , , . . . . C. RcspoiiscLs to Spec,ific, ‘l’lial~unoc~tirtical All’ct-cnt Volle!-s . , . , D. Respoiiws to Corticociirticnl All‘crcnt Vollcy , . . . . , . . . . , , . E. Rcsponscs to Local Cortical Stimulation . .. ... . , ... F. Respotises to Aiititlromic Stimit1;ition of tl IV. l’attenis of Unit Respciirsc~to Sp(3cific Thal;imocorticnl Arerent A.

68

76 89 91

95

97 103

105 107

. ......................................... 109 matoscnsory Arc,;i\ I ;inti I1 . . , , . . , . , . , . , . . . , , , . . . . . . 110 I3. hlotor Cortex . . . . . . . . . . . . . . . 117 C. Aiiditory Area I , . . . . , . . , , , . , 119 D. Visual Cortex . . . . . . . . . . . . , , . . . . . . . . . . . 120 V. VI. VII. 1’111.

Patterns of l’attcwx of Iiitegrative Discussion

IJnit Rrsponsc~to Ilircct 193 Unit R c y ~ o t r \ cto ~ Corticocortical Aflcrent \Tollcys . , 124 llcsporises ti, h l i d Corticipetiil \'alleys , . , . . . . . , . 12,5 and Summar!- . . . . . . , . . . . . . . . . . . . . . . . . . . . 126 Rcfcrenccs . . . , . . . . . . . . . . . . . . . . . . . . , , , , , . . . . . , . . , . . . . 131

A number of recent revicws have tlealt admirably with the gcnera1 properties and the analvsis of cortical potcwtials ( Albe-Fessard, 1957; Ruser, 1957; Breincr, 1958; I’iirpura, 1959). Emphasis on a particular method of analysis, for example niicroelectrode esploration, may he considered claiigcrous wlien applied to such a complex system a s thc cortex. Our kno\vletlgc~of cortical function can only be sccure when inferences tlrawn from many cliff erent approaches 67

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are in agreement. Even so, the use of the microelectrode uniquely demonstrates the different temporal patterns of activity of members of the cortical population. In addition, the neuronal dipoles rcsponsible for the surface waves can sometimes be localized by studying the difference in pattern of the population responses which are recorded at the surface and witliin the cortical depths. This review deals mainly with the use of microelectrodes in the isocortex a n d in the analysis of descending pprainidal tract activity. In 1940, Rensliaw et 01. introduced the microelectrode in the study of the cortes. They recorded axon-like spikes o f about 1 insec duration from the hippocainpiis, but recorded only slow waves from the isocortes when using microelectrodes of more than 15 p tip cliametcr. This pioiieering study is also of technical interest because of the excellent control of electrode depth, the attention paid to grid current flow through the electrode aiicl to the electrolyte coinposition of the contents of the recording micropipctte. Woldring and Dirkiii ( 1950) recorded 1)rief unit spikes by meaiis of microelectrodes resting on the pial surface. In 1952, prelimiiiary rcports from four l~aboratories appeared ( Amassian, 195213; Ainassiaii a n d Thomas, 1952; Bauingarten and Jung, 1952; Li et Nl., 19S2; Thomas and Jenkner, 1952 ) in which unit activity \vas described in various regions of the isocortes. The important steps were the iise of clectrodes with small tips a n t 1 the introdnction of methods of controlling ecrebrnl pulsations.

I . Technical Procedures in Obtaining, Localizing, and Handling of Unit Recordings

hlaiiy of the tcclinical procediires used are not common to all studies and m a y intleecl have an iniportiuit bearing on the differences in results obtained. The chef difl'crences in technical procedure include: ( ( I ) The method of control of cartliorcspiratory pulsations of the corks. ( 1 9 ) The type of ancsthesia and the control of artificial ventilation. ( c ) The size and type of microelectrode. ( r l ) The method of localizatio,n of the electrode tip. To somc estent these factors are interrelated. For example, optiinal control of cerebral pulsations is required when recording with glass micropipettes of about O.Sl.0 11 tip diameter in locally anesthetized animals which

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have been paralyzed and are artificially ventilated, because such preparations show large spontaneous fluctuations in blood pressure. However, unit activity can l x recorded fiiom an occasional cortical neuron in cats under chlor‘ilose anesthesia without controlling cerebral pulsations, provided that large micropipettes (4-12 tip diameter) are used. ( a ) A method of controlling cerebral pulsations is essential to permit the use of microelcctrodes small enough to secure an adequate sampling of the various types and sizes of cortical neurons and to hold such unit activity long enough for analysis. Two techniques are in common use: (1) the “closed h e a d preparation ( L i and Jasper, 1953; Mountcastle et nl., 1957; Amassian et al., 1959; Hubel, 1959) and ( 2 ) the use of a “pressor” which is lightly applied to the cortex after which the micropipette is inserted through a centrally placed hole in the pressor (Amassian, 1953a; Phillips, 1956a; Cohen et nl., 1957, Towe and Amassian, 1958). Cerebral pulsations may also be prevented by supplying the brain with a nonpulsatile source of blood (Krnjevic, 1956), but unit records obtained with this technique have not yet been published and its usefulness remains to be established. The “closed h e a d technique depends on the finding (Forbes, 1928) that vascular pulsations are minimal when cerebral vessels are viewed through a sealed window in the skull. It was necessary to design a fluidtight, airtight coupling between the movable electrode and the chamber which is fixed to the skull. Three designs of the closed head chamber yield excellent stability of cortical unit recording in both immobile (Mountcastle et al., 1957) and in partially restrained animals ( Ricci et d., 1957; Hubel, 1959). The latter finding is remarkable because inertia of the brain during rotation of the head would be expected to change the position of the microelectrode tip relative to the unit recorded. A modification of the “closed h e a d technique was introduced ( Amassian et al., 1959) to permit independent manipulation of three microelectrodes into the cortex via tunnels in a hard paraffin wax roof. The only advantage of the wax chamber technique lies in the ease with which multiple electrode penetrations can be made, but it is probably the least desirable method for single electrode penetrations. The use of a pressor on the cortex should be combined with cisternal drainage ( Amassian, 1953a). If artificial ventilation is

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used, a double pneumothorax (Woodbury and Patton, 1952) should be performed. It is difficult to compare the relative merits of the “closed h e a d and the “pressor” techniques. The general condition of the cortex would be expected to be closer to normal in the closed head preparations and after many hours in such preparations, the pial surface is grossly indistinguishable from its appearance immediately after removal of the dura. However, the sole indication that superficial neurons are in a more physiological state in the closed head preparation is the diffjculty encountered by Li and Jasper (1953) in obtaining records from superficial units in the open head. However, unit activity in neurons above 300 p has been studied with the pressor technique ( Amassian, 1953a; Patton and Towe, 1960). The most critical test, that of intracellular recording, so far favors the use of the pressor (Phillips, 1956a). Possibly the closed head preparation would give equally satisfactory results when applied to the largest cortical cells (Betz cells) rather than to the smaller cells in the sensory cortex. To summarize, the pressor combined with cisternal drainage and double pneumo-thorax is a simple method of sampling single cortical neurons over a wide area of cortex in immobile preparation. Ideally, the pressor is attached to a heavy duty manipulator which also carries a fine manipulator for driving the microelectrode. This obviates the need for aligning the tip with the hole in the pressor prior to each penetration. The closed head method is essential when long term stability of unit recording is required in a moving animal, or in one which is being extensively manipulated. ( b ) Preparations are either anesthetized with a barbiturate, or chloralose, or a combination of anesthetics, or are operated on under ether anesthesia which is subsequently discontinued. The animal is usually paralyzed and artificially ventilated. A local anesthetic is infiltrated into the operative sites. The use of a paralyzing drug is avoided in the encephale isole preparation (Baumgarten and Jung, 1952) of Bremer and in Hubel’s experiments (1959). A variant is the “pyramidal” cat in which the mesencephalon is thermocoagulated under ether anesthesia, but the cerebral peduncles are spared (Whitlock et al., 1953). Such preparations are immobile and exhibit a “sleeping” electrocorticogram when undisturbed. Early studies by Marshall et al. (1941) and by Marshall (1941)

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demonstrated the effect of anesthesia in prolonging recovery time in the somatosensory system. The problems in relating anesthetic depth to unit activity are the difficulties of precisely defining the anesthetic levels used by different investigators and the lack of adequate comparison of unit behavior in anesthetized and unanesthetized animals. All studies agree that either spontaneous or evoked unit activity is reduced under deep anesthesia (Li and Jasper, 1953; Amassian, 1953a; Mountcastle et al., 1957). Slowly adapting cortical unit responses to maintained somatic stimulation are observed only under very light anesthesia when the recovery cycle of the projection system is but slightly prolonged. Such responses were missed in all studies of the somatosensory cortex prior to Mountcastle’s in 1957. The capacity of the cortical unit to follow high frequency peripheral stimulation is markedly reduced by deepening the level of anesthesia ( Mountcastle and Powell, 1959b). Administration of small doses of sodium pentobarbital (15-20 mg/kg ) prevents activation of single pyramidal projection neurons by somatic stimulation in unanesthetized cats (Calma and Arduini, 1954). Similarly, surface responses in the somesthetic association cortex of chloralose-anesthetized animals are abolished by administration of sodium pentobarbital ( Amassian, 1954). Purpura and Girado (1959) abolished the relayed pyramidal response to stimulation of the contralateral cortex by administration of as little as 8-10 mg/kg sodium pentobarbital. Some units respond with a characteristic discharge pattern under light Dial anesthesia in which a period of about 100 msec elapses between single, or grouped high frequency discharges ( Amassian, 19534. Such discharge patterns, clearly analogous to the secondary repetitive surface responses described by Adrian (1941), were rarely encountered by Mountcastle et al. (1957), presumably because the level of barbiturate anesthesia used was lighter. Single cortical unit activity is readily evoked under chloralose anesthesia ( Amassian, 1953a; Patton and Towe, 1957, 1960; Imbert et d,1959). Pyramidal tract projection neurons often have a large receptive field which includes two or more limbs (Adrian and Moruzzi, 1939; Patton and Towe, 1957, 1960).The question inevitably arises whether such responses are “artifacts” in the sense that they either do not occur, or they occur less frequently in the unanesthetized animal. Calma and Arduini (1954) observed small

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and “more often” large receptive fields in pyramidal projection neurons of unanesthetized animals, but no quantitative comparison between the fraction of wide receptive field neurons in the population under chloralose anesthesia and in the unanesthetized preparation has yet been published. The possibility that the use of chloralose anesthesia quantitatively affects the data by increasing the fraction of pyramidal projection neurons which respond within a short time span to the peripheral stimulus cannot be excluded. Usually it has been assumed from Adrian and Moruzzi’s findings ( 1939) that chloralose anesthesia induces abnormally high frequency discharges in cortical neurons. While cortical neurons rarely discharge at more than 400/sec under barbiturate anesthesia as compared with 800/sec under chloralose ( Amassian, 19!S3a), the frequency of discharge in the unanesthetized preparation would be expected to reach an intermediate value. The intact preparation under light barbiturate, or chloralose anesthesia, or after discontinuation of ether anesthesia is usually immobilized. Use of depolarizing agents such as decamethonuim salts or succinylcholine may introduce complications because an increased discharge rate is recorded from spindle afferents and possibly may occur in other afferent fibers (Granit, 1955; Fujimori et al., 1959). Tubocurarine chloride has the disadvantage that it may cause a severe drop in arterial pressure, and cortical activity may be greatly reduced (Ochs, 1959). However, the arterial pressure and cortical unit activity are well maintained under Flaxedil ( Amassian et nl., 1960). Analysis of the expired CO, with a rapid infrared analyzer (Amassian et ol., 1959), shows that when a cat is ventilated to the point of obvious respiratory movements by the usual positive pressure method, the animal is often hypocapnic (expired C 0 2 down to 2% ). Either CO, can be supplied in the inspired air (Frank and Fuortes, 1955), or the animal can be ventilated with 100% oxygen and the ventilation reduced until the C02 level rises above 4%. While no systematic analysis of the effect of different CO, levels on cortical unit activity has yet been made, seizures followed by depression sometimes appear in the electrocorticogram of hyperventilated, unanesthetized animals which have been immobilized with Flaxedil ( Amassian ct al., 1960). To summarize, the ideal preparation for observing cortical unit activity is probably the chronic one (Hubel, 1959). While such

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preparations have yielded valuable data in the visual system, it is not clear how they could be adapted to the study of the somatosensory system where uncontrolled shifting contacts or bending of the joints would be expected to affect the system under investigation. In acute experiments, operation under ether anesthesia with local anesthesia, subsequent paralysis with Flaxedil and artificial ventilation with a high 0, mixture combined with control of the COZ level appears adequate. The use of a cat which has been previously deafferented at the future site of operation is a particularly humane approach to research (Poggio and Mountcastle, 1960). Initial preparation under ether followed by maintenance doses of a “short acting” barbiturate may be adequate for studies of initial stages of activation of primary sensory cortex (Mountcastle ef al., 1957), but should be avoided in studies of activation of motor cortex, or of association cortex. The cerveau isol6, encephale isol6, and the “pyramidal” cat have specific uses, e.g. where one wishes to study the relationship between unit discharge and “sleep” spindles, but should be avoided in general studies on the afferent systems because of the obvious interference with the reticulocortical modulation system. ( c ) Conventional glass micropipettes of 0.5 p tip diameter and filled with 3 A4 KC1 are used for intracellular recording from cortical neurons (e.g. Albe-Fessard and Buser, 1953; Phillips, 1956a; Li, 1959b). Either micropipettes filled with electrolyte, or metal electrodes insulated with enamel (Amassian, 1953a; Hubel, 1957), or insulated with glass (Dowben and Rose, 1953) may be used for extracellular recording. Metal electrodes have the disadvantage that they are a little more difficult to prepare than fluid-filled glass micropipettes, but they have the advantage that the peak-to-peak noise levels appear to be less than with fluid-filled pipettes of comparable efficiency in unit isolation. Hubel’s tungsten electrode has the additional advantage that it is hard enough to penetrate the dura without bending. Fluid-filled pipettes have a potential disadvantage because they must be filled with a hypermolar solution ( NaCl or KCl) to reduce their internal resistance when tips small enough to sample cortical neurons of all sizes are used. Theoretically KC1 should be replaced by NaCl if the microelectrode is recording extracellularly, but, in practice, a fine tip with gradual taper appears to be more important in avoiding injury than the com-

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position of the electrolyte ( Amassian et al., 1960). This implies that mechanical injury is more important than the effect of electrolyte diffusion outside the neuron, a conclusion which is hardly surprising if one considers the difficulty of approaching the soma without damaging one of the many dendrites orientated in three dimensions around it. Metal electrodes, especially those plated with platinum black, may replace a possible error due to electrolyte diffusion by another error, that being the proximity of a highly active catalytic surface to the neuronal membrane. Regardless of the type of elecbode used, the tip diameter should be 2 1.1or less (Amassian et al., 1955; Mountcastle et al., 1957), and the taper must be gradual to obtain a sampling from cortical neurons in the superficial layers, bo avoid damage to the superficial cortex in deep penetrations, and to avoid “pial” dimpling ( see below ) . ( d ) The position of the electrode tip within the cortex is determined either from the manipulator readings combined with the histological sections, or by electrolytic marking (Hubel, 1959), or by iontophoresing a dye (Rayport, 1957). Three sources of errors in localizing the tip position from the manipulator reading are (Amassian, 1953a): (1) Shrinkage of the cortex occurs owing to histological preparation. ( 2 ) Microelectrode penetration does not occur normal to the pial surface. This can be checked in serial sections. ( 3 ) The pia may be dimpled by the microelectrode during penetration, especially when the electrode tip is larger than 0 5 1 . 0 p. Renshaw et al. (1941)) directly measured this error by observing the displacement of a glass capillary next to the microelectrode when the latter was lowered into the cortex. Pial dimpling may be an important factor in the controversy over the reversal point of the somatosensory-evoked response (see below). It is presumably insignificant when progressive introduction of the microelectrode below the surface leads to progressive reduction and reversal of the primary evoked responses in the superficial and midcortical layers. Pial dimpling can be entirely avoided by localized removal of the pia (Phillips, 1956a; Mountcastle d.al., 1957). ( e ) Data handling. In addition to conventional optical enlargement of photographic records, use of certain electronic instruments has proved valuable in the analysis of single cortical unit responses. A combination of electronic chronometers is by far the most accurate and least time-consuming way of determining laten-

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cies and interspike intervals in repetitive cortical unit responses. In an early model (Amassian and DeVito, 1957; Towe and Amassian, 1958), synchronous with the delivery of the stimulus pulse to the animal, the output of a 100-kc crystal-controlled oscillator was gated into a set of decade counters. When the neuron discharged, one phase of its amplified action potential was used to close the gate for the first set of counters and simultaneously to open the gate for a second set of counters. Additional discharges of the same neuron performed analogous operations on further sets of counters. The maximum intrinsic error of each measurement is a count of two pulses ( 20 psec ) , but in a number of observations, the error of the mean is obviously less. Commercial instruments are readily available to perform the same function. The Berkeley Universal meter (model number 7360) is suitable because it can be used both for timing interspike intervals and for counting in computer operations. The number of timers used will depend on the number of interspike intervals to be measured. As a safety precaution, it is advisable to insert a fixed delay of 1 msec between the shock and the start impulse input to prevent the timing of a differentiated shock artifact. Similarly, a fixed delay (e.g., 0.2 msec) should be inserted between the end of the gate on a given timer and the start pulse input on the next timer to avoid timing a later portion of the rising phase of a given spike. (Fixed stable delays are readily available by using the positive-going phase of the sawtooth provided by the Tektronix Waveform Generator model number 162.) In addition to their advantages in handling the data, electronic chronometers help in collection of single unit data. Discontinuous variation in response latency (abrupt jumps in latency) is often critically related to strength of stimulation, and such critical levels of stimulation are readily spotted if the unit latencies are measured during the intensity series. It is probable that computers will be extensively applied to analysis of single unit data. Some correlation techniques for analysis of brain waves are given in reports from the Massachusetts Institute of Technology ( 1959) and Brazier ( 1960). Need for an aid to direct measurement of the records is apparent when neurons are spontaneously active, as they usually are when the preparation is lightly anesthetized or unanesthetized. To be sure, the sweep can be divided up into small equal periods and the incidence of spikes in the successive periods compared with and without

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stimulation ( Amassian et al., 1959) but this method is obviously time-consuming. As an alternative, the action potentials may be recorded on a tape recorder which is as free as possible from irregularity in tape transport mechanism. ( W e use the Ampes model number Fr 1100.) The action potentials are then played back through an amplifier, shaper, and converted into digital pulscx In the analysis of evoked activity a gate of any desired duration (e.g., 10 msec) is opened at a preset timc after each stimulus. In thc analysis of spontaneous activity thc gate is opened by arbitrary pulses whose period is approximately the same as the interstimulus periodicity. The gate permits transmission of the digitalized spike through a logical “and” gate. Neuronal responses occurring during the time of the gates are electronically counted. The tape run is systematically combed at different times after the stimulus or the arbitrary pulse. Ideally, the number of counts during the gate periods opened at a given time after the arbitrary pulses in spontaneous runs should approximate the total number of action potentials in the run divided by the number of gates times the duration of each gate. When the distribution in successive gate periods following stimulation departs from the expected number on a uniform random basis, one can conclude that the neuron is either excited or inhibited at the spccified time after the stimulus. Coincidences between simultaneously recorded neuronal responses can be readily detected. A neuronal discharge from one neuron initiates a gating pulse of fixed duration while discharge from the other neuron initiates a brief digital pulse. Gating and digital pulses are fed through an “ a n d gate and the coincidences are counted for that particular duration of the gating pulse. Such techniques have proved valuable in studying the relationship between simultaneously recorded reticular neurons.

11. Classification and Interpretation of Single Cortical Neuronal Spikes and lntracellular Waves

Analysis of cortical unit activity depends on a description of the properties of the individual spikes including polarity, amplitude, duration, presence or absence of a resting potential, extent of its potential field, interspike intervals during spontaneous activity, the probability of one or more responses to deliberate stimulation;

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initial latency, interspike intervals, and the amount of variability of the temporal aspects of evoked discharges; the effect of changing the intensity, position, and type of stimulus; the use of two or more stimuli to the same afferent source (temporal interaction); the use of stimuli applied to different afferent sources at different time intervals (time course of spatial interaction). In addition, an attempt is made to correlate the above properties with such spatial parameters as the depth within the cortex and the position of the penetration relative to the penetration yielding the maximal response to a given stimulus. Comparison of two or more single neurons simultaneously ( Amassian, 1953a; Mountcastle, 1957; Baumgarten and Schaeffer; 1957, Amassian et nl., 1959; Li, 1959c) reveals differences in properties of neurons which lie close to one another and also permits an estimate of the coherence between properties of neurons, e.g., initial latency of discharge. Ideally, one attempts to relate the unit responses to the simultaneously recorded population responses in order to determine whether the unit sampling is biased or random. The problem of comparing unit discharge and population responses in the cortex is that, unlike peripheral nerve (Gasser and Grundfest, 1939), no population response recorded from the surface, or the depths, can be attributed with certainty to discharge of a single type of cortical neuron. By contrast, assumptions are made as to the amount of bias in the unit sampling and a comparison is made between the population of single units and the population response. When lack of correspondence is observed [ e.g., between spontaneous activity recorded at the surface and unit activity (Li and Jasper, 1953)], one can conclude that the population response and unit discharges are largely unrelated. The one exception to the lack of validation of single cortical unit data would come from a comparison of population responses in the pyramidal tract and single unit records from pyramidal neurons which project into the tract (pyramidal projection neurons). The next step is to correlate the functionally defined types of cortical neurons with the morphological types of cortical neurons. Ideally, the identification of the actual cell recorded is made by dyeing the neuron (Rayport, 1957), but more often the investigator is able to identify only the lamina within which a morphological type of the neurons predominates or is uniquely distributed. A small fraction of cortical neurons project into the pyramidal tract and can be

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identified by antidromic invasion following a shock to the ipsilateral bulbar pyramid (Phillips, 1956a; Patton and Towe, 1957, 1960; Martin and Branch, 1958; Li, 1959a). For the great majority of cortical neurons, correlation of unit properties with the laminar distribution of the various morphological types of cortical neurons is the most that can be hoped for. Three types of single neuronal spikes can be recorded extracellularly in the cortex. Such spikes are recorded in the absence of a resting membrane potential. Extracellularly recorded spikes are attributed to a single neuron when they are alike in amplitude, polarity, duration, and are recorded at the same position of the microelectrode. Mountcastle et nl. ( 1957) rigorously analyzed the short-term variability in spike amplitude and reported a coefficient of variation of 5 % or less when using their closed chamber technique. The spike height may be reduced during high frequency repetitive discharge ( Amassdan, 1953a; Mountcastle et nl., 1957). That such discharges are derived from the same neuron is inferred from the direct relationship between spike amplitude and interspike interval ( Mountcastle et nl., 1957). In addition, intracellular records from Betz cells (Phillips, 195613) show a comparable reduction of spike height during high frequency repetitive discharge. When the position of the microelectrode is carefully changed, the spike amplitude may change without altering the pattern of response (Amassian, 1953a; Mountcastle et al., 1957), which implies that the spikes are derived from the same neuron. The types include: ( a ) Initially negative spikes with or without a smaller positive phase following the negative phase. The amplitude of the initially negative phase ranges from a little above noise level up to 5 mv ( L i and Jasper, 1953) but is usually 0.2-0.5 mv (Amassian, 1953a). Li and Jasper (1953) correlated the average size of the cortical neurons in a given layer with the amplitude of the negative spikes recorded from the layer. The incidence of initially negative spikes relative to the other types recorded is dependent on the size of the microelectrode tip. When using microelectrodes of 4-12 p tip diameter, initially negative spikes predominate in records taken from the gray matter ( Amassian, 1953a). The duration of the negative phase ranges from 0.3-0.85 msec (Amassian, 1953a; Li, 1955). The spatial field of the initially negative spike is not less than about

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100 p (Mountcastle et al., 1957) under particularly favorable conditions for such measurement (after removal of the pia). Evoked initially negative spikes are characteristically recorded only from regions of synaptic transfer such as the gray matter (Amassian, 1953a; Li and Jasper, 1953; Mountcastle et al., 1957). Their latency invariably exceeds the latency of the earliest primary response or the earliest slow wave recorded within the cortex (Amassian, 19534. Furthermore, at the cuneate nucleus, presynaptic latencies can be determined very precisely and the initially negative spike has a postsynaptic latency ( Amassian and DeVito, 1957). Evidently, the initially negative spike cannot be derived from a long tract axon or its terminations, but must be derived from the perikaryon, dendrites, or axon hillock region of a neuron. The short duration of some of the initially negative spikes, together with the spatial extent of the potential field, implies that a large portion of the neuron yields such spikes. ( b ) Positive-negative spikes upward of 1 mv in amplitude are predominantly recorded when the microelectrode tip is 3 p or less (Amassian, 1953a; Tasaki et nl., 1954; Li, 1955; Mountcastle et al., 1957; Martin and Branch, 1958; Towe and Amassian, 1958; Phillips, 1959). The initial positive phase ranges from 0.25 to 1.1 msec in duration and the negative phase ranges from 0.3 to 5.0 msec (Amassian, 1953a; Li, 1955). In common with the initially negative spike, the positive-negative spike of a millivolt or more amplitude is not recorded from the white matter and has a longer latency than the earliest primary response or deep slow wave ( Amassian, 19534. The long latency and the repetition of lateral geniculate positivenegative spikes following a single shock to the optic nerve prove that the positive-negative spike is a postsynaptic event ( Tasaki et al., 1954; Freygang, 1958). Finally, when the microelectrode is progressively lowered into the cortex, small initially negative spikes are frequently changed into larger positive-negative spikes ( Amassian, 1953a; Li, 1955; Mountcastle et al., 1957). Positive-negative spikes are often associated with an increase in baseline noise (Amassian, 1953a), which serves as a useful warning that one is close to a neuron. This kind of noise is identical with one type of noise recorded by Brock et al. (1952) when the microelectrode tip lay just outside the motoneuron and is to be distinguished from “synaptic noise” in intracellular recording. The

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transformation of positive-negative spikes into much larger monophasic positive spikes associated with a resting potential occurs either “spontaneously” (Li, 1955) or is caused by lowering the electrode slightly (Phillips, 1959). These observations together with the large amplitude of the positive-negative spike ( u p to 32 mv, Phillips, 1959 ) indicate that the positive-negative spike is recorded when the electrode tip is close to, impinges upon, or invaginates the cell membrane. Interpretation of the positive-negative spike is postponed until the discussion of the intracellular spike. ( c ) Initially positive spikes which are nionophasic from the time that they are first observed, are the least frequently encountered type in the gray matter. By contrast, such spikes are readily recorded in the white matter (Adrian, 1941; Marshall, 1941; Amassian, 1952a) and in the pyramidal tract (Adrian and Moruzzi 1939; Whitlock et al., 1953; Patton and Amassian, 1954). The spikes recorded from white matter and from the pyramidal tract are obviously derived from long tract axons. Such spikes usually show a prominent notch and are probably due to “killed end” recording from axons ( Marshall, 1941). 1. Intracellular Recording

Satisfactory intracellular recording from cortical neurons is notoriously difficult to achieve as evidenced by the sporadic records prior to 1956 (Albe-Fessard and Buser, 1953, 1955; Tasaki et al., 1954; Buser and Albe-Fessard, 1957; Li, 1955; Amassian et nl., 1955). Most of what is known about the intracellular spikes of uninjured cortical neurons is derived from the admirable study of Phillips (1956a) in which a series of 16 Betz cells were identified by antidromic invasion following a pyramidal shock and which provided suitable data over a period of 5 4 0 minutes. The resting membrane potcntial was not constant under light hexobarbital anesthesia but showed oscillations with, or without, related spike discharges. The maximum membrane potentials observed ranged from 48 to 69 mv. Phillips (1956a) noted the lower figure of the maximum membrane potentials recorded from Betz cells as compared with spinal motoneurons (Brock et al., 1952), and suggested that under light anesthesia, the Betz cell membrane potential was held at a reduced value by synaptic bombardment. Possibly, some-

s1

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what different values of the membrane potential might be obtained if microelectrodes were selected for minimum tip potential (Adrian, 1956). The action potential of the Betz cell showed an overshoot whose magnitude varied in the series. The largest action potentials recorded were 84 mv (Phillips, 1956a) and 90 mv (Phillips, 1959), but these figures are probably an underestimate because no compensation was made for inadequate frequency response of the recording system. The duration of the antidromic intracellular spike in Phillips’ illustrations (l95Ga, b, 1959) ranges from about 0.4 to 0.8 msec. Martin and Branch (1958) recorded intracellularly from 7 Betz cells and presented records substantially similar to those of Phillips ( 1956a). Intracellular records from cortical neurons might be derived from axons, “near the perikaryon” ( perikaryon, axon hillock region, dendritic stems), or from fine dendrites. In the spinal cord, Woodbury and Patton ( 1952) distinguished primary afferent spikes from those believed to originate in the motoneuron soma. Primary afferent axon spikes followed peripheral stimulation at high frequency while motoneuron recordings were identified by invasion following a shock to the ventral root. Primary afferent axonal spikes were brief and lacked the prepotential recorded from motoneurons. Frank and Fuortes (1955) and Frank (1959) give definitive accounts of the differences between axon and soma spikes, which are based on the study of many units, and the use of a negative capacity preamplifier to secure faithful reproduction of the spike form. Two populations of spikes were differentiated. Pure 0.2 axonal sites (dorsal and ventral roots) yielded spikes of 0.6 msec duration. A gradual decrement in spike amplitude was noted in the response to a second stimulus given less than 2 msec after the first stimulus. Very small slow potentials were recorded from such units. Another type of spike which had a duration of 1.5 t 0.3 msec was recorded from motoneurons (as proved by antidromic invasion), was accompanied by large slow potentials ( u p to 40 mv in amplitude during a stryclinine convulsion), and showed a sudden large drop in amplitude in response to a second antidromic shock which was brought progressively closer to the first shock. Such spikes were attributed to the motoneuron soma. Other spikes recorded within the spinal cord were allocated either to the axon or to the soma according to the criteria of spike duration and presence of a significant slow potential. Li (1959b) distinguished

*

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between the various spike forms recorded intracellularly in the cortex and came to conclusions analogous to those of Frank and Fuortes ( 1955). No frequency compensation system was used during spike recording. Spike potentials of 1.2 msec duration (range 0.73.0 msec) were usually recorded from gray matter and were associated with large prepotentials, which were exaggerated by topical application of strychnine solution. Li further noted an inflection at about 35 mv depolarization, similar to that described by Martin and Branch (1958). Such spikes were attributed to the cell bodies of cortical neurons. Other spikes of 0.5 msec duration were recorded with equal facility from gray and white matter, were not preceded by prepotentials, and were attributed to axons. hlartin and Branch (1958) show an intracellular Betz cell spike with a duration of 0.8 msec and illustrate others with prepotentials and a spike duration of about 0.6-0.7 msec. Slow potentials are also evident in Phillips’ (1956a, b, 1959) records although the spike durations are as much as 50% less than the motoneuron soma spike. If the criterion of a prepotential is accepted as evidence for intracellular recording in or near the cell body, the soma spike of cortical neurons apparently has a much briefer duration than the motoneuron soma spike (cf. Li, 1959b). The morphological entities responsible for the two portions of the cortical neuronal spike are not definitely known, but mily be inferred from data on the spinal motoneuron. Eccles (1957) summarizes the evidence for relating the initial portion of the spike ( I S ) to the activation of the initial axonal segment and the second portion (SD) to activation of the soma. Fuortes et al. (1957) use the noncommittal terms “A” and “ B and provide evidence for axon hillock and soma origins, respectively. Of particular importance is their proof that discharge in the ventral root fiber may occur following orthodromic excitation of a motoneuron which is hyperpolarized during the spike so as to prevent the appearance of the “B” portion. Fatt (1957) attributed the A-B spike to perikaryon and dendrites, respectively, but comparable A-B spikes have been recorded from dorsal root ganglion cells which lack dendrites (Crain, 1956).Common to all the interpretations is the notion that a critical depolarization, however induced, leads to firing of a low threshold portion of the neuron (th e “A” portion), which then causes sufficient depolarization to lead to discharge of the “B”

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portion. Voltage clamp experiments (Fig. 1) suggest that the “B” portion of the cell only involves part of the soma (Frank et al., 1959). A concentric microelectrode was used and was presumed to lie in the cell body. The pattern of inward current flow during voltage clamping was analogous to the conventional A-B spike. That a B spike was still observed implies that the clamped cell body did not lie between A and B areas, i.e., the B area involved that part of the soma close to the axon. Further information bearing on the site of production of the cortical cell spike comes from the study of the extracellularly recorded spike. It is assumed that the spike recorded by a microelectrode tip which is just outside the membrane reflects the voltage drop produced by flow of membrane current across the local external resistance. Tasaki et al. (1954) initially recorded one inflection on the initial limb of the positive phase of the extracellular positive-negative spike. They distinguished a brief axonal component from a longer duration cell body component during injury discharges. In addition, Mountcastle et al. (1957) recorded an earlier inflection marking the point of take-off of the rest of the positive phase of the spike. Two inflections were also observed in spikes recorded from the ventrobasal complex of the thalamus (Rose and Mountcastle, 1954) and from the lateral geniculate body (Freygang, 1958). The two inflections subdivided the positive phase into an initial small positive component and two larger positive components. Freygang’s (1958) analysis indicates that the first component results from the electrode tip sitting on a portion of the postsynaptic membrane which is the source for a synaptically created sink located elsewhere on the neuron. The second positive deflection is attributed to discharge of the initial axonal segment and the third deflection to discharge of a higher threshold, small area of the somatic membrane. The succeeding negative phase does not reflect spread of activity to the membrane under the electrode tip, but is due to the passive capacitative response of the membrane. The soma membrane is envisaged as mainly electrically inexcitable (cf. review on this concept by Grundfest, 1959) and is specialized for synaptic excitation. Freygang and Frank ( 1959 ) simultaneously recorded extracellular and intracellular spikes from the same motoneuron during antidromic invasion (Fig. 2 ) and found that the membrane current was outward in direction throughout the rising

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phase of the intracellular spike, i.e., the extracellularly placed electrode tip lay outside membrane which was inactive during the A and B components of the spike. However, injury to the cell membrane could not be excluded as an explanation of this finding. By contrast, Bennett et al. (1959) deduced that the portion of the large supramedullary cell body of the puffer which yielded a positivenegative spike was electrically excitable because the membrane current flowed inward just after the A-B inflection and long before the peak of the intracellularly recorded spike. It is uncertain whether positive-negative spikes recorded from cortical neurons are due to placement of the tip outside electrically excitable or inexcitable membrane, because intracellular and extracellular records have not been obtained simultaneously. The closest approximation is the recording of a positive-negative Betz cell spike at one depth, followed by intracellular recording from the same cell at greater depth (Phillips, 1959). The start of the negative phase of the extracellular spike corresponded practically with the end of the intracellular spike. It would be unwise to conclude that the electrode tip lay against electrically inexcitable membrane because the externally recorded A-B spike might have changed in contour following impalement of the cell. Martin and Branch (1958) compared intracellular and extracellular spikes which were recorded from different Betz cells. The negative phase of the extracellular spike commenced prior to the A-B inflection of the intracellular spike. However, the total duration of the extracellular spike chosen for FIG.1. A. Block diagram of experimental arrangement. ( x 1 ) Unity-gain, negative-capacitance cathode followers; ( cross iwutr. ) capacity neutralization circuits; ( x 5000) clamping amplifier; ( V and I ) direct-coupled amplifiers measuring potential of internal microelectrode and current throngh external microelectrode, respectively. “Comp” conipensates for contact and tip potentials, and “cal” provides calibrating pulses between preparation and ground. The gain of the x 5000 amplifier must be reduced at higher frequencics to prcvent oscillation. B. Currents through external microelectrode during clamp at different voltages. Clamping voltages are indicated (in millivolts) by numbers a t left. At 57 and 93 mv there is evidence of repetitive firing. C. Potentials recorded by internal microelectrode following antidromic stimulation. The upper record was made in the absence of clamp; the lower record W:IS made during voltage clamp. The square wave is a 20-mv calibration. Arrows indicate A-B inflection; l-msec time marks apply to records B and C. (From Frank ct al., 1959.)

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A

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illustration is unusually brief (0.28 msec) and it is possible that the spike was recorded from the axon hillock or even further down the axon. Phillips (1959) seldom observed splitting at the A-B inflection when the pyramidal tract was stimulated at high frequency and he attributed the high safety factor of the A-B transition to the gradual expansion of the axon hillock region. Rapid A-B transmission would help explain the brevity of the intracellular Betz cell spike. A

B

i/!2m W

2

O

r

n

V

I msec I

I

I

I

I

I

I

I

I

I

I

FIG. 2. Coniparison of intracellular and cxtracellular spikes obtained simultaneously by concentric micropipettes. Upper traces are extracellular spikes recorded by the outcr pipette. Lower trace in part A is the intracellular spike recorded with tlic upper trace. Lower trace in part B is an intracellular spike recorded with the upper trace and transfoniied by the equivalent circuit. Antidromic excitation, multiple swecps superimposed, threshold stimulus at start of each sweep, cell fired once. (From Freygang and Frank, 1950.)

Other “unitlike” responses were recorded with a duration as long as 8-15 msec (Li, 1959b), or up to 20 msec (Tasaki et al., 1954). Such responses were of much smaller amplitude (5-17 m v ) than the briefer axon or soma spikes and were associated with a small resting potential of 31j-50 inv (Li, 1959b). The long duration variety of positive-negative spike ( Amassian, 1953a; Tasaki et al., 1954) may be the extracellular counterpart of such i1itr;icellular spikes. Long duration cortical spikes arc unifomily attributed to

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dendritic activity, but this hypothesis cannot be accepted until such spikes are recorded under conditions where the latency can be precisely timed and the over-all frcquency response of the recording system is known. The most favorable analytical condition is provided by the recording of responses from antidromically excited Betz cells, but such experiments havc not yet revealed long duration “unit” responses (Phillips, 1959). It may also be noted that the unitlike appearance ( all-or-nothing quality ) of the “dendritic” responses of Tasaki et al. (1954) and Li (1959b) is compatible with the ability of dendrites, as a species of electrically inexcitable membrane, to show graded depolarization (Grundfest, 1959). If “dendritic” responses are electronically conducted from a region generating all-or-nothing spikes, they would be expected to have an all-or-nothing behavior in the absence of direct synaptic excitation during the period of observation. Miniature small potentials of 0.8-1.5 mv amplitude were recorded by Li (1959b) from cortical neurons and are analogous to the second type of synaptic noise that Brock et al. (1952) recorded from spinal motoneurons. Graded, slow depolarizing or hyperpolarizing waves have been recorded from cortical neurons ( Albe-Fessard and Buser, 1953, 1955; Amassian et nl., 1955; Phillips, 1956a, b; Li et al., 1956b; Buser and Albe-Fessard, 1957; Martin and Branch, 1958; Branch and Martin, 1958; Li. 195913; Phillips, 1959). Such potentials are analogous to those recorded from spinal motoneurons (summarized by Eccles, 1957) and differ only in the following respects: (1) cortical postsynaptic potentials (p.s.p. ) occur in the absence of deliberate stimulation ( 2 ) cortical p.s.p.’s have a long duration (over 20 msec in Amassian et al., 1955, about 60 msec in Albe-Fessard and Buser, 1955). Slow potentials recorded from spinal interneurons ( H unt and Kuno, 1959) similarly show long lasting depolarization. In both the cortex and the spinal interneuronal system, it is undecided whether the long lasting potential changes are due to prolonged transmitter action through temporal dispersion of the presynaptic inflow ( e.g. through intcrneuronal delays ), to repetitive discharge in presynaptic fibers, to prolonged transmitter release by a single presynaptic discharge, or to a peculiarity of the receptor membrane. The depolarizing p.s.p. is not momentarily reduced following the spike in some intracellular records (see Fig. 1 in Amas-

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sian et al., 1955; Li et al., 1956b). I n such neurons, the slow wave may originate in electrically inexcitable membrane which is prevented from repolarizing by persistent transmitter action. Finally, mention should be made of stablc resting potentials of 60-90 mv (Phillips, 1956a; Li, 1959b) which show no spontaneous fluctuations of meml.)rane potential and are unaffected hy neural stimuli or by administration of strychnine. Similar resting potentials wcrc obser\7cd by Frank and Fuortes (19.55) in the spinal cord and were attributed to gliul cells. 2. Criterici of Injur!y By injury it is meant that the patterns of activity observed (spike discharge or graded slow waves) are modified by the presence of the microelectrode tip. Two situations may b e distinguished: I n the first, the ni(~mbr:ine i s clearly affected by the prcsence of the electrode, but the tcmporal pattern of responses is unaffected and can still be used as a n indcx of the activity of tlie uninjured neuron. This situation obtains in “killed cncl” cxtracellnlar recording from axons ( Marsliall, 1941; Amnssian, 1952a). Alternatively, the pattern of response may be grossly affected either by intracclliilar or by extracellular recording from the coma. The w c l l - k n o n ~criteria ~ of injury (Amassian, 19532; Li and [asper, 1953; Tasaki et oZ,, 1954) include: an increase in the rate of resting discharge and rhythmic firing at a high frcquency of several hundred per second. T h c spike height in such trains is reduced in amplitude and m,ty show splitting. Injury discharge of positive-negative iniits is often associated with an increase in the amplitiide of the positive phase relative to the negative phase. Two more subtle forms of injury shoiild be mentioned. The first is manifested in positive-negative units by an increase in the number of spikes in the repetitive evoked response, an increase in evoked firing rate, and a reduction in latency of discharge. This sort of injury is unimportant when the pattern of response of the neuron is the same at different recording distances from the cell membrane. The possibility that the positive-negative spike is recorded from soma membrane, which is rendered inexcitable by a closely applied external electrode, has already been mentioned. This alone would not invalidate the use of positivenegative spikes as an index of the temporal pattern of discharge of

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the neuron. However, Mountcastle et al. (1957) found that, in some instances, the excitability of the unit was reduced when an initially negative spike was transformed into a positive-negative spike. This phenomenon has not been observed at lower levels [e.g. the cuneate nucleus (Amassian and DeVito, 1957)], perhaps because of the higher safety factor for synaptic transmission. Until the degree of injury in the prodtiction of positive-negative spikes is decided upon, it appears ncccsiiy, ac; in the past, to draw general conclusions about the behavior of cortical neurons from observations of both initially negative , i d positive-negative spikes.

111.

Microelectrode Analysis of Brain Waves and the Relationship of

Slow Waves to Unit Activity

In the general analysis of activity recorded from the surface of the cortex answers are sought to the following questions: Is the electrical activity attributable either to corticipetal axons, or to cortical neurons, or to both sets of structures? If the activity is due to cortical neurons, what part of tlic neuron is responsible for the EhIF? Is the activity composed of propagated all-or-nothing discharges, graded slow postsynaptic potentials, or afterpotentials? Finally, are the electrical waves conducted through the cortex by a combination of all-or-nothing propagation in axons and transsynaptic activation, or are they elcctrotonically conducted in a set of structures which clo not show self-regenerative changes? An attempt is made to fractionate the surface response into a number of components on the basis of thc following analytical procedures: (1) Changing the stimulus strength may reveal that a given surface potential is secondary to activation of fibers of one group (e.g. Bishop and Clare, 1951). ( 2 ) Use of high frequency stimulation may separate afferent from postsynaptic activity (e.g., Per1 and Whitlock, 1955). ( 3 ) Changing the position of the stimulus may reveal either that two sets of afferents are responsible for the surface activity (e.g., von Euler and Ricci, 1958), or that the expected temporal dispersion of responses which are supposedly due to more than one group of afferent fibers does not occur (e.g., Bremer and Stoupel, 1956). ( 4 ) Physical agents such as heating, or cooling (e.g., Bremer and Stoupel, 1957), or drugs such as the general anesthetics,

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strychnine, d-tubocurarine, etc., may be used in the analysis. ( 5 ) Interaction between the surface response and a response of known composition may be studied ( e.g., Chang, 1953b). Because there is disagreement about the nature of virtually all surface responses, this type of procedure has the least analytical value, although clearly of descriptive value. ( 6 ) The temporal relationship bptween components of the surface response and an identifiable response, e.g. pyramidal tract discharge, may be studied. ( 7 ) The pattern of reversal of the response in the depths of the cortex is studied with a microelectrode. (8) The temporal pattern of single neuronal activity may reveal sequential steps in activation of the cortex. Intracellular recording of slow membrane phenomena provide the basis for explanation of the postsynaptic components of surface records. Several factors must be considered in interpreting patterns of depth reversal: ( 1 ) Hyperpolarization of a superficial portion of a longitudinally oriented cortical neuron cannot be distinguished by pattern of depth reversal from depolarization of the deeper portions of the neuron, because the potential drops produced in the external volume conductor will have the same sign (cf. Purpma, 1959‘) . Furthermore, negative and positive waves will be recorded at different places in the external medium if different portions of a neuron are hyperpolarized to different degrees. A net depolarization of the perikaryon-axon hillock region is inferred when an increased spike discharge occurs, but this is proved only by intracellular recording. ( 2 ) The sites of maximin negativity or positivity do not necessarily indicate the positions of major “sinks” and “sources.” Assume, for example, that a cortical wave is produced by a series of synchronously created neuronal dipoles of limited spatial extent which are uniformly distributed in depth through the cortex. An exploring electrode might then encounter a zero potential about midway through the cortex although itself situated close to the highest concentration of active elements. The change in voltage with respect to small increments in depth might be more valuable in locating the dipoles. A rigorous analysis of the type performed by Howland et al. (1955) on the spinal cord needs to be done on cortical responses. ( 3 ) A single reversal point will not be observed when neuronal dipoles are sequentially formed in the axis normal to the surface of the cortex. Such movement of the dipoles may be due to

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electrotonic conduction in electrically inexcitable portions of the neuron, to synaptic spread within the cortex followed by generation of postsynaptic potentials at different sites within the cortex, or by temporal dispersion of the corticipetal volley. (4) Volume conductor spread from buried cortex is a further complication, especially when reversal patterns are studied in feline sensorimotor cortex.

A. SPONTANEOUSACTIVITY Dempsey and Morison ( 1942) observed interaction between rhythmic spontaneous S-l2/sec waves (barbiturate bursts, or spindles) and recruiting waves which were evoked by repetitive stimulation of the intralaminar thalamic nuclei. They suggested that a common group of neurons was implicated in both activities. Li et al. (1956b) showed that rhythmic spontaneous activity (58/sec) and recruiting waves had a similar configuration and usually had a similar pattern of depth reversal when the microelectrode was progressively lowered into the cortex. Li and associates’ (1956b) findings agreed with those of Dempsey and Morison (1942) in suggesting a similar cortical basis for rhythmic spontaneous waves in the 5-18/sec r m g e and recruiting waves. Using the “pyramidal cat” Whitlock ct cil. (1953) and Arduini and Whitlock (1953) detected a further similarity between spindle waves (‘i-lO/sec) and recruiting waves because high frequency discharges were recorded from single pyramidal axons during both types of wave. Unfortunately, this iinifying concept is inadequate. A careful analysis by Brookhart and Zanchetti ( 1956) revealed that recruiting waves could be elicited in the absence of pyramidal discharge either in the intact cat or after thermocoagulation of the midbrain. By contrast, both spontaneous bursts and the cortical responses to repetitive stimulation of the sensorimotor thalamic relays were accompanied by large pyramidal tract responses. The latter observation was confirmed (Purpura, 1958). Evidently, spontaneous burst activity recorded by different investigators has properties in common both with recruiting waves and with the responses to repetitive stimulation of the sensorimotor thalamic relays. I t is likely that a t least two types of spontaneous waves were studied. Dempsey and Morison ( 1943) distinguished spontaneous “projection” waves from

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the 8-12/sec waves. Spontaneous “projection” waves we1 e sensitive to anesthesia, were reduced by section of the great afferent pathways, and showed interaction with the cortical response (the augmenting response) to repetitive stimulation of either the ventrolateral thalamus or of the radiations. Two problems are immediately presented in describing tlie relationship of cortical unit discharges to spontaneous waves such as the spindle. (1) Are the spontaneous waves composed of synchronized unit discharges? ( 2 ) If the answer to this question is negative, do the unit discharges nevertheless bear a temporal relationship to spontaneous waves? Li and Jasper (1953) made the crucial observation that temporary arrest of artificial ventilation, or careful administration of barbiturates led to a loss of spontmeous unit discharges without necessarily reducing “a-like” waves. Admittedly, cortical neuronal dischargcs which contrihited to such spontaneous wave activity may not have been sampled in this study because of the small size of the ncurons. This seems unlikely because the microelccbodes used had tip diameters down to 1 1-1 and discharges of cells in a11 layers of the cortex are shown elsewhere in the study. Furthermore, small Golgi type I1 neurons which might have escaped the microelectrode woulcl not be expected to yield large spontaneous wcives at the surface because of their “closed electrical field” (Lorente de iY6, 1947). The dissociation of unit discharge and spontaneous spindle waves has been generally confirmed by other investigators and clearly indicates that spii”dle waves are due to either graded slow postsynaptic potentials or t o afterpotentiah in fibers. Avons within the dorsal columns are known to have very prominent negative afterpotentials ( Rudin and Eisenman, 1954), but the role of axoiial afterpotentials in spindle waves is difficult to evaluate. Manifestly, the only axonal afterpotentials which need be considered when spindle activity is dissociated from unit activity are those of the corticipetal axons. Assuming that the spindle-producing corticipetal axons have similar afterpotentials to those of specific thalamocortical axons, the trivial contribution of the latter to the primary surface positive response (see below ) implies that spindle waves are not composed of summed afterpotentials of corticipetal axons. Spindle waves under barbiturate anesthesia are most probably due to postsynaptic slow waves in the cortex ( L i and Jasper, 1953). This hypothesis can be proved only

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by systematically comparing intracellular slow potentials with spindle activity in population rccords. Spontaneous cortical unit discharge may either: (1) appear as an irregular tonic discharge nnrelated to spindle waves (L i and Jasper, 1953; Jung, 1953) or to low voltage fast waves (Calma and Arduini, 1954); ( 2 ) change in rate (usually an increase) during spindle activity as compared with the pauses between spindles (Li and Jasper, 1953)--however, the transition from sleep to awakening is usually accompanied by an increased rate of discharge (L i and Jasper, 1953); or ( 3 ) show single or grouped high frequency discharges during particular phases of the waves composing the spindle (Jung, 1953; Li and Jasper, 1953; Whitlock et al., 1953; Calma and Arduini, 1954). The rate of discharge is reduced during the period between spindles as compared with the period during the spindle, but reaches its maximum when the animal is aroused ( L i and Jasper, 1953). By contrast, a reduction in firing rate of pyramidal projection units was observed during arousal of the “pyramidal cat” by Whitlock et al. (1953) and during arousal of the intact cat by Calma and Arcluini (1954). The difference in results is most probably due to variations in the types of cortical neurons which were samplcd. Afore recently, Ricci et d.(1957) found that units recorded from the motor cortex of chronic preparations showed either increased firing, decreased firing, or were unchanged during a generalized alerting response. Hubel ( 1959 ) observed either a smootliing out of grouped discharge of visual units with little change in firing rate, or a marked diminution in firing rate. Several factors must be considered in assessing the significance of type (1) and ( 2 ) units. ( a ) The population spontaneous wave selected for comparison with the neuron should be derived from a volume containing the nciiron. Coinparison is most readily made when a wide area of cortex is activated within a brief time span. Thus most studies show an obvious relationship between convulsive waves and unit discharge (e.g. Wliitlock et al., 1953; Li and Jasper, 1953). However, Brookhart and Zanchetti ( 1956) noted differences in synchrony of spindle waves recorded a t different points on the motor cortex and found a relationship between spindle waves recorded only from certain areas and pyramidal neuronal excitability. Desynchronized low voltage fast activity obviously poses the most

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difficult problem in establishing a relationship. Clearly, multiple simultaneous population recordings from the cortex combined with cross correlation techniques are required to determine whether discharge of a given cortical unit is related to brain waves within its general area. Recently, by an ingenious display system, Green et al. (1960) detected a correspondence between unit discharge and the hippocampal theta rhythm, although the correspondence would have been missed by simple visual inspection of the records. ( b ) If spontaneous waves are viewed as summed depolarizing and hyperpolarizing postsynaptic potentials ( cf. Purpura, 1959) in cortical neurons which are situated at various depths and which are synaptically bombarded at different times, the relationship of the average surface or depth response to unit discharge would be expected to be obscure. Phillips ( 1956a) recorded spontaneously occurring depolarization waves from Betz cells. Hyperpolarization waves were also detected particularly after a depolarization wave. Various frequencies of Spontaneous discharge of single cortical neurons are reported depending on the state of alertness of the animal and the morphological type of neuron. Li and Jasper (1953) give figures of 100-150/sec during arousal and l-l2/sec at rest. Most of these cells were probably not pyramidal projection neurons. Calma and Arduini ( 1954) recorded spontaneous discharge frequencies of 20-100 ’sec from pyramidal axons in the unanesthetized cat which showed uninterrupted low voltage fast cortical waves. Phillips (1956a) gives mean frequencies of 1&50/sec for Betz cells under hexobarbital anesthesia. Martin and Branch (1958) give a range of 1-27/sec (mean 6.4/sec) for spontaneous discharge of Betz cells in cats which were initially anesthetized with Sodium Pentothal and subsequently converted into the “pyramidal” preparation. An analysis made of the distribution of interspike intervals in spontaneously discharging Betz cells revealed two distributions. In continuously &charging Betz cells, the intervals were not randomly distributed, but were grouped around the most probable interval of about 60 msec. Other Betz cells did not fire continuously, but fired in bursts separated by silent intervals of irregular duration. If intervals greater than 600 msec were ignored, the distribution of intervals within the bursts approximated the theoretical random distribution. Rhythmic discharge in continuously firing B e k cells was distinguished from random discharge within the burst dis-

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charges of other Betz cells. However, this distinction depends on the infrequency of short intervals in continuously firing Betz cells, and Martin and Branch (1958) suggest that a potentially random discharge in such cells may be given a rhythmic appearance because of brief inhibition following discharge. Such inhibition was probably lost following extensive midbrain lesions because the “bursting” pattern of random clischargc was usually observed then. This important type of analysis should be confirmed with smaller class intervals than 40 msec and with a much larger number of intervals. Simultaneous recording from several cortical neurons by two or more microelectrodes shows that spontaneous activity occurs asynchronously in the population ( Amassian et al., 1959; Li, 1 9 5 9 ~ ) . Nevertheless, Li ( 1959c) obscrved a temporal relationship between burst discharges of different cortical neurons. The tendency toward synchronized discharge was greatly increased by topical administration of strychnine. Enomoto and Ajmone-Marsan ( 1959) similarly demonstrated synchronized discharge during convulsive activity by observing the relationship between several units which were recorded with a single microelectrode. Units recorded from normal cortex and from chronic epileptic cortex differed in behavior (Schmidt et al., 1959). “Spontaneous activity” occurred more commonly in epileptic cortex. High frequency discharge occurred during random epileptic waves and during propagated seizures. The authors attribute such aberrant patterns of activity to an abnormality of the dendritic arborizations of the “epileptic” neuron. WAVES B. RECRUITING In most instances, spindles and recruiting waves in unanesthetized animals undergo similar phase reversals when the microelectrode is progressively lowered into the cortex ( L i et al., 1956b). A penetration in the anterior sigmoid gyms is graphed in Fig. 3 and shows reversal of both the small initial positive component and of the much larger negative component. A large positive phase was recorded at a depth of 1mm below the surface. The surface positive and negative components of the recruiting response are partly reversed between the surface record and the record at 600 p, The

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pattern of reversal of the surface negative component was tentatively attributed to depolarization of apical dendrites with the cell bodies and basal dendrites acting as “sources.” Inspection of Fig. 3 (Li ct al., 1956b) reveals no significant differencc in latency of the first zcro potential between positive ancl negative com-

2100 1

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20

30 40

50

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70

Time i n msec

FIG. 3. Recruiting responses from anterior sigmoicl gyrus to rcpctitive stimulation of nucleus centranum mcdinnum. Actual potential measiircments and time courses of thcsc responsrs arc sliown. (From Li ct a!., 195611.

ponents at different depths (cf. the reversal pattern of the primary surface response). The records provide no evidence of synaptic spread of activity, nor of dendritic “conduction” in the axis normal to the cortical surface to account for the diphasic wave. The appearance is that of a t least two temporally separated “standing” waves within the cortex. This might occur if a portion of a cortical neuron undergoes clepolarizntion followed by hyperpolarization as

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shown by Albe-Fessard and Buser (1955). However, Li et al. (1956b) recorded unit discharges during both phases of the reversed recruiting wave. High frequency discharges were observed during the surface negative component, recorded at depth as a large positive wave, implying tli‘it net depolarization occurred and was most prominent in superficial portions of the neuron. Li et al. ( 1956b) recorded graded positive-going waves with an intracellular microelectrode during a particular phase of the recruiting wave. There is no evidence available from their intracellular record that hyperpolarizing p.s.p.’s are responsible for the surface neg at’ive components of the recruiting wave as suggested by Purpura ( 1959) . A systematic comparison of intracellular and recruiting waves is clearly required. A long lasting hyperpolarizing p.s.p. was recorded after the surface negative wave and was associated with a reduction in unit discharge. Only single discharges were shown for neurons responding during the initial surface positive component, implying that deeper portions of the neuron were initially, but weakly depolarized. By contrast, Arduini and Whitlock (1953) recorded high frequency discharges in pyramidal projection units which were in phase with the initial surface positive component of the recruiting wave. Such associations may be due to mixed stimulation of recruiting and augmenting systems ( Brookhart and Zanchetti, 1956). C. RESPONSES TO SPECIFIC TIIALAMOCORTICAL AFFERENTVOLLEYS An initial surface positive wave is recorded from certain loci in somatosensory areas I and I1 regardless of whether it is elicited by tactile or by electrical stimulation of skin (hlarshall et nl., 1941; Adrian, 1941), or by elcctrical stimulation of peripheral nerves ( Amassian, 1952a), dorsal column nuclei, ventrobasal thalamic complex, or white matter (Per1 and Whitlock, 1955). The surface positive wave is followed by a longer duration negative wave except at the deepest levels of anesthesia. Central stimulation has the advantage of synchronizing the corticipetal volley and thus permitting a clearer display of sequential steps in the activation and inhibition of the various cortical laminae. This advantage is offset, however, by the difficulty in drawing conclusions about the natural functions of the neurons so investigated. The initial surface positive

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response to peripheral stimulation may have at least one inflection soon after it begins (Patton and Towe, 1957, 1960). More components are distinguishable in the surface positive response to central stimulation, e.g., the thalamic relays. Perl and 1Vhitlock (1955) distinguish a brief initial positive response ( S ) from two later waves ( W 1 and 2 ) . The S deflection was attributed to the incoming specific thalamocortical afferent volley because of its brief latency, high following rate, and relative insensitivity to deterioration of the preparation. The two subsequent waves were attributed to postsynaptic cortical responses. Depth analysis would be expected to help localize the dipoles responsible for the surface positive response, but instead has yielded conflicting results. Using electrodes with tip diameters less than 2 p, Amassian et al. (1955) showed that for large, short latency responses, the maximum voltage change toward negativity for a given increment of depth occurred at positions superficial to 400-600 p. The positive response is slightly reduced at 150 p depth, is obviously reduced at 250 p, and is reversed at 400 p (see Fig. 4 ) . Mountcastle et al. (1957) recorded maximum voltage changes superficial to 200 p (Fig. 5 ) , but in the example illustrated the surface positive wave was much smaller than the succeeding negative wave. Both studies agree in demonstrating marked changes in the primary response in layer 11, i.e. above the main site of termination of specific thalamocorticdl afferents in layer IV (Lorente de Nb, 1938), and in showing that the latency of the zero potential between positive and negative waves is shortened in the superficial layers. Nakahama (1958, 1959) observed that both contralateral and ipsilateral responses similarly reversed at about 500-700 p. Perl and Whitlock (1955; Fig. 7 ) observed reversal of the initial response to skin stimulation at 1100 11, but the positive wave was reduced in amplitude between 300 and 700 p. Although the final electrode tip position was histologically controlled, nonuniform motion through the cortex and “killed end” effects might have accounted for their results, especially as an electrode of 32 1.1 tip diameter was used in the penetration cited. Perl and Whitlock (1955) and Li et al. (1956a) studied the reversal pattern of the polyphasic surface positive response to stimulation of the thalamic relay nucleus with differing results. Perl and Whitlock (1955) found little change in the initial response down to 500 p and observed reversal to negativity at 1700-

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1800 p. Li et al. (1956a) noted that the zero potential latency was shortened at depths of 500 11 or more and they described complete reversal at 800-1200 p. In addition, the negative wave at 800-

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FIG.4. Responses at indicated denths in somatosensory area I1 to stimulation of somatosensory area I (dotted) a i d contralateral ulnar nerve (dotted). Chloralose-Syncurine anesthesia. Positivity clown. Time 10 mscc. ( From Amassian et al., 1955.)

1000 p was, in some experiments, several times the amplitude of the surface positive wave. Because this depth corresponds to layer IV, Li et al. (1956a) attribute the surface positive response to summed depolarizations of specific thalamocortical afferent ter-

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FIG. 5. Study of primary evoked potential during microelcctrocle peiictration of cortcx. Responses to electrical stimulation of skin at center of peripheral receptive field, on lower forelimb. Time, 500 cyclcs/sec. Column of records above left obtained on penetration; above right, those obtained on withdrawal at the depths indicated. Plots of amplitudes on penctration and withdrawal, and of onset and peak latencies on penetration, below. Each point on thcsc charts mean of 20 responscs. (From Mountcastle et al., 1957.)

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minals and Golgi type I1 cells which are particularly prominent in this layer. Their data cannot be reconciled with those of either Amassian et al. (1955) or of Mountcastle et al. (1957), and it must be concluded that either the cortical mechanism of the response to a shock to the thalamic relay nucleus differs from the response to a peripheral somatic stimulus ( b u t see Per1 and Whitlock, 1955), or that microelectrode penetration failed to occur uniformly in the experiments of Li et al. An “apparent velocity of spread” may be calculated from the progressive shortening of the latency of the zero potential between positive and negative components at increasing depths within the cortex. The \docity of spread is linear above a depth of 400 p and is 0.05 in ’sec in the penetration illustrated in Fig. 4. This value approximates the velocity of transynaptic spread of the “deep” response which was estimated at 0.05-0.25 m/sec (Adrian, 1936) and 0.15 m/sec (Burns, 1950). [By contrast, dendritic “conduction” velocities in cat (Chang, 1952) and rabbit (Chang, 1955a) were estimated by different methods a t 2 m/sec and O.GO.21 m/sec, respectively.] This implies that neuronal dipoles are initially created close to the site of specific afferent endings and are transynaptically created at successively more superficial levels. This hypothesis has two implications that can be tested. The first is that the earliest single neuronal discharges within the population should occur below the superficial laminae. This has been confirmed by Amassian (1953a, b ) , Li et al. (1956a), Patton and Towe (1960), but is questioned by Mountcastle et al. (1957). The second is that a deeper reversal point unaccompanied by shortening of the zero potential latency, should be observed when propagated responses of cortical neurons are abolished. Li et al. (1956a) and Mountcastle et al. (1957) obtained large surface positive responses at anesthetic depths which abolished cortical unit discharge, but apparently did not compare the patterns of depth reversal at various anesthetic levels. Amassian et al. (1955) suggest that persistent depolarization of basal dendritic arborizations contributes to the surface positive response. Mountcastle et al. (1957) suggest that local p.s.p.’s rather than discharge of neurons are responsible for the surface response. Such p.s.p.’s are probably serially generated in the more superficial portions of cortical neurons which are situated above the site of specific afferent termination. The succeeding surface negative wave was attributed to synaptic activation

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of superficial portions of apical dendrites ( Amassian et al., 1955). However, the surface negative wave observed under deep anesthesia cannot depend on transynaptic spread from within the cortex. Possibly, specific afferent fiber activity leads to a depolarizinghyperpolarizing sequence of p.s.p.’s as in the records of Buser and Albe-Fessard ( 1957). Von Euler and Kicci (1958) observed a completely different pattern of depth reversal in the auditory cortex. The positive responses to clicks, or to medial geniculate stimulation, reversed similarly at a depth of about 10oO p regardless of whether the surface positive wave was followed by a negative wave. The temporal features of both waves were unchanged in the depths. Surface positive and negative waves were differentiated by changing the position of the stimulus to the medial geniculate body and by interaction with superficial cortical responses. Von Euler and Ricci attribute the surface negative wave to superficial cortical neurons which may be activated by a different set of thalamocortical afferents from those responsible for the surface positive wave. However, their findings might equally well be explained if specific auditory afferents generate depolarizing and hyperpolarizing p.s.p.’s. Analysis of the visual primary response has given conflicting results. The issues are well discussed elsewhere ( Albe-Fessard, 1957; Bremer, 1958) and will be touched upon only briefly here. All are agreed that following a shock to the optic nerve a t least four positive deflections ( 1 , 2 , 3 , 4 ) are recorded from the visual cortex. The positive deflections ride on a positive wave which is followed by a negative wave. Repetitive firing of geniculate neurons (Tasaki et al., 1954) and temporal dispersion of the afferent inflow (Chang and Kaada, 1950) are unnecessary conditions for the appearance of the surface potential sequence, because the surface responses to stimulation of optic nerve, lateral geniculate body, and white matter are similar in appearance (Bishop and Clare, 1953a; Bremer and Stoupel, 1956). However, Malis and Kruger (1956) recorded a biphasic response to optic nerve stimulation from the white matter following the removal of the visual cortex, and deduced from the identity of the intervals (0.9 msec) between the peaks of the white matter complex and between deflections 1 and 2 in the cortical record that the first two cortical deflections reflected afferent activity. Bremer and Stoupel (1956) claim that only one

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afferent wave is recorded from the white matter, but inspection of Fig. 7B in their article reveals a small inflection close to the termination of deflection l and prior to the later positive wave. However, it seems dangerous to draw conclusions from minor deflections in records composed of “killed end” and triphasic tract responses in unknown degree (cf. Patton and Amassian, 1954), and the lack of temporal dispersion of deflections 1 and 2 provides a compelling argument against slower fibers being responsible for deflection 2 (Bremer and Stoupel, 1956). Bremer and Stoupel (1957) further distinguish deflections 2 and 3 from afferent deflection 1 by the lability of the latencies of 2 and 3 at different temperatures, but conclude that deflections 2 and 3 are due to electrical activity of unmyelinated intracortical portions of afferent axons rather than to postsynaptic structures. Bremer and Stoupel (1956) were evidently impressed by the lack of increase in deflections 2 and 3 following strychninization of the cortex. However, the early relnyed response in the pyramidal tract to cortical stimulation is not increased by strychninization; it may actually decrease ( Amassian et nl., 1955).Thus, the lack of increase in size of deflections 2 and 3 is insufficient evidence for their presynaptic origin. Bishop and Clare’s analysis (1953a) indicates that all deflections after the first are due to postsynaptic cortical activity and this appears to be the most likely hypothesis. Bishop and Clare ( 1953b) distinguish major postsynaptic deflections (such as 3 ) , which are attributed to pyramidal cells, from interposed minor deflections (such as 2 ) , which are attributed to Golgi type I1 cells. The period between major and minor deflections is about 0.7 msec, that is, a single synaptic delay. Activity is propagated toward the surface over an alternating chain of pyramidal and Golgi type I1 neurons. This elegant scheme awaits confirmation by single unit analysis. Bishop and Clare (1953a) interpret the succeeding negative wave as the sign of conducted activity in apical dendrites of pyramidal cells. However, it is difficult to see how one can stimulate dendrites intracortically without also activating axons. Such axons may transsynaptically excite the cortex and thus mimic the appearance of conduction in apical dendrites. TO CORTICOCORTICAL AFFERENTVOLLEYS D. RESPONSES

Curtis (1940) performed a depth analysis of the transcallosal response recorded under barbiturate anesthesia. The surface posi-

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tive wave commenced its reversal at a depth of 100 p and was attributed to the superficial ramifications of corticocortical afferents. The succeeding surface negative wave was magnified by administration of convulsants and was attributed to summated responses of descending internuncial axons. More recent depth analyses have yielded different reversal patterns, but are agreed that postsynaptic components of the responses to specific thalamocortical afferent volleys, to interareal afferent volleys, or to callosal afferent volleys have virtually indistinguishable patterns of depth reversal ( Amassian et al., 1955; Perl and Whitlock, 1955, Nakahama, 1959). The interpretation of the interareal response ( Amassian et al., 1955) is identical to that given for the somatosensory primary response. [The somatosensory interareal response does not depend on conduction through the gray matter as described by Sencer (1950), but depends on corticocortical afferents which traverse the white matter ( Amassian, 1952a).] The interpretation of the transcallosal response is complicated by the “spectral” distribution of the data reported by different investigators. The most superficial reversals were encountered by Curtis (1940) when recording from cortex which had been treated with 6.5% sodium pentobarbital. Nakama (1959, Fig. 8 ) showed that the positive w7ave in cats under pentobarbital anesthesia was markedly reduced below a depth of 350 p and was reversed a t about 500 p. Purpura et nl. (1960) showed that the positive wave in the unanesthetized cat was markedly reduced below 400-500 p. Peacock (1957) showed that the positive wave in cats under pentobarbital was markedly reduced below 800 p and was reversed below 1300 p. A still deeper reversal pattern was reported by Perl and Whitlock (1955). If the thesis is accepted that the important errors in depth analysis lead to an overestimate of the depth of reversal ( Amassian, 1953a; Mountcastle et aZ., 1957), then more weight should be given to the supeificial reversal patterns reported by some investigators. Alternately, different investigators may have stimulated mixtures of different types of callosal fibers and thus may have studied different responses. Significantly, Grafstein ( 1959) distinguishes between callosal fibers responsible for the positive component, the negative component, and for the spreading burst responses. An electrical stimulus delivered to the cortex may orthodromically excite corticocortical neurons and may antidromically excite

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corticocortical axons which project to the site of stimulation (Chang, 1953a). However, von Euler and Ricci (1958) consider that contamination of corticocortical afferent volleys by antidromic volleys is unimportant.

E. RESPONSES TO LOCAL CORTICALSTIMULATION Adrian ( 1936) distinguished between the localized surface negative response and the spreading surface positive response to stimulation of the cortex at weak and strong intensities, respectively. Chang (1951, 1952), Burns (1950, 1951), and Brooks and Enger ( 1959) characterize the negative wave as decrementally spreading at a velocity of up to 2 m/sec. Purpura et nl. (1960) showed that the negative wave was progressively reduced below the surface and virtually absent at 600 p. Their findings indicate that the surface negative wave is in fact a superficial cortical response (SCR). The SCR is viewed either as a directly elicited response of apical dendrites (Chang, 1951) or as a postsynaptic response of apical dendrites which is secondary to the direct stimulation of afferent axons ( Eccles, 1951; Purpura and Griindfest, 1956). A compelling argument against Chang’s hypothesis is provided by the finding that the negative wave can be recorded several millimeters beyond the known limits of distribution of apical dendrites (Purpura and Grundfest, 1956). However, the evidence for a postsynaptic origin (as opposed, for example, to an origin in the negative afterpotentials of presynaptic terminals) is based on the use of “selective” synaptic blocking agents (summarized by Purpura, 1959). While it is outside the scope of this review to discuss this type of analysis in detail, so much reliance has been placed upon the use of drugs in the analysis of cortical potentials that a brief review of the conflicting evidence seems appropriate. Purpura and Grundfest ( 1956) origiiially showed that the SCR was reversibly abolished by intravenously injecting a large dose (3.0 mg/kg ) of d-tubocurarine. This dose is many times that required for paralysis of skeletal muscle and it can be expected to produce a severe drop in arterial pressure (Ochs, 1959). On the assumption that d-tubocurarine acts selectively on junctional transmission, Purpura and Grundfest ( 1956) conclude that the SCR depend on transsynaptic excitation of the apical dendrites. More recently, the reduction in SCR has been

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I

II

FIG. 6 . I. A. Blockade of dendritic response waked by stimulation of cortical surface. Bipolar stimulating electrodcs were about 1.0 mni awiiy from recording lead on anterior suprasylvian g p s . Iiiclifferent electrode was in subcortical white matter. 1: Initial response, entirely siirface negativt, rising out of shock artifact; 2: 50 scc aftcr injecting 3.0 ing/kg rZ-tubocnir;arine into fenioral vein; 3: 20 sec later; 4: at 5 min; and 5: a t 20 min. Horizontal bar, 20 msec. B. Electrical inexcitability of synaptically blockaded dendritic response. Stimuli applied 0.8 imn below, cortical surface in anterior signioid gyrus and recording lead on surface clirectly above. Indifferent lead on bone over frontal sinus. 1: Initial response is a positive deflection, followed by dendritic negativity; 2: 45 scc after injection of 2 iiig/kg d-tiibocurarine only positive component remained; 3 : recovery after 90 sec. Horizontal bar 20 msec. C. Direct and synaptic coniponents of antidromic and orthodroniic activity in pyramidal system. 1-3: responses at cortical surface to stiinulating pyramidal tract in medulla. Indifferent electrode on frontal sinus. 4-6: Activity in tract on stimulating cortex. 1,4: Initial rcsponses. 2,5: 5 min aftcr injection of 3.0 nig/kg (1-tubocurarinc. 3,6: 20 niin later. Horizontal b a r 10 msec for rccords 4-6. (From Pnrpiira and C:iuiclfest. 1956.) 11. Blockading eflects of d-hibocurarine on surface negative dcndritic responses evoked 2 mm and 5-6 nlln from stimulating site. “Near” rcsponses on left; “far” responses on right. Within 15 sec after intravenous administration of 3.3 mg/kg d-tubocnrarine both responses are reduced and nearly abolished at 92 sec. Recovery is virtually complete after 13 min. [From Fan and Feng (1957) Actn Physiol. Sinicci 21, 423.1

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attributed to changes in the amount of shunting of the electrical stimulus (Brinley et al., 1958) or to the direct mechanical effect of shrinkage of the cortex away from the electrodes due to a fall in arterial pressure (Ochs, 1959). Purpura (1959) rejects such explanations for the reductions of the SCR, because directly excitable responses are unaltered by d-tubocurarine. However, examination of the published records does not bear this out. Figure 6 (Purpura, 1959), Fig. 4 in Purpura and Grundfest (1956), and Fig. 1C in Grundfest (1958) appear to show responses from the same experiment and can be discussed together. The antidromic cortical response to pyramidal tract stimulation may be contaminated by lemniscal stimulation (see below). The initial positive spike reflects direct activity in large pyramidal neurons, but is reduced by about 30% following injection of d-tubocurarine. The latency of this positive deflection is too brief to permit synaptic relay in its production. The conclusion is inescapable that either d-tubocurarine is not a selective blocking agent, but also blocks direct conduction, or, more likely, that the efficacy of the pyramidal stimulus may be reduced by a drop in arterial pressure and thereby may cause a change in the position of the pyramidal tract relative to the stimulating electrodes. Preservation oi the direct orthodromic pyramidal response to cortical stimulation cannot be used as evidence against a mechanical explanation for loss of the SCR. One would expect a phenomenon observed at weak stimulus intensities such as the SCR to be sensitive to small changes in stimulus intensity. While the explanation for the SCR given by Purpura and Grundfest (1956) is plausible, and may prove correct, the evidenae obtained so far is not compelling. Li (1956b) reported that the negative wave reversed in the superficial layers, and he observed cortical unit driving when the cortex was stimulated with weak shocks a t 3/sec. Further unit analysis is required to determine whether the SCR is complicated by neuronal discliarge in the superficial layers.

F. RESPONSES TO ANTIDROMIC STIMULATION OF TRACT

THE

PYRAMIDAL

Antidromic invasion of pyramidal projection neurons is readily elicited by stimulation of the pyramidal tract (Phillips 1956a; Patton and Towe, 1957, 1960; h4artin and Branch, 1958; Li, 1959a). The recording of the antidromic population response of such neurons

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would be expected to help in the interpretation of other surface records. However, stimulation of the bulbar pyramid may be accompanied by inadvertent stimulation of the medial lemniscus. This can be avoided by section of the lemniscus (Landau, 1956), and the antidromic surfacc response then consists of a positive spike and wave followed by a slow negative wave. Landau attributes the positive spike and wave to apical dendrites acting as ~‘SOII~OCS’’ for “sinks” in the somata of two groups of pyramidal neurons and the negative wave to the apical dendrites subsequently acting as “sinks” for deep “sources.” The surface activity is attributed to depolarization of different portions of the pyramidal neuron at different times. However, pyramidal axons have collaterals which are distributed within tlie gray matter (Ramon y Cajal, 1911; Lorente de Nb, 1938; Chang, 1955b). Such collaterals are presumably activated by an antidromic impulse in pyramidal axons and may affect tlie electrical record. Chang (195513) reported that impulses returned down the pyramidal tract following an antidromic shock. The latency of such discharges was never less than 5 msec. Although Cliang proved that the returning discharge originated in cortex, the possibility W J S not excluded that the stimulus also excited the medial lemniscus (Landau, 1956). [A further possibility is that pyramidal collaterals repetitively discharge in a manner similar to primary afferent fibers during the dorsal root reflex (Frank and Fuortes, 1955).] I’hillips ( 1956a, 1959) observed both depolarizing and hyperpolarizing p.s.p.’s following stimulation of the pyramidal tract. Phillips ( 1959) carefully controlled the shock intensity and the location of the stimulus to the pyramidal tract, but points out the pitfalls in attributing the p.s.p. sequences to the effects of pyramidal recurrent collaterals. Towe and Jabbur (1959) dissected off a strand of the pyramid and stimulated it without danger of spread of current to the brain stem. From the motor cortex, they recorded ‘1 brief short latency positive wave which was sometimes followed by a very small, brief (0.5 msec) negative wave and a prolonged positive wave. The latency of the peak of the negative deflection is about 1-1.2 msec (see Fig. 1B in Towe and Jabbur, 1959). By contrast, peak negativity occurs about 10 msec after the pyramidal shock in Landau’s ( 1956) experiments. The input-output functions of the first positive spike and the negative wave are clearly different as shown in Fig. 7C. The findings of Towe and Jabbur can be recon-

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ciled with those of Landau if the slow surface neg at'ive wave is attributed to a cortical event which requires activation of a critical number of pyramidal fibers. Purpura and Grundfest ( 1956), Grundfest (1958), and Purpura (1959) observed a marked reduction in the surface negative wave following administration of d-tubocurarine and they concluded that the negative wave reflected transsynaptic activation of apicd dendrites by cortical interneurons which had previously been activated by the pyramidal collaterals. Their records (see Fig. 6 ) also show the abolition of the early

--

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E'IC. 7. A. Uppcr trace i n niiti,rior siginoid, lower posterior sigmoid. liesponse to medullary pyrainidal stiinulris. B. Posterior sigmoid response abolished by section of medial leinniscris in midbrain. C. Another preparation. Anterior sigmoid response after lcmniscirs section. Arbitrary voltage scale values as indicated. Positive wave appvars bcfore early positive spike attains half maximal intensity. Calibrations: 200 pv and 5 msec. ( From Landau, 1956.

second positive deflection followiiig administration of tl-tubocurarine. However, Landau's (1956) data indicates that this deflection is due to directly conducted activity in small pyramidal neurons.

IV. Patterns of Unit Response to Specific Thalamocortical Afferent Volleys

Either electrical or phy\iological stimuli are used. In general, use of electrical stimulation permits great precision in timing and accurate quantitation of thc stimulus intensity. An electrical stimulus can often be used undcxr conditions when there is a danger of spread of a mechanical stimulus. The problcms stemming from use of electrical stimulation include: ( 1) An artificially synchronized volley is presented to the nervous system. This may help in demonstrating sequential steps of activation of the cortical laminae, but

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simultaneously introduces the danger of introducing an abnormal temporal pattern of afferent inflow with perhaps an abnormal spread of cortical excitation. ( 2 ) The electrical stimulus a t threshold intensity excites afferent fibers of largest diameter. However, the largest afferent fibers are not necessarily connected with sense organs of the lowest threshold. The afferent volley set up by an electrical stimulus will therefore be biased toward conduction in large fibers as compared with the volley induced by a physiological stimulus. ( 3 ) Electrical stimulation indiscriminately excites afferent fibers with different functions and cortical projection patterns. Afferent fibers from cutaneous pressure and tactile receptors are presumably excited by an electrical stimulus to the skin, but Mountcastle (1957) showed that touch and pressure receptors activate different columns of cortical neurons. An even worse situation is encountered when a nerve trunk is stimulated because excitatory and inhibitory afferent fibers derived from widely separated portions of the periphery are stimulated together. Electrical stimuli can rarely, if ever, aid in the study of cortical representation of modalities. However, local electrical stimulation of the skin is permissible in studying the mechanism of localization because such stimuli are readily localized in man. ( 4 ) A given afferent fiber usuallv discharges repetitively in response to physiological stimulation of receptors (Adrian, 1931), but at the intensities usually used, a brief electrical stimulus (e.g. 50 psec) evokes only a single discharge. It is necessary in interpreting patterns of cortical unit response to peripheral stiniulation to know the pattern of activity in corticipetal afferent fibers. Early cortical responses in, for example somatosensory cortex, are assumed to be mediated through the specific thalamic relay nuclei. This assumption becomes tenuous when applied to late firing units and conventional analytical procedures should be used to distinguish delays due to transcortical spread from those due to delayed corticipetal excitation. A.

SOMATOSENSORY AREASI

AND

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1. Response to n Single Electrical Stimulus The three main patterns of behavior observed are: ( a ) Cortical units which rarely discharge in the absence of stimulation may respond once or repetitively to the stimulus. The unit usually responds

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during the primary response. Less frequently, discharge occurs about 100 msec later. ( b ) Spontaneously firing units may show an increased rate of discharge following the stimulus. Such units are commonly observed in very lightly anesthetized preparations. ( c ) Spontaneously discharging units may show a reduced rate of discharge following the stimulus. Cortical neurons often discharge repetitively in response to a single electrical stimulus applied either to a nerve trunk or to the skin (Amassian, 1953a; Mountcastle et al., 1957; Towe and Amassian, 1958; Mountcastle and Powell, 195913). Such repetitive discharge has been observed at intermediate levels in the ascending system such as the dorsal columns and the cuneate nucleus (Amassian and DeVito, 1957), the ventrobasal complex of the thalamus (Rose and Mountcastle, 1954), and the white matter (Marshall, 1941; Amassian, 1952a). A t ascending levels in the feline somatosensory system, the effect of increasing the stimulus intensity above threshold usually is to reduce the latency of the first discharge, to increase the probability that at least one discharge will occur, and to increase the number of discharges in the repetitive response. Shortening of the initial latency of discharge failed to occur in only two out of forty feline cortical units (Mountcastle et al., 1957). A comparable shortening of latency of discharge occurs also in the monkey (Towe and Amassian, 1958) and probably occurs in all three cytoarchitectural divisions of the postcentral gyrus ( Mountcastle and Powell, 1959b). The amount of reduction in latency ranges from a fraction of a millisecond to more than 7.5 msec ( Mountcastle et nl., 1957). In the monkey, the reduction in latency occurred in two phases (Towe and Amassian, 1958). The latency was at first greatly reduced when the stimulus intensity was increased gradually above threshold. Subsequent increase in stimulus strength led only to a slight reduction in latency of discharge. Kennedy and Towe (1958) describe additional patterns in which the latency is either gradually reduced at increasing stimulus strengths or, rarely, the latency is increased when the stimulus intensity is increased above ten times the threshold value. The latter effect is attributed to mobilization of higher threshold inhibitory pathways. Li et nl. (1956a) observed a marked change (SO to 7 msec) in latency of the cortical unit response to stimulation of the thalamic relay nucleus at several intensities, which implies that cortical

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synapses share the temporal lability of lower somatosensory synapses. Simultaneous recording of cvoked responses of several cortical neurons (Amassian p t al., 1959) permits an estimate of the coherencc of the latencies of discharge. The correlation between latencies of discharge of neurons separated by a distance of 1-15 mm is usually weakly positive and seldom significant ( unpublished observations; Amassian et al., 1960). This implies that there are important sources of independent variability in the somatosensory projection system. Li et al. (195Ga) elicited monosynaptic activation of cortical neurons by stimulating the thalamic relay nucleus. Such neiirons were situated 800-1200 ci below the surface and thus lay close to the site of termination of specific thalamocortical fibers. Later discharges were also observed at this level, but only late discharges were observcd at more superficial or deeper levels. Similarly, the earliest responses of superficial cortical neurons (above 350 11) to stimulation of nerve occurred several milliseconds later than the earliest activity recorded at greater depths ( Amassian, 1953a ) . Such observations suggested that the superficial laminae were activated by transynaptic spread through the cortex from the site of specific afferent termination, but Mountcastle et 01. (1957) and Powell and Mountcastle ( 1959) criticized this inference on the groimd that the maximum point within the receptive field was not plotted for each neuron. It is difficult to see why the optimal point of stimulation would have been regularly missed when data from either nerve or from thalamic stimulation were pooled. Some late firing superficial cortical units discharged at high frequency ( Amassian, 1953a; Li et al., 1956a), which implies that the axons that were stimulated were related to the maximum point in the receptive field. However, the effects of electrical stimulation may be complicated by mixed 1956a). Powell and Moimtcastle excitation and inhibition ( Li et d., (1959) conclude that within a period of 2-4 msec after the activation of the first cortical neuron all the responding neurons within a vertical column begin to discharge. When the peripheral stimulus is increased above threshold, the probability that at least one discharge occurs may either rapidly reach 1.0 (Towe and Amassian, 1958; Mountcastle and Powell, 1959b), or may gradually increase throughout the intensity series

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(Towe and Amassian, 1958). The latter behavior was encountered when the stimulus was applied near the edge of the peripheral receptive field. This implies that members of the cortical unit population do not have identical input-output functions when a given region in the periphery is stimulated at different intensities. Simultaneous recordings from two cortical neurons reveals such differences in input-output functions (Aniassian et al., 1959). When the stimulus was made stronger, hlountcastle et al. (1957) observed an increase in average number of repetitive discharges in thirty-five out of forty feline cortical iinits. The average number of spikes evoked by a strong stimulus v as u s i d l y less than 4. The response to nerve stimulation may incliitlv a5 many as 7 repetitive discharges (Amassian, 1953a), and evcm longer trains were observed following thalamic stimulation ( Li ct nl., 195Ga). Successive interspike inter\ als in the repetitive response of cortical neurons to nerve stimulation do not usually show the pattern of regular increase during the train ( Amassian, 1953a) as d(~scribedfor tlialamic iinits (Rose and Mountcastle, 1954). Complcx repetitive firing was attributed to the activity of cortical interneuron chains. The peripheral fiber groups responsible for the different fcaturcs of the repetitive response have not been determined. The surface p-iinary response in anesthetized preparations is elicited by stirnulation of group I1 cutaneous fibers (Mark and Steiner, 1958), h i t observations on the primary response may liave little bearing on tliv input-output functions of unit discharge. The pattern of cortical unit response is altered by changing the position of the peripheral stiniulus ( Amassian, 1953a; h4ountcastle et al,, 1957; Towe and Amassian, 1938; Mountcastle and Powell, 1959b). Stimulation at ccrtain sites within the receptive field evokes responses with a shorter latcncy and with a higher probability of at least one discharge and with an increased number of repetitive discharges than does stimulation of other sites. I n the monkey, 73 out of 110 cortical neurons wliicli responded to somatic stimulation responded to separate stimulation of at least two digits (Towe and Amassian, 1958). Mountcxtle and Powell (1959b) find that the position of the stimulus within the receptive field is more sensitively refleded by the number of repetitive discharges than by the probability that at least one discharge will occur.

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2. Responses t o T w o or More Electrical Stimuli Delivered to the Same Input Mountcastle et nl. (1957 ) and Moiintcastle and Powell (lS59b) showed that the pattern of recovery following a stimiilus was markedly affected by the anestlietic level. Under very light anesthesi‘i, the response to a second stimulus given 10-20 msec after the initial stimulus was facilitated as shown by an increase in number of repetitive discharges. By contrast, in more deeply anesthetized cats and monkeys, the response to a second stimulus was depressed. Equilibrating and “cut-off patterns of response to high freqiiency repetitive stimulation occurred at very light and moderately deep levels of anesthesia, respectively. In the “cut-off pattern of response, the unit initially responds to the first few stimuli and subsequently ceases to respond at all during the period of stimulation. In the equilibrating pattern, each stimulus elicits a response at stirndation rates of 50-100/sec. At higher rates of stimulation, responses fail to occur to every stimulus, but those which occur bear a consistent relationship to individual stimuli within the train. A similar preservation of phase relationship between stimulus and response was noted by Towe and Amassian (1958). Neurons which respond repetitively to a single peripheral stimulus, eventually respond once per stimuIus when the stimulus frequency is progressively raised ( Amassian, 1953a; Momitcastle et al., 1957, Towe and Amassian, 1958). A neuron may yield equilibrating and “cut-off” responses to repetitive stimulation of the center and the periphery of its receptive field, respectively ( Mountcastle and Powell, 1959b). Li c’t al. (1956a) describe a complex cycle of facilitation, depression, and facilitation following stimulation of the thalamic relay. Facilitation of some unit discharges was obscrved when the thalamic relay nucleus was stimulated at 10;scc. The “ciit-off” type of behavior was also observed. 3. Interaction between Volleys f r o m Two Inputs Inhibition may be defined as the failure of a neuron to respond to a previously effective test volley due to the action of a conditioning volley which does not usually set up a propagated impulse in the same neuron. This operational definition is unaffected by disagreements concerning the mechanism of inhibition, but does not

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distinguish between an inhibitory mechanism located at the neuron and inhibition of the presynaptic inflow. Inhibition in cortical units was detected following eithcr electrical stimulation of nerve trunks ( Amassian, 1953a) or skin ( Mountcastle, 1957; Towe and Ama 1958; Mountcastle and Powc~ll,1959b) and was manifested by a rediiction in probability of occurrence of at least one response, or by a reduction in the number of repetitive responses to the test stimulus. Inhibition occurred in somc nnits when both afferent sources were simultaneously stimulated and even occurred when the inhibitory volley was set up after the cwitatory volley (Amassian, 1953a; Towe and Amassian, 1958). Ncvcrthcless, in both the above reports and from Mountcastle and Powell’s ( 195911) data it is clear that inhibitory interaction is most pronounced when the excitatory stimulus trails the inhibitory stimulus by more than a few milliseconds. The duration of inhibitory interaction can be reduced either by increasing the intensity of the test stimulus, or by selecting a more distant inhibitory input (Towe and Amassian, 1958).

4. Response to Physiological Stimulation Mountcastle (1957), Mountcastle and Powell (1959a, b ) , and Powell and Mountcastle ( 1959) thoroughly analyzed the representation of cutaneous and deep receptors in somatosensory area I. In 8476 of vertical penetrations through feline somatosensory area I, single neurons in layers 11-VI responded either to mechanical stimulation of hairs, to steady pressure applied to the skin, or to deformation of deep tissues. Thus neurons in a “vertical column” were activated exclusively by one of three modalities of stimulation. The modality specific vertical columns were intermingled for any given topographical region, but “skin” columns tended to occur more frequently in the posterior portion of area I while “deep joint” columns occurred more frequently in the anterior region. Responses occurred exclusively to contralateral stimulation. The extent of the peripheral receptive field was approximately the same for neurons within a given column. Responses to cutaneous stimulation were either rapidly or slowly adapting (cf. Adrian, 1931, 1941). The extent of the peripheral rtnceptive field was approximately inversely proportional to the distance of the center of the field from the tip of the limb. Further evidence supporting the columnar hypothesis of modality representation was obtained in the monkey. Sixty-six

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per cmt of responding units in cytoarchitectural area 3 \vme activated by cutaneous stimuli, but only 30% of units in area 1 were activated by such stimuli. About 90% of the units in area 2 were activated by deep stimuli. ( T h e anteropostaior disposition of “skin” and “deep” columns apparently differed in eat and monkey. ) In the monkey, units responding to movement of joints showed either slow or rapid adaptation to a maintained displacement. W i e n two units were simultaneously recorded a t a given position of tlie electrode, an inverse relationship in firing rate of the two units was noted during passive flexion and extension of the joint. This plienomenon was attributcd partly to unloading of peripheral receptors and partly to afferent inhibition. Inhibitory receptive fields were plotted by stimulati~iga rcgioii of skin which surrounded the excitatory receptivc field. Tlie excitatory field was usually, but not invariably, symmetrically surrounded by the inhibitory field. The response to ytimulation of deep rcceptors was also inhibited by stimulation of skin. Inhibition due to cutaneous stimulation was highly sensitive , ) . However, inhibition to anesthesia ( hIonntcastle and P o ~ e l l1959b is readily demonstrated by the method of single conditioning and testing shocks in anesthetized animals ( Towe and Amassian, 1958). This implies that the form of inhibition desciibcd by Mouiitcastle aiid 1’owell ( 1959b ) when using continuous mechanical stimulation may depend on “cut-off type units ( defined above in Section IV, A ) . A small fraction of postcentral neurons (12,693) showed quite diiCerent properties ( Mountcastle and Powell, 1959b ) . Such neurons had large reccptivc fields and some were activated by ipsilatcral stimulation. Carreras and Levitt ( 1959) isolated both small and large receptive field units in somatosensory area 11, but the units isolated in a given vertical column were modality specific. Poggio and Rlountccistle ( 1960) relate the moda1it)r specific small receptive field unit to an excitation pathway via the medial lemniscus and ventrobasal tlialamic complex. The posterior thalamic group ( see Wliitlock and Perl, 1959) and spinothalamic system are related to wide receptive field cortical neurons. However, a difficulty arises because many units isolated in the posterior group were not modality specific, but tlie large receptive field neurons in area I1 were modality specific ( Carreras and Levitt, 1959). Furthermore, the receptive field and ultimately the rcspoiisivity of posterior group neurons are reduced by deepening the anesthetic lcvel (Poggio and

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Mountcastle, 1960). The primary responses and the widespread interaction phenomena detected in somatosensory area I1 of deeply anesthetized cats ( Amassian, 1952a) cannot depend on transmission through a subcortical relay such as the posterior group which is sensitive to the anesthetic level. It might be supposed that the differential distribution of cutaneous and deep receptor columns in the different cytoarchitectural regions of the postcentral gyrus conflicts with Woolsey’s (1958) plan o f Organization in which the body surface is mapped over tlic cntire postcentral gyrus. However, Woolsey’s maps are based on the distribution of surface primary responses in anesthetized cortek and do not necessarily indicate the sites of neuronal discharge. Cohen et al. (1957) recordcd unit responscs in somatosensory cortex either to tactile, or to gustatory, or to thermal stimulation of the tongue. A total sample of- 5 “taste” neurons was encountered at a depth of 2 mm while the morc numerous “tonch” neurons lay 0.7-1.5 mm below the surface. These findings do not necessarily indicate a different plan of organization of the representation of the tongue, but may reflect the difficulty of obtaining an adequate sample from all laminae when gustatory stimuli are used.

B. MOTORCORTEX Malis et nl. (1953) recorded early surface responses to peripheral stimulation from the prccentral gyrus of monkey. Such responses persisted after removal of the postcentral gyrus and thus probably depended on specific thalamocortical afferent activity. Single cortical neurons of cytoarchitecturally defined motor cortex may respond after a brief latency to stimulation either of nerve ( Amassian, 1953a, Fig. 1) or of the ventrolateral thalamic nucleus (Li, 1956b). Li (1956b) also observed inhibition of spontaneous activity following thalamic stimulation. Imbert et al. (1959) distinguished between wide receptive field units recorded from feline pericruciate cortex and small receptive field units recorded posteriorly from classic senlsory cortex. The posterior group of units fired after a short latency (8-10 msec) and corresponded to the units recorded by Mountcastle et 02. (1957). The units recorded from motor cortex responded 15-25 insec after somatic stimulation, but responded 30-50 msec after auditory and visual stimulation. (Com-

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plex excitation pathwctys were presumably implicated in the responses to thc latter kinds of stimuli.) I n all the above \tuclies, pyramidal projection neurons were not distinguishcd from other ncurons in the motor cortex. Such neurons can be wmpled either by recording from tlie pyramidal tract or can be labeled in cortical itnit recordings by demonstrating ankidromic invasion. The pioneering study by Adrian and Moruzai (1939) inclicated that, under chloralose anesthsia, pyramidal projection neurons rcspomled repetitively at high fieqnency to a peripheral stimulus and had wide ~ and receptive fields. Calina and Artluini ( 1954) found both 1 : ge small receptive ficlcl projection neurons in the unanestlwti-/ed cat. Some units were inhibited by somatic stimulation. Patton and Towe (1957, 1960) present a detailed analysis of the latency of Brtz cell response to stimulation of the contralateral forepaw. The cats were anesthetized with chloralose. The units were allocated to somatosenwry area I, but it is likely that somatosensory area I u7as defined in these experiments on the basis of tlie short latency of the evokecl responses rather than by cytoarchitectural criteria. The Betz cell rcsponded to stimulation of the contralateral forepaw by 1-11 discharges at frequcncies up to 700/sec. The mean number of spikes per burst was significantly higher for Betz cells (3.6) than for cortical cells (1.8) which could not be labeled by antidroinic stimulation. Of the Betz cells, 44.6% commenced firing prior to the peak of the surface positive response and 51.47 commenced firing bctween tlie peak of the positive and negative waves. The distribution of latencies for other units was similar except that superficial units tended to discharge later than units in layers I11 and IV (cf. responses in somatosensory cortex). Population responses of the pyramidal tract are readily evoked by somatic, visual, and auditory stimuli ( Ascher and Buser, 1958). The above studies agree that pyramidal projection neurons discharge soon after the arrival of the corticipetal volley but do not permit an accurate estimate of the minimum number of cortical synapses in the pathway. Branch and Martin (1958) obtained latencies of 0 . 5 3 0 msec for Betz cell responses to stimulation of the ventrolateral thalamic nucleus. Latencies were usually 2 3 msec. Li (1958, Fig. 2 A ) shows a response with a latency of 0.8 msec. I n ncither study was it shown that units which discharge with latencies of less than a millisecond fail to respond to high frequency thalamic stimulation, This test is essential to eliminate the

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possibility that local collaterals of pyramidal projection axons are excited by the thalamic stimulus (Branch and Martin, 1958). The population re5ponse in the pyramidal tract to stimulation of the ventrolateral thalamic nucleus also has a short latencv ( Brookhart and Zanchetti, 1956; Purpura, 1958). Purpura (195s) &olished the short latency pyramidal r c y m s e by the administration of only S mg/kg of sodium pentobar1)ital and thereby proved the transynaptic nature of the rcsponse. Purpura’s data indicatc that few if any pyramidal axons dischargc lcss than 2 msec after the thalamic stimulus. Rif inimal conduction times from thalamus to cortex and from cortex to bulbar pyramid prob‘ibly exceed a total of 0.8 msec. The minimum cortical delay for the pyraniidal relay is unlikely to exceed 1.2 msec, and it is prol~ablylcss because the earliest relayed discharge in the bulbar pyramid is not necessarily carried by the most rapidly conducting pyrcimidal axons. This estimate raises the possibility that the relayed discharge depends on a single cortical synapse, but the assumptions are too tenuous to eliminate a disynaptic link. Hrookhart and Z,inoheitti ( 1956) demonstrated marked changes in pyramidal excitability during the augmenting response, but found no increase in escitabilitv unless the thalamic shock led the test cortical stimulus by more ‘than 3 msec. Their data imply that, at least during the augmenting response, short latency pyramidal responses are polysynaptically mediated. Spontaneous discharge of Beti cells is inhibited for periods up to 20 sec following repetitive stimulation of the ventrolateral thalamic nucleus (Branch and Martin, 1958). This inhibitory effect was unaccompanied by hyperpolarization or by reduced excitability of the Betz cell, which indicated that other neurons were primarily inhibited.

C. AUDITORY AREA I Erulkar et ul. (1956) recorded units from the first auditory field which responded either to clicks, or to tones, or to both kinds of stimulation. Under light anesthesia, 34% of the units failed to respond to a sound [cf. Powell and hlountcastle (1959) who observed evoked responses in almost 9054 of the units recorded in somatosensory cortex]. Sixty per cent of responsive auditory units were driven by clicks. The responses to intense clicks were either early

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( 5-12 msec) , or occurred at an intermediate latency ( 1 5 4 0 msec ) , or occurred late (84-250 msec). A click stimulus just above threshold intensity usually evoked responses with a longer latency and fewer repetitive discharges than did a greatly suprathreshold click (cf. analogous behavior of somatosensory units). The effect of tonal stimuli was either to suppress spontaneous discharges, to reduce the response to a click, or to cxcite the unit. About 407; of units responded exclusively to tones. Every unit sensitive to tones responded optimally within a restricted range of frequencies, hut the degree of aclaptioii varied within the series. Katsuki et al. (1959a) reported that tuperficial cortical units rapidly adapted to tonal stimuli and responded to a broad range of frequencies. Units isoly long lasting related at depths of- about 1-2 nim ~ i s ~ i d yielded sponses to tone bursts and responded only to a narrow band of frequencies. Superficial and deep units also showed, respectively, “apparent facilitation” ( d u e to beats) and suppression of- the test response when a background tone at diff a e n t frequency w‘is presented (Katsuki et al., 195913). Deep units were tentatively attributed to corticipetal axons because of the similarity of their behavior to that of medial geniculate neurons. Erulkar et nl. (1956, Fig. 12d) illustxated slowly adapting behavior of a typical positive-ncgative soma spike and did not report that such units were restricted to the deeper layers although they secured an adequate sampling from all layers below 200 p. The level of anesthesia probably differed in the two studies and there is the further possibility that cooling of the exposed cortex occurred during the experiments of Katsuki et al. (1959a). Both studies agree that the tonotopic organization dcscribed by Tuntuii (1950) for dog cortex is somewhat modified in the cat becausc neurons situated within the same srctor may respond optimally to quite different frequencies.

D. VISUALCORTEX A series of studies from the University of Freiburg give detailed accounts of the responses of different visual units to diffuse illumination of the retina (Baumgarten and Jung, 1952; Jung, 19S3, 1958; Jung et al., 1957). Five types of behavior were observed: ( a ) Units showed no response to light, but spontaneously discharged a t 8-15/sec. ( b ) Units were activated by light and inhibited b y dark-

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FIG. 8. Neurons of type 2. Average frequency of discharge following stimulation of the optic nerve, and distribution of latency periods. a. Contralateral optic nerve shock of a type 2a neuron. Ordinate: Average discharge frequency of reaction from 15 single shocks. Abscissa: time in milliseconds. b. Type 2b. Ordinate: Average discharge frequency of reactions from 20 single c. Distribution of primary latency shocks. Abscissa: time in milliseconds. periods of 76 neurons of type 2; @: ipsilateral optic nerve shock; 0: contra) lateral optic nerve shock. The latency period of the second discharge of 26 neurons was included if it was constant. (From Griitzner et al., 1958.)

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ness. ( c ) Units were inhibited at both the commencement and the termination of visual stimulation. ( d ) Units were inhibitcd b y light and bec'tme active during darkness. ( e ) Units responded a long time after the start ot the visual stimulus, but showed a markcd off response. All five types of- neuroii were found in layers 11, 111, and IV, and in some instaiices neurons of- different type lciy in close proximity to one mother. Grilsser and Grutzner (1958) and Grutzner et nl. (1958) analyzed tlie visual unit rcsponse to one or more shocks clclivcred to the optic nerve. Four types of behavior were observed. T > p e 2 units discharged after a brief latency. The envc,lopc of latencies presented in Fig. 8 (Grutmer ef nl., 1958) has a polyphasic contour which is reminiscent of the multiple deflections in the snrfaco primary response ( cliscnssetl in Section 111, C ) . However, the sampling was insiiflicient to provide a critical test of Bisliop and Clare's (1953b) hypothesis of the visual primary respoizsc. Other units fired after 1' very long latency ~ h i c hwas attiibiited to an excitation lmthway via the unspecific thalamic projection system. Some visual units followed very high rates of optic nerve stimulation ( u p to 500 ' s e c ) . A rather different picture of the organization of the striate am1 is provided by the stiitlies oi H ~ h (l1959) and H ~ i b e land Wiesel (1959). Diffuse illumination produced little or no response in most visual units. By contrast, most tinits responded to a restricted light source. Such xtivity was inhibited by illumination of- a small I (@on outside the excitatory receptive field. Some units responded to both stationary and moviiig spots. Others responded only to a moving spot. I n gene1al, optimal responses occun-ecl when the stimulus was specific in form, size, position, and orientdtion. This was: ath-ihuted to the side-by-side arrangement of ewitatory and inhibitory receptive fields. Hubel and Tliiesel ( 1959 ) suggest that the numerous units which responded to cliifuse stimulation in tlie studies of Jung and his collaborators were lateral geniculate fibers. This exp1,ulation for thc discrepancy between the two stndies may be correct, but it is difficult to see liow it can account for the recordings made by Jung ( 1953) in the superficial layers. Furthermore, typicdl soma spikes are illustrated in many of the records of Jung and his collaborators.

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123

Patterns of Unit Response to Direct Cortical Stimulation

Units respond to direct cortical stimulation by a complex sequence of increased and reduced discharge ( Creutzfeldt et al., 1956). Inhibition of spontaneous activity consistently occurred for periods of 150-400 msec following stimulation. Inhibition was often followed by a period of increased activity. The period of inhibition was occasionally preceded b y a phase of short latency discharge. Phillips ( 1956b) analyzed the intracellular responses of Betz cells to long duration pulses. The latency for the first discharge was reduced and the number of repetitive discharges was increased by increasing the strength of stimulation. The Betz cell spike was fragmented at the A-B inflection during high frequency discharge. The cell was hyperpolarized for many milliseconds after the stimulus. Spontaneous activity was depressed during the period of hyperpolarization. Adrian and Moruzzi (1939) were the first to record from pyramidal axons and thus pioneered a new type of analysis of cortical neurons. Two studies ( Patton and Amassian, 1954; Zanchetti and Brookhart, 1955) have yieldccl comp,irable data and can therefore be discussed together. A brief shock delivered to the motor cortex of cat or monkey evokes a complcx response in the pyramidal tract. The response is separable into a direct clomponent ( D wave) followed by one or more relayed coiuponents ( I waves). The brief latency of the D wave (0.5-0.7 mscc) is accounted for by conduction time in pyramidal axom and cannot include a synaptic delay. The D wave differs from I \ \ ' ~ v e sin following repetitive stimuli delivered a t several 1iundrc.d per second and is less affected by cortical injury or by asphyxia. I waves are not rccordecl when the white matter is stimulated. The D wave response to cortical stimulation is probably not due to stimulation of axons in the white matter because the respoiise to a second shock shows well marked changes in amplitude follon ing a subthreshold stimulus. The cui-rent concepts of cell excitability (see Section 111) imply that the impulse is initiated intracortically at the axon hillock region. The D-I interval and the period of subsequent I waves are usually about 2 msec. The response to a second cortical shock may exhibit peaks of facilitation with a comparable period. Such observations suggest that pyramidal projection neurons are bombarded by impulses from

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cortical interneuron chains. If it is aslsumed that an elongated neumn is more readily excited by a given shock than is the compact Golgi type I1 neuron, then D and I wave periodicities can be attributed to a chain composed of alternating elongated cell-Golgi Type I1 cell linkages (Patton and Amassian, 1960). The elongated cell is most probably a pyramidal cell, but whether the pyramidal cell projects into the pyramidal tract or, for example, into the gray matter (type 4 of Sholl, 1956) is unknown. T h a e is evidence both for and against the possibility that recurrent collaterals of Betz cells mediate discharge of other B&z cells (see Section IV). Single pyramidal axons yield either single responses or repetitive discharges following a brief shock to the cortex (Patton and Amassian, 1954). The latency of the first discharge may correspond to either the D or the I waves. The I waves are accounted for by polysynaptic re-excitation of some pyramidal projection neurons and polysynaptic spread to othtrr projection neurons which are not directly excited. Such spread may occur either through gray matter or white matter. In the isolated cortical slab preparation ( R i m s , 1950, 1951), spread can only occur through the gray matter. A recent microeleetrode analysis of the spreading burst response in such preparations ( B L I ~et~ Snl., 1957) revealed that the neurons whose activity was essential for tangential spread of the biust response had large cell bo'dics which were usually, but not exclusively, distributed in layer V. Burst activity was also recorded from chronically isolated cortex which had lost the projection type pyramidal neuron. This indicated that another type of neuron mediated the spread of the burst response.

VI. Patterns of Unit Response to Corticocortical Afferent Volleys

Units responded to an interareal afferent volley by 1-7 clischarges at high frequency ( Amassian, 1953b). Superficial cortical units located at a depth of 150-300 IL (layers II-upper 111) responded 7-50 msec after the distant cortical shock, but deeper layer units responded either after monosynaptic or polysynaptic delays. Some units were activated both by cortical and by peripheral stimuli. The responses of superficial neurons were attributed to plmisynaptic spread from deep corticocortical afferent terminations

MICROELECTRODE STUDIES OF THE CEREBRAL CORTEX

1%

rather than t o the direct actions of terminations in the superficial layers (Lorente de N6, 1938). Per1 and Whitlock (1955) observed in sevmal instances that a unit which responded either to tactile or thalamic stimulation could also be activated by stimulation of the contralateral cortex. Creutzfeldt et al. (1956) obtained a range of 1.5-25 msec for the latency of discharge of units activated by callosal aff ewnt volleys. These authors alslo demonstrated delayed depression of spontaneous imit activity following stimulation of the contralateral cortex. L,aCimcr and Kennedy (1958) give a range of 1-100 msec for t he latencies of units activated by stimulation of the contralateral cortex. Out of a total of 97 units, 38 fired before peak positivity of the surtacc response. Thirty-three units fired between the peaks of the positive and negative components. Purpura and Girado (1959) carefully analyzed the relayed responses in the pyramidal tract to stimulation of the contralateral motor cortex. The relayed pyramidal tract relsponse was complex and had several components, but the earliest response had a latency of 3 msec. Pyramidal projection neurons weye activated prior to the peak of the surface positive component of the transcallosal response. Minimum oonduction times in callosal and pyramidal axons are estimated at 0.5 and 0.7 msec. [The latter figure is approximate and is derived by doubling the latency of the first zevo potential in Peaoock‘s record (1957, Fig. 4 ) of the callosal tract response.] Subtraction of total conduction time in axons from the latency of the relayed pyramidal discharge yields an estimate ot 1.8 msec for the “minimum” cortical delay. This type of calculation probably leads to an overestimate (see Section IV, B ) and it may be concluded that pyramidal projection neurons can be activated either by specific thalamocortical afferent, or by corticocortical afferent volleys after similar cortical synaptic delajs.

VII.

Integrative Responses to Mixed Corticipetal Volleys

Jasper and Ajmone-Rilarsan (1952) observed that the surface negative component of the primary visual response to stimulation of the lateral geniculate body was markedly affected by prior stimulation of the intralaminar system. It was inferred that interaction between specific and intralaminar systems occurred following tran-

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synaptic conduction in the cortex. IIutual facilitation of the sllrface negative components of recruiting and augmenting responses occurred when stimuli werc altcrmtely tlclivei ed to mcdinl and lateral thalamic nuclei (Rrookhart ef al., 1957). L,i ( 195Ga) distiiiguished between a group of units which \vere monosynaptically excitcd by specific thalamocortical afferent volleys and another group which responded to “uiispecific” thalamic afferent volleys. Unit responses to specific thalamic volleys were facilitated by stimulation of the centromedian nucleus of the thalamus. J ~ m get nl. (1957) and Jung ( 1958 ) demonstrated reciprocal interactions between volleys set up by visual stimulation and by intralaminar thalamic stimulation, but in addition repoi-ted that some cortical neurons were activated by either type of stimulus. Such observations do not invalidate the important distinction made by I,i (1956a), but suggest that specific and unspecific thalamic afferent volleys ultimately reach a similar group of neurons through different cortical pathways. Recently, Hubel et nl. (1959) reported that about 10% of the iinits rcoorded from auditory cortex only responded to the auditory stimulus if the animal “pnid attention” to the sound somce. Siich lxhavior may depend on interaction between specific and unspecific systems. The surface responses of the association mrtex of cat ( Amassian, 1954; Albe-Fessard and Rouged, 1955; Buser and Borenstein, 1956a, b, Buser, 1957) are probably related to the centromedian thalamic nucleus ( Albe-Fessard and Rouged, 1958). Units recorded from the anterior portion of the lateral gyrus have wide receptive fields which may include all four limbs (Amassian, 1954). The same association neuron may respond to stimulation of a limb nerve or to a click. VIII. Discussion and Summary

An attempt is rnade in most microelectrode studies of the cortex to correlate physiological and neuroanatomical data. Specific thalamocortical fibers and corticocortical afferent fibers are distributed quite differently within the cortex as shown by Loreiite d e Nb (1938). Chang (1953a) traced callosal fibers into layers I, 11, and 111 of the cortex in young mice and rats. Such afferents terminated by simple twigs or free arborizations as compared with specific thalamooortical fibers which had profuse end bushels in layer IV.

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Callosal fibers terminated on gcmmules which were situated on distal portions of the apical dendrites of pyramidal cells (paradenchitic synapsis) and wcrc bclieved to cause facilitation, but not discharge, of pyramidal iicurons ( Chang, 1953). Specific thalamocortical fibers terminated by pericorpuscular synapsis which wz considered to be much more effective in securing discharge of the postsynaptic neuron. Chang ( 1953b) subsequently suggested that callosal volleys might activate superficial cortical neurons and thus implied that the difference in efficacy of paradendritic and pericorpuscular synapsis applied only to the pyramidal neuron. Nauta (1954) found that corticocortical fibers ended in all layers of cat cortex. The most profuse endings occurred in layers 111-VI (cf. Chang, 1953a). Pericelliilar endings were infrequently observed. Unfortunately, microelectrode recording has so far failed to provide data which fit in with the anatomical differences between specific thalamocortical fiber and corticocortical fiber terminations. The patterns of depth reversal of the surface responses reported by different investigators are sufficicntly diversified to fit in with almost any anatomical scheme. The difference in results is perhaps attributable in part to the variability of transsynaptic spread from the middle laminae toward the cortical surface. Possibly, a block may be produced by anesthesia or by exposure of the cortex. In other studies, “pia dimpling” and “killed end” effects probably account for deep reversal patterns. Such factors would lead to the appearance of a “standing” wave rather than a “slowly traveling” wave. One of the few points of general agreement is that postsynaptic components of surface responses to either specific thalamic afferent or to corticocortical afferent volleys have indistinguishable patterns of depth reversal. The behavior of single cortical neurons would be expected to provide a morc significant test for differences in afferent termination, but has failed to do so. Short latency relayed responses OCCLLI- in the pyramidal tract to stimulation of either the thalamic relay nucleus (Brookhart and Zanchetti, 1956; Purpura, 1958) or of the contralateral cortex ( Purpura and Girado, 1959). However, it should not be inferred that the actions of specific thalamic and corticocortical fibers are identical. Grundfest ( 1958) suggests that the difference between denchitic anid somatic synapses may be quantitative; p.s.p.’s genemtod on distant portions of the dendritic field suffer greater electrotonic losses than those generated close to

the electrically excitable portion of the neuron. Probably, there are important quantitative differences between the actions of specific thalamocortical and corticomrtical volleys, but one would cvpect such differences to be obscured when electrical stimuli are used. By contrast, corticipotal fibers which mediate the surface recruiting response have little or no effect on the pyramidal projection system and associated interneurons (Brookhart and Zanchetti, 1956). Other cortical neurons discharge during the recruiting response (Li et al., 1956b, Li, 1956a; Jung et al., 1957; Jung, 1958). Confluence between specific and diffuse projection systems is viewed in ternis of facilitation (Li, 195610) and of neuronal sharing ( J m g et al., 1957). Regardless of how specific and cliff use projection systems intvract, there is genmal agreement that the initial effects of the two systems on the cortex arc dissimilar. Morison and Dempsey (1942) surmised that recruiting responses were mediated by the “unspecific” thalamocortical fibers which were described by Lorente de N6 (1938). The difference in the actions of specific and unspecific fibers was correlated with the difference in the sitcs of termination of thr two types of fiber (Brookhart and Zanchetti, 1956; Li, 1956~). This does not explain why cortimeortical fibers act so differently from the diffuse projection system, but nevertheless have a distribution within the cortex which is similar to that of the unspecific fibrm. Evidence from several sources suggests that an interlinked system of Golgi type I1 cell-pyramidal cell-Golgi type I1 cell is a building block in the cortical synaptic net. Bishop and Clare (195313) proposed this scheme to account for the postsynaptic portion of the primary response in visual cortex. A similar scheme provides an explanation for the periodicity of the relayed pyramidal response to cortical stimulation ( Patton and Amassian, 1960). Possibly, the cortical delays for the relayed pyramidal response to specific thalamic volleys ( Brookhart and Zanchetti, 1956) and to transcallosal volleys (Purpura and Girado, 1959) can also be explained by an afferent fiber-Golgi type I1 cell-pyramidal cell linkage. It is not inferred that the pyramidal cells in the chain are either of the same type or are situated in the same lamina. There is ample evidence ( Amassian et al., 1955; Purpura, 1958; Purl)ur:t and Girado, 1959) that the coupling between specific thalamocortical afferent inflow and discharge of pyramidal projection neurons is much more sensitive to barbiturate anesthesia than is the response

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of other ppamidal neurons. Ho\vcver, there is enough ambiguity concerning the shortest 1,itcncy for xtivation of pyramidal projection neurons (see Sectioii IV, B ) to suggest that the scheme depicts one out of many patterns of coitical activity. The advantages stemming from the use of discrete physiological stimuli are well illustrated by the studies of Mountcastle and of Hubel (see Section IV, A ) . IIubel and Wieisel (1959) showed that neurons of striate mrtex are preterentially driven by specific forms, sizes, positions, and orientations of the visual stimulus. This remarkable specificity is apparently due to the arrangement of excitatory and inhibitory receptive fields of the unit. Difhise illumination of the retina was relatively ineffective in driving most units because of mutual antagonism between the two fields. Mountcastle suggests that the basic pattein of organization in somatosensory cortex is a vertically oriented cylinder of cells that is activated by a specific type of stimulus delivered to a particular region 04 the body. Thus, the vertical cylinder of cells responds either to tactile stimulation, or to pressure on the skin, or to manipulation of deeper structures such as joints. Thc latencies of the initial responses of cells w i t h n a given column are dispersed over a range of %5 msec. This range pennits only a few intracortical synaptic delays, but the possibility that i n t e m m o n a l chains influence repetitive discharges of cells in the column is not excluded by Mountcastle's data. I t is inferred that stimulation of a given point in the periphery causes short latency repetitive discliarges of some cortical neurons and longer latency responses ivitli few repetitive discharges in cells situated in surrounding columns. However, repetitive trains in somatosensory units cannot be important in local sign. Repetitive responses are lost during high frequency stimulation of the periphery yet such stimuli are readily locnliied by man (Towe and Amassian, 1958). It is apparent that one is left with the problem of identifying the mechanism which determines the position of the columns of active cells within somatosensory cortex. The job is made easier when the zonc of active columns is surrounded by columns of inhibited neurons ( bloiintcastle and Powell, 1959b), but the general problem rern'iins. A similar difficulty is encounteTed with the hypothesis developed to account for two point discrimination. A discussion of the behavioral literature is relevant, but the lack of space pennits a reference onily to the studies of Ruch et al.

(19:3S) and Blum et (11. (1950). Hot11 sti1dic.s indicate tliat the monkey lias considcrahle tactile acuity follo\ving rcino\ral of the postcentral ~ ) I w . ( Tliis result is important 1)ecarise tactile acuity \vould 1ini.e 1)ccii lost if tlie postcentral gyriis iiiiiqiic>ly traiisiiiitted tlic afferent iiiflo\v to anotlicr region for analysis, Iiitcrprc~t~ition of the data \vould then 11n\e bcen umbigrtous. ) At prcseiit, it appexs unlikely tliat discriminativc ;ilAitics arc :iny 1 1 1 0 1 ‘ ~ tlepcwlciit on topogriiphicdly orgmizc‘cl cortc>s tlian they arc on not-itol)o~‘apliically organized ci r t c s . Tempord factors m i \ ’ bc iiivokctl to .Ivoid the conceptiial difficulty implicit in spatial tlicorivs of rcpx,sentation, but thc first prol,l(wi prc~sciitetlis to clistingiiisli thc, “iiitcnsity” from t I ie “posit i o 11’’ coclc ( A ti1 ; ~ s isan, 19.53a; A n i a s s i ;in ; i n cl \T al lcr, 1935). IIo\vever, tlre inpiit-oiitpnt functions of sitii~iltuneo~isl\~ recortled cortical nc~ironsdiifcr ( :Imassian c’t (//., 1‘3%) . It is most ~uilikc.lytliat t\vo poi~tions of tlic 1)otly stimrilatctl at \xrioiis intensitic,s could induce an identical temporal pattern of activity in a population of cmtral wiiroiis. Tlic major pro1)lcins fiiciiig such studicLs are to idcntify tliosr particular rclatioiisliips bct\\.cwi unit responses of tliffcrent iic~iiroiis\vliich persist in the face of ;I cliangc in stimiilus intensity and to prove that snch patterns of activity engentlcr specific rcspotisvs 1)y the organism. A further dimension is atldcd by stiit1ic.s directed to\\-ard the labile :icti\ritics of the higher lc\-cls. liicci ct rrl. ( 1937 ) distingriisIi(~1 niiic: pattcl-ns of motor cortical unit response during tlic conditionctl response. They conclude that “the tlc\~elopii-i(~nt of coiitrol over ii~c~lrvaiit rcy)nses may I i c more important tlinn nciv connection fol-mation.” Brooks ( 1‘360) reduced the gap lx,t\\.cwi the "stable coliimii” and \\,ilk rcceptivc field units by s h v i n g that the rwcptivc field of tlio p x m idal projection neiiron cmlargcd \\.it11 repeated testing. Tho qiic.stion may \\,ell be asked ~ l i e t h t the ~ r infrequent esceptions to the modality spc,cific vertical cylinder may not ultimately I)cx of great \ d u c ~ in I i n clerstan d i 11g 11i gli cr level ac tivi tics. Classic neriropliysiology \\’as c1irc.ctc.d at cyllaining beIia\.ior in teniis of cause ;ind effect relationsliips at s!napses. Tlic nc~ctlfor a statisticul interprc,tiition of catise and effect Ijectime appircii t \vhen it \\’as tlisco\~crcdtliat stimulation of a g i \ ~ nset o f fibcrs I d to discharges of neurons A, R, C, rtc., \\-it11 probabilitic~sP,,, Pit, P, -,etc. Rut \\,hat can be said of the caitse-effect relationsliip Iwtnwti t\\w spoiit;iiieoiisly active neurons \vliich are rc+med by a given pvriph-

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era1 stimulus? The possibility must be entertained that future studies will reveal only temporal relationships between discharges of neurons situated between inflow and outflow systems. Whether a modification of the search for a cause-effect relationship is as necessary a condition for the understanding of “integrative” neurophysiology as it proved for theoretical physics remains to be determined. REFERENCES

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EPILEPSY’ By

Arthur A. Ward, Jr.

Division of Neurosurgery, University of Washington School of Medicine, Seattle, Washington

I. Introduction

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

11. The Epileptic Focus . . . . . . . . . . . . .

Activity of Neuron Sonia . . . . Activity of Dendritt-s . . . . . . . The Epileptic Neuron . . . . . . . . . . . . . . . . . . . . Field Effects . .............................. Possible hlecha kndritic Synchronization . . . . . . . . 0 t h hfechanisms for Production of Seizure Activity . . . . . The Seizure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Propagation of the Seizure . . . Precipitation of Seizures . . . . . .................. Concluding Remarks . . . . . . . . . . . Refercnces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

A.

B. C. D. E. F. 111. IV. V. VI.

I.

157 160 165 169

181

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Introduction

The word “epilepsy” we owe to Hippocrates although our English forefathers called this clirease the “falling evil.” The latter name presents the most common feature of the disease, the former only a pseudomythical pathology. However the older term has persisted. This is perhaps related to the fact that, to the observer of such an attack, it is most easily ascribcd to some unseen operator and the attack itself must be the work of some malignant agent. This pseudomythical etiology for epilepsy has been culturally preserved down to the present in spite of the fact that a rather large body of factual data regarding this disorder has been accumulating for many years. Gowers (1901), in his superb monograph entitled “Epilepsy and Other Chronic Convulsive Diseases,” stated that “All our present 1 Supported, in part, by a grant (B-193) from the National Institute of Neurological Diseases and Blindness of the National Institutes of Health, U.S. Public Health Service.

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knowledge suggcsts that the primary disturbance of function which underlies the epileptic fit occurs in tlie cerebral cortex” His basic premises, which have not yet been accepted into the gcncral body of cultural knowledge, require surprisingly little fundamcntal modification in the light of recent knowledge. Altliougli the clinical manifestations of an epileptic seizure can appear in a multitude of patterns, it is still a funclamental postulate now, as in the time of Gowers, that a cortical seizure discharge originates in a cluster of nerve cells. The manifestations of the seizure will then reflect the functional organization of the neurons involved by the propagating seizure discharge. Although some seizures may not arise in this fashion (Penfield and Jasper, 1954), a thorough understanding of scizures of focal onset is necessary before more complex phenomena (such as seizures of subcortical origin, petit mal, etc.) can be investigated. The concept that seizures can arise in a focus and then propagate to involve additional circuits within the brain is not of recent origin but the ultimate confirmation has come only with advances in neurosurgical technicjiie which have permitted the removal of the epileptogenic focus with resulting cessation of seizures. Tho most complete documentation of this fact comes from the impressive surgical experience recorded by Penfield and Jasper ( 1954). Although it is true that an epileptic seizure represents an exquisite neurophysiological experiment performed by nature in which the seizure discharge is tlie stimulating electrode displaying the functional localization in the cerebral cortex, discrete data regarding the basic underlying mechanisms is difficult to obtain in the human. Variables are difficult to control and the human organism cannot be manipulated in the same fashion as an experimental animal. Thus the clues provided by nature must be analyzed in the experimental laboratory (Ward, 1952). Although seizure activity can be induced in neuronal populations in a variety of ways in experimental animals, we have concentrated in our laboratories on seizure activity induced in the monkey by the intracortical injection of alumina because we have felt that this experimental preparation most closely approximated human epilepsy. Seizures induced by chemical convulsants ( strychnine, pentylenetetrazol, etc. ) or electrically induced after-discharge may share certain properties with epilepsy but obvious dificrences

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exist. Less is known regarding the relationship between seizures induced by local freezing of cortcx and epilepsy. Nims and associates (1941) confirmed earlier reports that local freezing of cortex induces chronic, recurrent seizures. They also confirmed the observation that this tendency is greatly increased if morphine is given at the time of freezing. There appears to be a species susceptibility in that convulsions after cortical freezing occur easily in the dog, somewhat less commonly in the cat, while clinical seizures in the monkey are said to be dificult to induce altliough electrographic abnormalities are present. This technique has recently been reactivated by Morre11 (1959; Morrell and Florcnz, 1958) and important data is being obtained which may, in the future, provide additional insight into mechanisms underlying epilepsy. The elemental problem in epilepsy deals with the determination of the locus or loci of origin of the seizure discharge. The clinical observations were extended to experimental seizures arising in the motor cortex or primates by Kopeloff and his co-workers (1942), who first developed a method of procliicing focal, recurrent seizures in monkeys. They have shown that the local application of alumina cream to the cortex of animals results in the developmcnt of an epileptogenic focus which behaves much the same as epileptic foci in man. The method which we have used can be briefly summarized. The cortex is exposed under aseptic conditions and commercial aluminum hydroxide gel is injectecl intracortically into the sensory or motor hand and face areas with a tuberculin syringe and a no. 27 hypodermic needle. To prevent the formation of adhesions between the cortex and the overlying tlura, care must be taken to prevent spillage of the alumina onto the pial surface and, in many instances, it has been found desirable to cover the area of exposure with a patch of polyethylene film. These maneuvers minimize dural adhesions in the vicinity of the cpileptogenic focus and facilitate the subsequent exposure of the cortex. In such monkeys, spontaneous clinical seizures appear after a variable delay, usually 30-60 days. Electroencephalographic examination reveals abnormalities in the form of spikes or sharp wave foci prior to overt epilepsy. The clinical seizures are characteristic of the location of the scar. Seizures occur spontaneously, the frequency being roughly related to the degree of cortical scarring induced. If a large number of intracortical injec-

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tions are performed, the monkey may subsequently go into status epilepticus and expire. The seizures can often be precipitated by exciting the animal, while studies utilizing an activity cage which identifies the occurrence of generalized fits indicates that many of the spontaneous seizrircs occiir during thc night, as is the case in many patients with epilepsy. In studying a complex phenomena like epilepsy, the physiological events can arbitrarily be divided into an inquiry regarding ( a ) the abnormalities which generate the discharge at the epileptogenic focus, ( 0 ) the local spread or the changes occurring whicli precipitate the interictal activity into a seizure, and ( c ) the propagation of the discharges during a major seizure.

II.

The Epileptic Focus

Our knowledge regarding epilepsy has largely come from the clinical and experimental effort directed against focal seizures. This does not imply that there is complete agreement that all clinical seizures can necessarily be reduced to this common denominator, although certain workers share that hope. Be that as it may, from the experimental standpoint focal seizures represent the simplest problem to attack in an already complex field. If it is postulated that the seizure originates in a given cluster of neurons, it should then be possible to define how this cluster of neurons differs, hoth morphologically and functionally, from “normal” cortex. Extensive microscopic examinations of brain scars removed from human epileptic patients have been carried out by Penfield and his co-workers (1954). Such scars are surprisingly similar to those experimentally produced in the epileptic monkey by alumina ( LVard et d., 1948). In those areas where the damage by alumina (monkeys) or trauma (man j is maximal, the nerve cells have vanished and have been replaced by gliosis. The fibrils of these piloid astrocytes, as Penfield states (Penfield and Jasper, 1954), “transmit the actual pull of the contracting cicatrix upon neighboring brain which is held together by its vasoastral framework.” In the more marginal regions, depopulation of neurons has occurred, the remaining cells being enmeshed in the astrocytic gliosis. These remaining cells may show certain minor abnormalities in chromatic stains while rather

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drz.matic changes are present when the neurons and their processes are visualized by means of tlic Golgi-Cox stain (Ward, 1961). It would appear from other data that the epileptic discharge arises in these neurons around the margin of the scar although, as yet, there are no confirmed morphological criteria which will permit one to identify which cell visualized under the microscope is an “epileptic” cell. The abnormalities wliich are responsible for the generation of the seizure discharge must be expressed by alteration of neuronal fur ction. Such alterations of frinction can occur in the all-or-none membrane of the cell body or soma, or in the graded response membrane of the dendrite. Thesc will be considered separately.

A. A c n v r ~OF~ NEURONSOMA

In recent years, microelectrocle techniques have been devised which record the electrical activity of single neurons within the central nervous system. The majority of the data reported for cerebral cortex deals with the electrical activity recorded by a microelectrode introduced into the cortex with a micromanipulator until the electrode tip (2-8 my) lies close to but presumably not touching the cell membrane. In normal cortex, spontaneous all-or-none spike discharges of the soma can be recorded in this fashion (Fig. 1 ) . The rate of discharge varies depending upon the type and depth of anesthesia and the input to the cortc.1. The rate of spontaneous discharge may range from 2-14 sec (hlountcastle et nl., 1957) in animals uncler anesthesia to rates in the vicinity of 50/sec in animals under local anesthesia and immobilized by a neuromuscular blocking agent. In cats immobolized by elcctrocoagulation of the midbrain, Martin and Branch (1958) found the over-all mean frequency of spontaneous Betz cell discharge to he 6.4/sec. If a neuron in sensory coriex is stimulated into activity by a sensory volley produced by a shock applied to a periplieral nc’rve, the cortical cell will respond witli a repetitive burst of discharges. The number of discharges to a single volley and the frequency of discharge will again vary with anesthesia (Tasaki et al., 1954; hlonntcastle et al., 1957). Under optimal conditions, the cell may fire as many as 8 times a t a frequency of 100-200/sec in response to a single volley. The descriptions of Li and Jasper (1953) and Albe-Fessard and Buser (1953)

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regarding the spontaneous behavior of cortical neurons obtained with extracellular recording are also consistent with the descriptions of Phillips (1956) and Li (1959a) of Sonia activity recorded with an intracellular electrode. In contrast to this random but well-ordered activity, a variety of patterns of neuronal hyperactivity may be observed in the cortex of epileptic monkeys. The studics of \l’ard and associates (1955)

FIG. 1. Spontaneous dischaqe of single neuron in noriiial uiiancsthetized monkey cortex; time mark: 10 insec. (From Schmiclt et d.,19.59.)

and Schmidt and associates ( 1959) indicate that the most frequently encountered interseizure pattern of activity of the epileptic neuron is rhythmically recurrent bursts of high-frequency discharges. A wide diversity of pathological patterns of discharge have been recorded. Figure 2 illustrates a pattern of rhythmically recurrent bursts of high-frequency discharge. These were recorded in the cortex of epileptic monkey under local anesthesia and immobilized with Syncurine. Random bursts with varying frequency of soma discharge have also been observed as shown in Fig. 3. These bursts

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recurred continuously during tlie 90 minutes that this cell was under observation. The sustained nature of these high-frequency discharges over long periods of time distinguish such unit discharges from the injury discharges evoked by penetration of the cell membrane by the electrode tip. \Then tlie cell membrane is damaged, the characteristic activity which is recorded is composed of a highfrequency discharge with progressive decay of amplitude which is readily differentiated from the constant repetitive pattern observed in the presumably intact “cpilcptic” cell for long periods of time. In other instances, the interseizure hyperactivity of neurons in the epileptogenic focus may consist of long trains of high-frequency discharge waxing and waning in frequency (Fig. 4 ) . The tonic hyperactivity may be punctuated by brief bursts of higher fre-

FIG. 2. Interictal activity of \in+ cells in epileptic cortex of monkey. These rhythmic high-frequency bur\ts of short duration continued for over 1 hour.

quency discharges of 800/sec or more, and may be interrupted by brief silences. Although there are many superficial similarities between the seizure activity generated in tlie monkey by the intracortical alumina technique and human epilepsy, it must be demonstrated that this same kind of activity also occurs in cells of the human epileptogenic focus. This matter has been studicd by means of the microelectrode technique during tlie course of operations for human epilepsy (Ward and Thomas, 1955; \Vard et al., 1955). Although the human data is very limited, the patterns of interictal firing of human epileptic cells appear to be the same ( LVard, 1961) as those recorded in the monkey. The similarities and differences between the activity of such epileptic neurons in a spontaneously discharging focus and the hypersynchronous activity evoked by various drugs, or that which occurs during electrically induced after-discharges, are not well

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established. Studies of the activity of single cortical cells during chemically precipitated seizures have been reported ( cf. Enomoto and Ajmone-Marsan, 1959), and it would appear that such cell discharges differ from those recorded from epileptic neurons in a scar. A comparison of soma discharges induced by strychnine with those

FIG.4.Spontaneous interictal activity of epileptic neuron. Note long “tonic” trains of high-frequency discharge with occasional pauses and higher frequency bursts; time mark: 50 msec. (From Schmidt et al., 1959.)

appearing spontaneously in an epileptic monkey was made by Thomas and associates (1955) who pointed out that, whereas the waveform of the action potential in the epileptic cortex was not significantly different from that recorded in normal cortex, a very marked alteration was frequently observed in experiments where strychnine had been applied topically to the cortex. In the strychnine experiments, the soma action potential often showed a pro-

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gressive augmentation of the positive phase which often coincided with the appearance of high-frequency bursts of unit firing. They concluded that there was no evidence that anything like this btryclinine effect occurs in the chronic epilcptic focus. Although the patterns of activity recorded from the epileptic focus differ rather dramatically from any patterns of spontaneous unit activity we have recorded from the cortex of either cat. monkey, or man, Martin and Branch (1955) have described spontaneous unit activity in the cat which they considered similar to tliat recorded from “epileptic neurons.” These records were obtained in cats immobilized by coagulation of the mesencephalir reticular formation. The interesting observations they report consist of regular high-frequency bursts of discharge recorded from a B ~ I Lcell. The frequency at the beginning of each burst was about 1000/sec, declining to about 300, sec at the end of the burst. As they pointed out, such observations were relatively infrequent. They concluded that such activity may be the normal discharge pattern of a small number of cells in the cortex; or this type of discharge may be restricted to animals with midbrain lesions. This pattern of regular bursts of high-frequency discharge decrementing in frcquency has not been seen in our experiments on epileptic monkeys. Since soma activity occurring in bursts is characteristic of some preparations with extensive midbrain lesions, these unusually high-frequency unit discharges may be explained on this basis. They do not appear to represent an exception to the observation that the patterns of neuron firing recorded from cells in the epileptogenic focus are peculiar to the epileptic focus and are not seen in normal preparations. Summary. The activity of cell bodies of neurons propagating all-or-none spikes has been monitored in the epileptogenic focus of monkey and man by means of the microelectrode technique. These studies indicate that, within the epileptic focus, the majority of cells are found to b e spontaneously active. Their interictal activity is characterized by autonomous, high-frequency bursts in varying patterns which differ dramatically from the spontaneous activity generated by normal cells and also differ, in certain regards, from the activity evoked by convulsant drugs.

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B. ACTIVITY OF DENDRITES 1. Normal Deizclritic Function

It is generally agreed that the dendrites of the neuron generate graded electrical responses in contrast to thc all-or-none responses of the axon or the cell body. Such graded responses are characterized by the absence of an absolute refractory period and a second response may thus always sum with the first (Bishop, 1956). \Vliereas, first, the intensity and timc course of the all-or-none impu Ise are independent of stimulus magnitude and, second, conduction of excitation is the major function of membranes which genera te such spikes, intensity and time course of the graded response of dendrites are directly related to the stimulating input, and the graded response is essentially a local response of postsynaptic membrane. It has been further proposed by Grundfest (1957) that corticd synapses are electrically inexcitable. He points out that the major phenomena which distinguish central nervous activity from that of nerve or muscle activity can be accounted for by two properties of synaptic electrogenesis: (1) that it is chemically excitable, not electrically; and ( 2 ) that either depolarizing or hyperpolarizing electrogenesis can occur, depending on the subclass of synaptic membrane. Although complete experimental verification of this hypothesis is currently incomplete, no significant body of data has b e m presented which would be inconsistent with this formulation. Furthermore, these postulates have a very important bearing on concepts relating to the propagation of the epileptic discharge. There is general agreement that the response of dendritic membrzne to synaptic activation is characterized by the absence of an absolute refractory period in contrast to the well-known absolute refractory period of axon or cell body (Bishop, 1956; Grundfest, 1957; Tasaki et al., 1954). However, it is less clear whether or not the graded response of dendrites may be propagated in a decremental fashion over the dendritic tree of a neuron. Eyzaguirre and Kuffler (1955b), on the basis of indirect information, concluded that conduction of an impulse from soma into at least the larger dendrite branches can occur in the stretch receptor of the crayfish. However it must be recognized that the dendrites of this specialized neuron differ in many ways from cortical dendrites including the fact that the dendrites of the stretch receptor are not covered by

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synaptic boutons as is the ca5e in the cerebral cortex. However, Fatt ( 1957) has presented evidence that antidromic activation of spinal motoneurons results in conduction up the apical dendrite at relatively low velocities (0.7-1.0 m’scc). He further points out that there is no cvidence to indicate tliat the impulse dies out 1)efore reaching the terminal dendritic arborizations. Bishop ( 1956), on the other hand, feels that conduction in dendritcs does not usually take place except under very abnormal conditions. If clectrogenic activity in dendritic membrane can be evoked chemically bllt not electrically, as proposed by Grundfest ( 1957), no mechanism for propagation of an impulse over the dendritic membrane would appear to exist. Thus Grundfest (1957) makes a strong case for the concept that postsynaptic potentials generated in dendrites are standing potentials and are nonpropagating except by the niechanism of passive clectrotonic spread. It is implicit in his proposal that the electrically inexcitable membrane is that patch of dendritic membrane which is subsynaptic. Thus, if the entire dendritic membrane is invested with synaptic boutons, the entire dendritic tree will be electrically inexcitable. On the other hand, if the local morphology is such that appreciable areas of dendritic membrane are free of synapses ( a s in the stretch receptor of the crayfish), electrical excitability and the ability to propagate impulses might well be present. The inability of dendritic membrane to sustain a propagating impulse is also confirmed in cerebral cortex by the data obtained by von Euler and Ricci (1955). Thus, although the data is incomplete, a hypothetical model of a cortical neuron can be formulated. The axon consists of membrane which responds in all-or-none fashion, is electrically excitable, and appears to be designed to transmit the message it receives at one end unaltered to the other end. The membrane of the Sonia of the cortical neuron probably shares the same property of all-or-none response and is thus also electrically excitable. However, it is possible that those patches of postsynaptic membrane on the cell body which are covered by synaptic boutons may consist of graded response membrane. The dendrite, at the other pole of the neuron, is composed ( a t least largely) of graded response membrane, is electrically inexcitable, and its membrane responds to chemical transmitter substances liberated by the synaptic boutons which cover essentially all the dendritic surface. It is also probable that

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the terminal axonal segment leading up to the synaptic bouton of the presynaptic axon may also consist of graded response membrane. Thus the dendritic membrane generates standing postsynaptic Fotentials as a consequence of chemical stimulation by its synaptic: input. This synaptic electrogenesis then can affect the electrically excitable membrane of the cell body by electrotonic spre
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of cell body activity with a microelectrode immediately beneath tlie surface electrode. Such a record is seen in Fig. 5. Thcse two strips of record compare the activity of the all-or-none spike activity (in the upper trace) with the activity recorded with a surface electrode a few millimeters above it on the pia. Although the “epileptic” cell is spontaneously discharging with occasional high-frequency bursts, there is no apparent relationship between tllr soma activity and the dendritic activity recorded by the pial electrode. This lack of consistent correlation between dcndritic and cell body activity in epilepsy (Schmidt et al., 1959) also characterizes tlie soma

FIG.5. Simultaneoiis records in epileptic cortex from an intracortical microelectrode (upper trace ) and an ovcrlying surface macroelectrode ( lower trace ). Note poor correlation of single cell activity and slow dendritic potentials; time mark: 10 msec. (From Schmidt et al., 1959.)

discharges in normal cortex ( L i and Jasper, 1953; Jung, 195.3; Mountcastle et al., 1957). However, under the action of certain convulsant drugs, a constant relationship between dendritic activity and unit spikes may be present (Eiiomoto and Ajmone-Marsan, 1959), which again serves to distinguish seizure activity induced by drugs from epileptic activity. Nevertheless the classic epileptic spike as recorded from either the pial surface or the scalp is generally considered to be characteristic of epilepsy. It may arise locally at the epileptogenic focus; or it may b e “broadcast” to normal cortex by conduction from a distant primary source, cortical or subcortical. At times epileptic spikes may be associated with bursts of discharges of the cell body

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wfile at other times no correlation may be present (Ward, 1960a). A iypical epileptic spike as recorded in the EEG is shown in Fig. 6 as it appears when recorded within the cortex by a microelectrode. In this instance, unit firing is obviously associated with the paroxysmsl spike. Even when this occurs, however, there is no constant relationship to the phase of the slow potential when multiple examples are analyzed. The lack of consistent relationship between discharges of the neuron soma and the slower potential fluctuations of the cortical surface would be consistent with many clinical ob-

FIG.6. Epileptic unit potentiJs occurring during the course of a paroxysmal “epileptic spike”; time mark: 10 mscc. (From Schmidt et al., 1959.)

servations. Epileptic spikes arising in motor cortex are not always accompanied by soma discharge as evidenced by the fact that twitches of peripheral muscles ordinarily do not accompany such random spike discharges. The common observation-that dendritic circuits in the cortex may be flooded with input that generates very widespread spike-and-wave discharges in some patients with petit ma1 epilepsy without discernible alternations of behavior-would also be consistent with the formulation that the activity of cell bodies need not be extensively altered during a burst of such dramatic potentials in the electroencephalogram. An epileptic spike such as that recorded in Fig. 6 presumably represents a relatively well-synchronized discharge of the graded

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response membrane. Possible mechanisms which may be playing a role in such synchronization will be discussed in subsequent sections where data will be presented to indicate that the basic defect in epilepsy may relate to distorted dendritic function. One reflection of this pathological dendritic function is the epileptic spike. Summary. It is generally agreed that the dcndritcs of the neuron generate graded responses in contrast to the all-or-none spike responses of the axon or cell body. Although there would appear to be no consistent correlation between the local dendritic activity and thc discliarges of the cell body, the epileptic spike as recorded in the EEG or directly from the cortical surface is one of the most characteristic electrographic signs of the epileptic discharge. Even within the epileptic focus, however, there is no consistent correlation between such epileptic spikes and the high-frequency discharges of the soma. On the basis of data to be presented in the next section, there is reason to postulate that the basic defect in epilepsy may relate to distorted dendritic function. One reflection of this is the epileptic spike. C. THEEPILEPTIC NEURON

Schmidt et al. (1959) postulated that the autonomous activity which characterizes the epileptic neuron is due to a relatively enduring dendritic depolarization with a resultant difference in potential between the cell body and its dendrites. Under these circumstances, the membrane potential of the soma recovers rapidly (acting as a source) with resulting current flow from the soma to the depolarized dendritic tree which acts as a continuing sink. If this current flow continues to reach threshold, a high-frequency discharge will occur of the type illustrated in Fig. 4. It was further proposed (Ward ct d., 1956) that the dendritic depolarization in the epileptic cell might be the result of mechanical deformation of the dendritic tree by the fibroglial scar known to be present. Thus the relatively enduring dendritic depolarization produced in the epileptic cell by mechanical deformation from the “actual pull of the contracting cicatrix” ( Penfield and Jasper, 1954) might account for the autonomous activity of the epileptic neuron and the highfrequency discharges recorded by the microelectrode.

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The fundamental assumption upon which this hypothesis is based is that cortical dendrites may be depolarized by mechanical stimuli. That other types of nerve membrane may be stimulated by mechanical deformation is easily confirmed by anyone who mechiinically stimulates his ulnar nerve by striking his “funny bone”; the repetitive responses of nerve evoked by stretching have been carefully studied by Sat0 ( 1952). Local mechanical deformation of the membrane in a nerve ending has been shown to produce a geners tor current which evokes nerve impulses (Loewenstein, 1959). Furthermore, Svaetichin ( 1958) has demonstrated that mechanical stimulation of single ganglion cells will evoke responses. As he points out “the conditions seem to some extent to imitate a synaptic aci ivation; the electrode tip simulating a synaptic ending which in this case mechanically changes the membrane structure and causes a graded reduction of the perikaryon membrane potential ( EPSP). This results in a continuous outward current flow through the M-membrane (of the axon) and a repetitive firing.” Since Alanis and Matthews (1952) have shown that local pressure on neurons in the ventral horn in frog spinal cord results in presumed dendritic degolarization with increased rates of neuron discharge, it is reasoriable to assume that central neurons can be excited into activity by mechanical stimulation, at least under certain circumstances. This would also be consistent with the formulation of elcctrokinetic membrane processes presented by Teorrel (1959) who presents evidence for the equivalence of electrical and mechanical stimuli in evoking membrane depolarization in his model membrane. That dendritic depolarization might account for the high-frequency discharge recorded from epileptic neurons is suggested by sttdies on crustacean stretch receptors. Here the dendrites of the sensory cell are stretched and mechanically deformed when tension is applied to the muscle to which they are attached. This matter has been extensively studied by Kuffier (Eyzaguirre and Kuffler, 1955 a, 13). Figure 7 illustrates the response of such a neuron recorded by means of an intracellular electrode. In Fig. 7A, as stretch is applied, slow depolarization of the dcndrites occurs with a consequent reduction of the membrane potential of the soma indicated by the slower upward deflection. When this depolarization reaches threshold (indicated by the level of the dotted line), a spike discharge ocixirs. With maintained stretch and continuing dendritic depo-

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larization, a continuous discharge occurs. In Fig. 7B, a greater strerch produces greater dcndritic depolarization and generates a higher rate of cell discharge. There are certain similarities, at least qualitatively, to the high-frequency discharges of the epileptic cell illustrated in Fig. 4. ‘That mechanical deformation of neurons in the cortical scar might play a role in the inccytion of the epileptic process is also sugsested by clinical observation. It is widely recognized that a seizure may be precipitated in tlie human by manipulation of durn adherent to underlying cortex during the surgeon’s efforts to free the dura from the cortical scar in the course of operations for epilepsy. However, except for observations of this kind, controlled data dcaling with physical distortion or displacement of dendrites in cortex is difficult to obtain. It is possible that the precipitation of :seizures in epileptic patients by acute water intoxication may be secondary to the gross brain swelling that occurs with increasing traction on the scar. However, shifts of electrolyte also occur under the.je circumstances which might also change thresholds of excitable meinbranes in the epileptic focus. Ward and Morlock (1959) attempted to increase the dentlritic stretching in epileptic monkeys with focal alumina cortical scars by inducing increases in brain volume with Diamox. It had previously been shown that increases in brain volume follow intravenous injection of Diamox, but continuous EEG monitoring in the epileptic monkeys failed to show an). significant alteration of epileptic activity in the EEG in any of these animals, and no clinical seizures were observed. However, multiple variables again are present since Woodbury and Esplin (1957) have shown that Diamox also results in a higher gradient of soc.ium ion across the membrane with a rise in threshold as well as an increase in y-aminobutyric acid. Both of these actions of Diamos, ass x i a t e d with elevations of cell thresholds, might well mask any cff oct of increased dendritic stretch, and consequent dendritic depolarization, if such occurred. Tlius the role of mechanical deformation of the dendrites in producing those properties which we have associated with epileptic nenrons must await further experimental documentation. However, there is a body of data which would tend to confirm the postulation that abnormal dendritic function characterizes tlie epileptogenic focus. Rose and Mountcastle (1954) as well as Tasaki

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et al. (1954) have shown that the duration of repetitive firing of single neurons corresponds to the duration of the dendritic response. Thus the prolonged high-frequency bursts of soma discharges recorded from epileptic neurons might well be associated with pathological actviity in their dendrites. With the onset of a seizure discharge, von Euler ef al. (1958) have reported a massive dendritic depolarization. It is well known that epileptic foci have a lower threshold for electrically induced after-discharge and it would appear that this is related to an cnhancecl superficial cortical response (Eidelberg ct al., 1959). White and associates (1960b) h a \ e also presented data which indicates that the capacity of cortical neurons to exhibit seizure discharges is directly related to distortcd function of cellular structures mediating the direct cortical response. They have monitored the direct cortical response to local electrical stimulation of cortex by means of implanted electrodes in monkeys made epileptic by the alumina technique. An initial depression of tlie aniplitude of tlie local cortical response after operation is followed, some weeks later, by an augmentation of the dendritic response and epileptic spike activity which then appcars in the EEG. Spontaneous seizures appear shortly thereafter. Their results thus indicate that increased excitability of graded response membranes is associated with the development of the epileptogenic focus in the monkey. Summay. The hypothesis is presented that the autonomous activity which characterizes the epileptic neuron is due to a relatively enduring dendritic depolarization which might be the result of mechanical deformation of the dendritic tree by the fibroglial scar known to be present. The cvidence that mechanical deformation of dendritic membrane may result in its depolarization in both peripheral as well as some central structures is summarized. Since it is known that the duration of repetitive firing of normal neurons corresponds to the duration of dendritic depolarization, one can conclude that the prolonged high-frequency firing of epileptic neurons is a reflection of abnormal dendritic function. This conclusion is supported by the data indicating enhancement of the direct cortical response at the epileptic focus. Since the direct cortical response has been assumed to be a postsynaptic dendritic response, a lowering of dendritic thresholds appears to be associated with the development of epileptogenesis.

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D. FIELD EFFECTS If dendritic depolarization is continuously present at the epileptic focus, it might well lie reflectcd by continuous local negativity -i.e. a standing negative potential at the epileptic focus. This matter has been studied by Ward and Mahnke (1960) using nonpolarizable electrodes and d.c. amplifiers. In two monkeys having spontaneous seizures, the cortex at the time of the acute experiment exliibited epileptic activity in the electrocorticograni. In both of these, thi j epileptogenic focus was also characterized by negative standing poientials of large magnitude. In one animal the focus was 7 mv negative as compared with its hoinologous site in the opposite hemisphere; in the other animal the focus was 9 mv negative to noimal cortex. The reference point was on the occipital bone. In boih cases, these sites in the area of the anatomical scar were the mc st negative points monitored by multiple placements of the d.c. electrode. They were more negative than any other point in the same or opposite hemisphere by a t least 3 mv and differed from some points by as much as 12 mv. This is in rathcr dramatic contrast to the relatively homogeneous distribution of standing potentia Is of millivolt dimensions in normal cortex. In one monkey there was a steep voltage gradient of increasing negativity along the cortex aniounting to 1 mv/mm as the area of peak negativity was approached a t the focus. At this point very large surface negative spiking was evident. As the d.c. electrode was moved away from the focus, the decrease in negativity wa5 correlated with a decrease in the magnitude of the spikes. Control observations includccl an animal that had received a minimal injection of alumina into liis sensorimotor strip. This animal did not have any observed seizures; did not have the obvious fibroglial scarring that the others displayed; did not produce an abnormal EEG at the time of craniotomy; and did not have a standing potential offset between the injected area and other cortical sites. Thus these preliminary observations would appear to indicate that the epileptogenic focus is characterized by a negative standing potential of appreciable magnitude. This standing potential would be consistent with persistent dendritic depolarization as predicted by the hypothesis previously presented. Of even greater interest is

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the role such standing potential fields might play in the pathological physiology of the cortex in epilepsy. In order to assess this factor, it is necessary to summarize the currently available data regarding the role of standing potentials in determining neuronal excitability. It is known that artificial polarization of neural tissue can have profound effects on the excitability of that tissue. Goldring and O’Leary (1951) found that cortical paroxysmal activity could be induced by passing a 1- to 3-ma current across the cortex with the surface positive to subcortex; and that the cortical paroxysm could be stopped by polarization of opposite polarity. The technicjiie of polarization has been also widely applied to the other portions of the central and peripheral nervous system to manipulate tlu-esholds of neuronal excitability. Since the observations of Libet and Gerard (1941), it has been known that standing potentials are characteristic of neural tissues. The role that such normally occurring standing potential fields play in the modulation of normal neuronal activity is, however, less clear, although an increasing body of data dealing with the role of d.c. phenomena in tlie normal and pathological physiology of the cortex is being accumulated ( O’Leary and Goldring, 1960). Since naturally occurring standing potential fields exist and these must be associated with currents flowing across cortical neurons, it then becomes pertinent to inquire whether these are of sufficient magnitude to influence significantly neuronal thresholds. Data regarding the sensitivity of neuronal membrane to such fields is available and changes in the strength and direction of electric fields have been shown to influence both peripheral receptor discharge and neuron activity at a number of sites in cord and brain. A behavioral demonstration is provided by Gqmnarchus niloticris, a fish possessing weak electric organs that apparently generate an electric field in the water surrounding the fish. Lissman (1958) proposes that the fish detects objects a t a distance by perceiving the tlistortions in the field produced by any object with a conductivity differing from that of water. Experiments with moving fields indicated that the fish was sensitive to a potential gradient of 0.03 pv/cm in the water about it. The current density in the fish a t threshold was calculated to be 2 x amp/cm2. This extraordinary sensitivity of the receptor organs in this fish indicates the lower limits at which some neural membranes may be excited by exceedingly minute

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cuprents. Even in the brain, Strumwasser and Rosenthal ( 1960) have shown that the rheobase for stimulation of normally inactive cells is '3etween 1-3 x l0W9 amp, while the threshold for modulation of spontaneously occurring activity i s 2.5 to 3.5 times lower. Terzuolo anli Bullock (1956) have further shown that the discharge of a sensory cell can be modified with voltage gradients in the surrounding medium of 0.1 1nv/lO0 nip. It may be more than a coincidence that this is identical to tlie voltage gradient of 1 mv/mm extending radially from the epileptogenic focus in tlie experiments on epileptic monkeys reportctl by \\'ard and hlahnke (1960). Thus one might assume that d.c. fields of the magnitude recorded can modify neuronal discharges of cells within these fields. Current flow from adjacent cortical tissue into such a negative electrical sink may b e the ineclianism underlying the hyperexcitability of epileptic neurons. It h a s been shown by Matthews (1937) thc t a d.c. current passed in a direction to produce depolarization of anterior horn cells (catlioclc on cord) will indiicc a steady rhythmical discharge of such cells with a frequency directly related to the current intensity. Such observations have been extended by Baeron and Matthews (1938) and more recently by Alvord and F u x t e s (1953) who pointed out that direct currents flowing in the proper direction for d e p o l a r i h g the soma membrane of extensor motonenrons result in repetitive firing with properties similar to those elicited by either orthodromic or repetitive electrical excitation. In addition, Morita (1959) has shown that d.c. polarization of a sense organ will evoke spikes. Thus it would appear to be possible that pathological dendritic function might generate slowly fluctuating d.c. fields of suffieicnt magnitude to induce autonomous high-frequency firing of epileptic neurons similar to that recorded with the microelectrode from the epileptic focus in both monkey (Fig. 4 ) and man. Summmy. If dendritic dcpolarization is continuously present a t the epileptic focus as postulated in the previous section, it might we'l be reflected by continuous local negativity at the epileptogenic focus, This has been confirmed by observations of negative d.c. fields which appear to characterize the epileptic focus in the monkey. The remaining question which arises is whether the d.c. vo1:age gradients or current flow of the magnitude recorded at the epileptic focus are able to alter neuronal thresholds of cells within

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these fields. Observations obtained on varying types of neuronal membrane indicate that these can be evoked into activity by imposed voltage gradients of 1mvlmm or currents as low as 8 x lo-" amp/cm2. Thus the data would indicate that, if pathological dendritic function can generate slowly fluctuating d.c. fields of this magnitude, they might be effcctive in inducing an autonomous highfrequency discharge like that recorded from epileptic neurons.

E. POSSIBLE MECHANISMS OF DENDRITE SYNCI-IRON~ZATION There is general agreement that the epileptic spike as recorded from either the scalp or the pial surface of the brain rcprescnts the summed activity of graded rcsponse membrane. Because of its large magnitude, it would be reasonable to assume that relatively large areas of dendritic membrane must b e depolarized to generate such potentials. Furthermore, during the onset of a spontaneous scizure, massive potentials are generated with sufficient synchrony that summation occurs and exceedingly large rhythmic rcsponses are recorded. Since it would appear that, in the case of focal cortical seizures, such massive dendritic waves are locally generated in the cortex, it is pertinent to inquire into possible mechanisms by wliich such relatively synchronous discharge of graded response membrane is accomplished. The waveform of either the intcrictal spike or the rhythmic spikes which cliaracterize the onset of a spontaneous scizurc suggests that thcre is rather rapid recruitment of graded rcsponse membrane. The mechanisms by which dendritic depolarization in one cortical neuron can influence adjacent dendritic membraiic are less clear, Of possible mechanisms, the local circuitry of the cortex is one which first comes to mind. Cajal (1952) proposed in 1904 that cortical neurons can communicate with each other by means of axon collaterals. He pointed out that axon collaterals are not unique for cortex but are also present in ganglionic masses throughout tlie central nervous system, and he further proposed that these structures might serve to diffuse the discharge (Chang, 1955). However, since it is now known that synchronization of dendritic activity can occur in the epileptic focus without evidence of soma or axon firing, it would appear to b e unlikely tliat axon collaterals are tlie means by \vhich synchronization of graded response membrane occurs in

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the epileptic process. Bok (1956) has studied the “network of fibers in tlie cortex and descrihed miiltiple crossings which are present diffusely throughout the cortex in both man and animals. He raises tE e question “why should tliis enormous fiber network, covering more than 90% of the space in our cerebral cortex, exist if its crossings did not act as synapses?”. Certainly if these are synapses, tl ey have an exceedingly simple and unspecialized morphology, but Bok goes on to point out that this is the only type of synapse ovcurring in the most primitive animals having a nervous system. He felt that this might correlate with the fact that the cerebral cortex is the youngest p a t of the central nervous system. Unfortunately there has been no experimental verification of his hypothesis that these local interconnections in cortex serve any transmissional function. However onc cannot exclude the possibility that SL ch structures might play a role in the elaboration and synchronization of graded response activity which appears to characterize cpileptic cortex. In addition to the possildity that cortical neurons interact by means of their circuitry in ~7hichone cell is “ w i r e d to another, there is also the possibility that they interact by means of field effccts. The formal aspect of these two possibilities has been disciLsseclin an intriguing fadlion hy Cragg and Temperley ( 1954 ) who suggest that the memlx-ane potential of a neuron can be altered by changes in the membrane pottmtial in any of tlie neighboring ncwons-not merely in certain neurons arranged in circuits with the one in question. As anatomical evidence, they point out that tlie ~c~lum formed e by the centers of 8 cell bodier in cortex contains the dendritic processes from 4000 other cclls. With such dense packing they feel that interaction could only fail to occur if fibers were arranged in some definite scheme (like an electronic circuit) which wl~uldprevent indiscrimin‘itc synapsing; this would also ensure that the extracellular current generated by an active neuron did not pass through the meinbrancs of its neighbors. Bullock (1958) a1jo suggested, but without dircct evidencc, that neurons may influence each other withoiit tlie mediation of spikes in certain circurnstances; Bennett ef al. (1959) have presented data to indicate that both hyperpolarization and tlepolarization of one cell may cause polarization in adjacent cells. The potential induced in adjacent cells is much reduccd and slovwl, as would be expected from

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electrotonic conduction. They felt that these data suggest the existence of electrical connections between cells which might be responsible for synchronization of neuronal responses. That field effects are responsible for synchronization has also been suggested by Bremer ( 1955) who lias shown (Bremer, 1941) that synchronization of seizure discharges can occur across complete transections of the stryclininized spinal cord. Enomoto and Ajmone-Marsan (1959), in a study of cortical seizures induced by topical convulsant drugs, described a massive hypersynchronization taking place through the entire cortical thickness, which they suggested might be due to “ephaptic” spread, likening this phenomenon to the transmission which occurs at an ephapse or artificial synapse created between the cut ends of two nerve fibers. Green (1958) has also reported that repeated activation of hippocampus leads to an increasing depolarization in the dendritic field. When this occurs, it is apparently possible for seizure discharges to spread, not only by the ordinary axonal route, but from cell to cell presumably via the dendrites. Further confirmation that electrotonic or “ephaptic” interaction may play a major role in the synchronization of tlic seizure discharge is provided by the evidence presented by Bernhard ( 1958). He reports a direct correlation between the anticonvulsive action of certain local anesthetic drugs and their effect on the ephaptic type of central neuron interaction which “shows that ephaptic interaction is essential to building up and spread of rhythmic epileptic cortical activity.” Although neither the hypotheses nor tlie data are explicit in this regard, it is implied that, if graded response activity in one dendritic tree can evoke graded response activity in adjacent dendritic arborizations on the basis of “field effects” or “ephaptic” excitation, this must be achieved by electrotonic conduction. If the adjacent dendritic tree is excited in this fashion, its denclritic membrane must be electrically excitable. Howevcr Grundfest ( 1957) has presented the thesis, with supporting arguments, that the dendrites in normal cortex are electrically inexcitable. Tlie essential feature of his thesis is that those patches of dendritic postsynaptic membrane covered by synaptic boutons are chemically and not electrically excitable. Studies with the electron microscope would appear to indicate that the cortical dendrites are possibly totally invested by synapses (Gray, 1959; Palay, 1956). Armstrong and

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Young ( 1957), by other liistological techniques, have estimated that each cortical neuron is covered by 8000 end-feet. Thus, if Grundfest is correct, at least the major portion of dendritic membrane in normal cortex would appear to be composed of postsynaptic membrane and t h i s electrically inexcitable. Moreover Eccles (1957) has pointed out that differences in mechanisms of firing between spinal motoncurons and the stretch receptor cell may arise because the latter is not covered with synaptic knobs. However, epileptic cortex is clearly not normal cortex and it is possible that the pathological process which produces the epileptogenic foc-us interrupts sufficient presynaptic elements so that the epileptic cell is a t least partially devoid of synaptic knobs. No morphological studies of epileptic cortex have been carried out with suitable staining teclinicjues to demonstrate the state of de ndritic synapses. However, some limited electrophysiological obsewations which may bear on this point have been carried out. It is well known that all-or-none discharges of cortical neurons in sensory cortex can be evoked with relative ease in normal cortex. Such evoked activity recorded by a microelectrode is obviously generated by an afferent barrage exciting the cell via axodendritic and, presmnably, axosomatic synapscs as well. In contrast Schmidt et nl. (1’359) have remarked that, in epileptic cortex, evoked unit activity of this type has rarely been ohtained in the vicinity of the scar in postcentral cortex. This is in striking contrast to the augmentation of evoked unit activity in stryclininized cortex (Thomas et al., 1955). Furthermore, Abdulla and Magoun (unpublished observations ) have noted that “evoked cortical potentials, of a graded response or dendritic nature, are especially prone to occlusion in experimental co-tical seizures.” In addition, Smith and Purpura ( 1958) have stated that spikes induced by local freezing of cortex will occlude signals generated by synaptic systems involved in the direct cortical, transcallosal, and specifically relayed responses, but not those responsible for thalamocortical recruitment. If it is confirmed that epileptic neurons are less easily evoked into activity by afferent stiinulation than normal cortical cells, a reduction of synaptic input might be one of the causes. If the dendrites of the epileptic cell are at least partially denuded of their normal envestment with synaptic end-feet, this exposed dendritic membrane might, if previously electrically inex-

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citable, now be responsive to current fluxes. They would then be responsive to electrotonic field effects from neighboring activity and an opportunity for relative synchronization and augmentation of dendritic activity woiild be present in the epileptogenic focus. These are just the phenomena which characterize epilcptogenic cortex. In any discussion of mechanisms for local synchronization of cortical activity, we should not forget that the cortex is not solely composed of neurons. Nurnberger and Gordon (1957) point out that neuronal elements constitute only about 147;-;?of the cellular population in tlie striate cortex in monkey and only 18% in man. It is known that glia have membrane potentials of the same order of magnitude as those of neurons (Phillips, 1956; Li, 1959a). Tasaki and Chang (1958) have demonstrated that glia can respond to electrical stimulation with a graded response lasting u p to 4 seconds. Furthermore, such electrical stimulation of the glia evokes ii slow mechanical contraction which lasts 7 to 16 minutes (Chang and Hild, 1959). On tlie basis of this data, Li (195%) has suggested that the electrical events in the glia may play a role in synchronization of unit activity in cortex. Certainly in epileptic cortex (where the dendrites of the epileptic neurons may be unusually responsive to electrotonic fields and where, in addition, thcy may be rcsponsive to mechanical deformation), both the electrical responses of glia as well as their physical contraction must not be omitted from consideration. Szimmar!y. Since massive dendritic potentials are characteristic of epileptic cortex, it must be determined how dendritic activity in one neuron can influence adjacent dendritic membrane. The anatomical data is reviewed as well as the observations indicating that local field effects can produce changes in excitability in adjacent cells, If such elcctrotonic activation of dendritic membrane is to occur, the dendritic membrane must be electrically excitable. Whereas normal dendritic membrane appears to be largely covered with synaptic end-feet ( and may thus be only chemically excitable), the suggestion is presented that the dendrites of epileptic neurons are relatively devoid of enveloping synaptic structures ant1 may then be electrically excitable. This would be consistent with the observations indicating that synchronization and augmentation of dendritic activity are characteristic of the epileptic focus. Since glia

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c a i generate graded responses and undergo physical contraction in response to electrical stimulation, their possible role in synchronization of cortical activity must not be neglected. F. OTHERMECHANISMS FOR PROD~JCTION OF SEIZUREACTIVITY In the previous sections, somc of the possible mechanisms that might be playing a role in tlic epileptic process have been presented. They were chosen from many possibilities because either direct or inferential data suggestcd their pertinence to the construction of a hypothetical modcl of the epileptic cortex. However additional possible mechanisms exist which future experimentation may indicate are important to an understanding of epilepsy. The first of these possible mechanisms deals with spontaneous or pathologically induced alterations of the membrane potential. Fatt (1954) has pointed out that, a t the myoneural junction, there is a continuous display of randomly occurring pulses each exhibiting all the characteristics of junctional activity. These pulses, obtained in the absence of any propagated impulse in the nerve, presumably arise from the intermittent relcase of small quantities of acetylcholine from the nerve terminals. Spontaneous potentials have been observed during the coww of intracellular recording from cortical neurons (Li, 195C3a). If such events are associated with spontaneous fluctuations in threshold, a shift of this process in the direction of increased excitability might result in autonomous t1i:;charge. Furthermore, it may bc that the firing level of a neuron is not a fixed level indepcndtxnt of the state of activity of the cell as has been previously assumed. Kolmodin and Skoglund (1958) have shown that, with increasing frequency of firing of spinal motoneurons, there is a successive lowering of the critical membrane potential for spike initiation. Such phenomena coupled with ionic shifts (Shanes, 1958) may play a role in the generation of the high-frequency burst activity characteristic of epileptic neurons. A second possible meclianism involves alterations of the local circuitry of the cortex. Li and Chou (1960) have suggested that a differential loss of inhibitory neurons in the cortex might play a role in hypersynchronous activity. This would be consistent with the observations reported by Phillips (1956) in which, by means of intracellular recording, he showed that the occasional

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brief high-frequency bursts of natural impulses were followed by a wave of hyperpolarization. He suggested that this might be the result of a cumulative inhibitory feedback eff ect. Such mechanisms might well be operating to quench a seizure (Gastaut and FisherWilliams, 1959) while, on the other hand, a deficit of such negative feedback systems might play a role in the genesis of the seizure discharge. Certainly alteration of input to epileptic cortev can precipitate seizures in susceptible epileptic humans or in aluminainjected epileptic monkeys as evidenced by tlie frequent observations in both species where seizures are precipitated by stress. A third possible mechanism which may be playing a role in epileptogenesis is the phenomenon of denervation hypersensitivity to acetylcholine. Although this phenomenon is well documented in the peripheral autonomic system, crucial experiments dealing with denervation hypersensitivity to acetylcholine in the central nervous system are difficult to devise. Cannon and Haimovici (1939) pointed out many years ago that spinal neurons ipsilatera1 to a hcmisection of the cord are more excitable than contralateral cells. They considered that this effect was due to sensitization of the spinal neurons following partial denervation produced by the heinisection. Drake and Stavraky (1948) as well as Spiegel (Spiegel and Szekely, 1955; Chavez ancl Spiegel, 1957) have subsequently presented evidence that denervation hypersensitivity to acetylcholine may occur in the brain itself. Since Brenner and Merritt (1942) postulated some time ago that a disturbance in acetylcholine metabolism might be etiologically important in certain varieties of epilepsy, it might be that, when any group of neiirons in the central nervous system are deafferented by any means, such cells become hypersensitive to acetylcholine; this phenomenon might play a role in the liyperactivity displayed by epileptogenic cortex. Infcrcntial data dealing with this hypothesis arc available. It is known that, if a slab of cortex is neuronally isolated, a burst response is evoked with unusual ease which propagates widely throughout this abnormal cortex (Burns et al., 1957). Echlin (1959) has shown that such “isolated” cortex is easily precipitated into epileptic-like activity by topical acetylcholine in concentrations which have no eff ect on surrounding normal cortex. Furthermore, Teasdall and Stavraky (1950) have shown that 6 to 9 months after section of the corpus

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callosum the threshold to Metrazol convulsions in cats is reduced, wh ch might be considered due to denervation sensitization. However Kristiansen and Courtois (1949), in their careful studies of isolated segments of cortex in the monkey, point out that, leaving the pial blood supply intact, high-voltage bursts of fasi activity appear in the slab within a relatively short period of time following the surgical isolation procedure. I t is thus difficult to conceive that any central clencrvation hypersensitivity to acetylcholine is the responsible factor in such activity generated in the isolated cortical slab. Furthermore, Forster (1951), on the basis of studies with DFP, believes that the current evidence indicates that acetylcholine plays no role in the genesis of convulsive seizurzs. This would be consistent with the observations of Kennard (1957) who has studied the cffect of temporal and frontal lobe ablations on the incidence of seizures in monkeys rendered epileptic by injection of alumina into the sensorimotor cortex Following such surgical ablations, she reports that the number and severity of clinical seizures increased considerably, and that several animals developed epilcpsia partialis continua. However the extreme potentiation of epileptic tendencies was always gre,atest during the first few clays after operation and then subsided, but Ieaving a chronic state of increased frequency of fits. Again the potentiation of the scizures appears too promptly to be due to denervation sensitization. In fact, an alternate hypothesis could be advanced that such potentiation of the epileptic process by these lesilms might be due to a rechiction of inhibitory input to the epileptogenic focus. 4 fourth and final approach to an understanding of epileptogenesis is a consideration of biochemical factors. It would be inappropriate, in this setting, to attempt to summarize the neurochemical observations bearing on epilepsy, particularly since an e1eE;ant review of this area of kno~vledgehas been recently presented by Tower (1960). It is sufficient to say that epileptogenic cortex appears to be biochemically characterized by an impairment of aceiylcholine-binding, a “metabolic” loss of glutamic acid, and a failure to maintain tissue potassium concentrations. Changes in redox potential and cortical pH also occur in cortex actively engaged in a seizure discharge which are similar to those observed dur ng seizures induced by electrical or chemical means (Ward

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et a[., 1948). In passing, it should be noted that biochemical observations on the effects of convulsant drugs are not necessarily pertinent to the problems of epilepsy. We have already pointed out that this applies to electrophysiologica1 observations as well. The most pertinent studies, biochemical or otherwise, are those on epileptogenic cortes. Preferably these should be carried out on human material such as the data reported by Tower and Elliot (1952). I n spite of the progress which has been made toward understanding the neurochemical correlates of epilepsy, no unequivocal formulation of a primary biochemical “lesion” can be presented to account for the observed changes in the function of epileptic cortex. It may well be that the observed biochemical changes passively reflect the grossly altered function of these cells. Certainly the energy requirements of the epileptic neurons must be appreciably different from normal cortical neurons and this must be reflected by metabolic alterations. If a biochemical defect were the primary mechanism of epileptogenesis in epileptic cortex, an increase in reaction velocities by induced hyperthermia might increase the epileptic activity. This matter has been studied by Schmidt et al. (1956) in epileptic monkeys subjected to artificial hyperthermia. The experimental results would tend to indicate that clinical seizures of cortical origin are not precipitated by temperature elevation alone. This would be consistent with tlre conclusion that a biochemical lesion is not the primary mechanism underlying epileptogenesis. There are additional possibilities which have, as yet, not been adequately investigated. It might be proposed that the hyperactivity of the epileptic neuron might be the consequence of rcduccd availability of inhibitory transmitter substance as thc result of a defect in its metabolic cycle. There is evidence to indicate that Diamox, a compound having anticonvulsant properties for certain types of subcortical seizures, produces an increase in tissue y-aniinobutyric acid ( GABA ) (Woodbury and Esplin, 1957). This might be taken as suggestive evidence that there is a relative deficiency of GABA in the circuits generating the seizures in such cases. If GABA is an inhibitory transmitter substance, a defect in its metabolic generation might play a role in certain types of epileptogenesis. Since it would appear that GABA can have excitatory

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as well as inhibitory actions (Malinke and Ward, 1960), it would not appear that there is any current evidence to indicate that epileptogenesis is related to a faihire of metabolic production of inhibitory transmitter agents. Since pathological changes in glia appear to b e characteristic of the epileptogenic focus, one might wonder whether alterations of their function might play a metabolic role in the generation of abnormal neuronal activity. If, in fact, astrocytes serve as metabolic pipelines between the vascular system and the neurons, defective metabolic transport via pathological glia might be a factor in the genesis of epilepsy. No studies on the point have been undertaken. Thus it would appear that, as yet, there is no biochemical formulation of mechanisms which can account for the generation of a cortical epileptogenic focus. Summary. In addition to the mechanisms presented in previous sections for which some direct or inferential evidence exists to indicate their pertinence to epilepsy, other possible mechanisms should be mentioned. Since spontaneous fluctuations of membrane potential and excitability may be present under certain circumstances, pathological derangement of the mechanisms underlying such spontaneous activity might play a role in the generation of the autonomous hyperactivity which characterizes the epileptic neuron. Furthermore alterations of local circuitry ( i.e. loss of inhibitory feedback) may be important. The possible role of denervation hypersensitivity of acetylcholine is discussed. Finally the possible role of metabolic factors is summarized. Although a body of data regarding central denervation hypersensitivity to acetylcholine has been accumulated, its role in the genesis of epileptic activity is as yet unclear. Insufficient data is available regarding the remaining possible mechanisms to permit any useful formulation of the role they may play in the genesis of the epileptic process.

Ill.

The Seizure

In the previous section, the interictal activity at the epileptogenic focus in the cortex has been described and some of the possible mechanisms presented which might be pertinent to the

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formulation of an hypothetical model of the epileptic neuron. It appears that abnormal activity is continuously and randomly occurring at the epileptogenic focus, yet an overt seizure appears only at relatively infrequent intervals. It is the purpose of this section to describe some of the events which transpire during a

FIG.8. Activity of epileptic neuron during propagated seizure. Time mark: 10 msec. (From Schmidt et al., 1959.) A. Increasing rate of soma discharge with high-frequency decremental bursts.

seizure and discuss some of the possible mechanisms which may play a role in precipitating the interictal activity into a frank seizure. Extensive studies have been made of the electrical activity gen-

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erated by graded response membrane during a seizure as recorded from the scalp by the EEG or directly from the surface of the brain in both man and the experimental animal. When one records from the cortical surface of the epileptic monkey, spontaneous seizures are seen to originate in the region of the cortical scar

FIG. 8. B. High-frequency decremental bursts continue superimposed on rhythmic slow wave activity.

and spread to involve homolateral as well as contralateral cortex (Kopeloff et al., 1942; Ward et al., 1948; Schmidt et al., 1959). As recorded with the ink-writing oscillograph, the seizure discharge is seen to resemble the familiar "tonic-clonic" sequence recorded in man.

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Microelectrode studies of single cell activity during such seizures provides additional insight into this phenomenon as reported by Schmidt and associates (1959). In many instances, the cell under

FIG.8. C. Seizure merges into “clonic” phase with massive, rhythmic, dendritic potentials.

observation may be involved in a characteristic fashion during the course of this seizure. Prior to onset of the propagated disturbance, spontaneous activity will be augmented. With the build-up of repetitive dendritic waves, this hyperactivity becomes

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extreme, culminating in a very high frequency discharge at rates to 1000/sec. Figure 8 presents successive samples recorded during the course of such a scizure. During the onset (Fig. 8 A ) there is a progressive increase in unit discharge which gradually

UF

FIG.8. D. Scizurc abruptly cmtls. In final trace first discliargc of cpilcptic neuron occurs 2 minutes aftcr c ~ i dof seizurc.

merges into a continuous high-frequency decremental firing ( Fig. 8B 1. This merges into the so-called “clonic” phase characterized by massive build-up and synchronization of “dendritic” waves (Fig. 8C) during which the unit discharge is often not recognizable. One can postulate that, at this stage, the soma is in a state

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of cathodal block as a consequence of the excessive depolarization. As the seizure ends (Fig. SD), clonic bursts slow and cease abruptly, to be followed by electrical silence during recovery. In the final trace of Fig. SD, the first spontaneous discharge of the unit has reappeared some 2 minutes after the end of the seizure. This slowly returns to preictal level of activity. The activity of the same cell has been recorded in this fashion tlirough the course of repeated seizures of this type without evidence of cell damage over a period of many hours. Other cells may fire continuously at high frequency during a seizure as seen in Fig. 9. Not all cells respond in this fashion during a seizure. In some, the seizure appears to start elsewhere and the cell under obscrvation is secondarily involved in activity of this type. At times, a cell under observation exhibiting high-frequency burst activity during the interictal period may cease to fire with the onset of the scizure and remain silent until several minutes after the seizure has ceased. The cortical standing potential has also been monitored during such spontaneous seizures in the monkey by Mahnke and Ward (unpublished data). During each seizure, a negative baseline shift of almost 3 mv develops after the seizure is well underway and this d.c. shift slowly progresses to peak value. It then shifts more quickly in the positive direction toward the original value as the tonic phase of the seizure ends, the rate of shift in the positive direction being greatest in the epoch containing tlic: end of the seizure. Goldring and O’Leary ( 1951) reported slow potential changes with seizures in rabbit cortex while Vanasupa and associates (1959) have described the d.c. potential changes which are characteristic of seizures produced by a number of convulsant drugs, and point out the significance of an abrupt negative shift as a sign of impending seizure. Since all the data would appear to indicate that an extraordinary synchronization and massive summation of graded response activity occurs during a seizure, the recorded d.c. shifts of appreciable magnitude may well play a major role in the production of such activity. In Section 11, E, possible mechanisms of dendritic synchronization were discussed in some detail including the evidence for tlie role of field effects in the generation of such phenomena. During tlie onset of a seizure, progressive recruitment of neuronal

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activity in the epileptogenic focus would appear to occlir. Altllough such local spread might b e the result of the local connections between cells (including axon collaterals ), certain aspects of the rhythmicity of the dentlritic activity (Fig. 8C) would not be easily accounted for on this basis. If local fields of sufficient inagriitude are generated during the onset of the fit, these might dl activate terminal axons of fibers entering the epileptogenic area. Synaptic activity generated by these elements would, in turn, result in depolarization of soma or further depolarization of dendrites This positive fecdback would, in these circumstances, become selfregenerative and might account for the irresistible progrvss of physiological events which transpire once a seizure has been initiated. However no objective data on this point are available. Even less is known regarding the mechanisms responsible for the cessation of a seizure. It has been assumed that seizures stop because of “neuronal exhaustion.” In partial confirmation of this concept, it is known (\Vard et al., 1948) that there is a profound fall in oxygen tension in cortex engaged in seizure activity. The drop in oxygen tension, which reaches a nadir after thr end of the clonic phase, may b e as low as can b e obtained by 3 minutes of inhalation of 100% nitrogen. This leads to tlie conception of the seizure as a sudden increase of metabolic activity so great that even the belatedly increased blood flow is inadcquate to sustain oxygen tension, and anoxic metabolism supervenes. The consequent accumulation of fixed acids and a depletion of energy stored in phosphate bonds is so great tliat maximal discliarges of neiiroiial groups, one into another, are no longcr able to excite it at all. At this point, the electrical seizure ceases but the cortex, loaded ~ i t hlactic acid, still consumes abnormal quantities of oxygen for tlie utilization of lactic acid and the eventual regeneration of its phosphate bonds. In this series of events, acidity itself m a y participate, since cortex rendered acid by any means shows an increased 1937). threshold (Dusser de Barenne ct d., It has also been suggested that, in addition to metaholic eshaustion, the seizure may b e actually quenched by an active inhibitory process (Jung, 1949). Since inhibitory internelirons have been demonstrated in cortex, this concept \vould be consistent with the suggestion of Branch and Martin (1958) that the arrcst of the discharge after electrical stimulation may also bc due to activation

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of inhibitory interneurons. One might even speculate that the positive shift of the cortical standing potential occurring at tlie termination of a seizure might represcnt summed inhibitory ( hyperpolarizin; postsynaptic) potentials. Szcniniary. During a seizure, studies of the activity of single neurons indicate that varying patterns of gross hyperactivity occur asscciated with the progressive recruitment of massive dendritic potmtial5. The possible role of field effects in the recruitment of local neuronal activity in the cpileptogenic focus is discussed. This woLld be consistent with tlie observation that there is progressive neg,ativity of the cortical c1.c. potential during the onset of the seiz-ire. ‘The cessation of the seizure may be the consequence of local metabolic exhaustion but active inhibition by involvement of inhibitory interneurons may also play a role.

IV. Propagation of the

Seizure

Once a seizure is initiated at an epileptogenic focus, it would appear that it spreads in two fachions. The first of these is a slow, creeping invasion of adjacent cortex; while the second type of propagation is rather rapid sprcad of the seizure discharge to more distmt portions of the brain. Since different mechanisms may be invcllved in these two types of propagation, they will be considered separately. ‘The slow spread of the sciziire discharge across the cortex has been extensively confirmed by clinical observations. Hughlings JacLson (1370) described this phcnomcnon in one of his patients: “his right hand began to twitch, the thumb and index finger taking the lead. . . . Next the whole arm twitched, but it did not rise; the exact sequence of involvement of its several parts was not ascertained, as the man was drcsscd. In about two minutes from the first, the right side of the face began to twitch . . . the right eye was closed, the right cheek clrawn up. . . . The time occupied was abolit ten minutes. Seven of these minutes were measured by the wat-h, the rest were guessed.” This is the march of the Jacksonian convulsion spreading down the motor cortex. Jackson recognized that the propagation of the seizure was slow since the spread of the

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seizure over a relatively few centimeters of cortex required a period of several minutes. It would be difEcult to conceive that such a slow spread through the cortical feltwork occurs as the result of conduction of the seizure discharge over rapid all-or-none fibers such as axon collaterals or even U-fibers. Electronic field effects might be involved in this phenomenon. Bremer (1941) has reported that synclironization of seizure discharges can occur across complete transections in the strychninized cord while Rosenbluetli and associates ( 1942) showed that a “seizure clonus” can be driven in an isolated portion of the cerebral cortex by repetitive stimulation of the main mass of cortex. Gerard and Libet (1940) have also shown that, in frog brain, seizure activity can be transferred from one region to another by simple contiguity. Although such field effects may play a role in the synchronization of the seizure discharge, they do not satisfactorily explain the slow propagation of the seizure across the cortex. However the phenomenon of spreading depression ( Leao, 1944) has many characteristics in common with the march of thc Jacksonian seizure. Marshall, in his elegant review of spreading depression (Marshall, 1959), points out that this phenomenon propagates acress the cortex at 2-3 mm per minute, is always associated with a slow change in the d.c. potcntial of thc cortex, and the “depression” phase of EEG activity may be replaced by various bizarre forms of hyperactivity (Bures and Buresova, 1960). The negative shift of cortical potential, the EEG changes, and the time course are also seen in epilepsy. Spreading depression (SD) is a fully graded reaction which occurs only in the presence of patlrology, and Marshall (1959) feels that this reaction may be a case in which the potential syncytial properties of dense neuronal interconnections and contiguity combine fortuitously with cellular lamination and dendritic organization to produce a propagated syncytial type of reaction, There appears to be no evidence of necessary invoh ement of specific transsynaptic activation reactions or circuit activity in the propagation of SD. Whatever the mechanism, it cannot be uniquely attributed to apical dendrites, because SD can be readily evoked on the dorsal hippocampus where basilar dendrites are oriented in the upward direction. SD can be evoked in suitable experimental preparations by electrical, chemical, and mechanical

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stimuli, by focal injury, or anoxia. It is also pertinent to point out that the propagation of SD and the accompanying epileptiform activity can be markedly depressed by various anesthetic drugs of which ether may be the most effective (Leao, 1944). Although ether is currently less fashionable in the treatment of clinical status epileplicus, there are some who feel it may be more effective than the bar'3iturate drugs. Ether is also more effective than barbiturates in depressing SD. As Marshall (1959) points out, a clear understanding of the unique phenomenon of spreading cortical depression is not yet available and thus any tantalizing association with such clinical entities as epilepsy must await further study. [n addition to the slow spread of the seizure discharge through the cortical feltwork, it is known that the seizure discharge may be widely propagated throughout the brain. Studies in the epileptic monkey by Ward et al. (1948) have shown that the epileptic discharge spreads to other cortical areas in the same and opposite henlisphere as well as to subcortical structures. They felt that this spread occurred only to those cortical or subcortical structures to which the discharging region sends axons or collaterals. They point out that the initial seizure discharge remains localized but, as the EEG potentials increase in amplitude and decrease in frequency, recipient areas begin to exhibit synchronous potentials which are usually initially positive. This regeneration of the fit in a distant region is clearest in the spread from one hemisphere to the other over well-known corticocortical connections. Any area in which such a disturbance has been generated has the same ability to provoke seizures elsewhere in the same manner in which its own seizure was provoked. The ability to do so does not appear to be lim ted to the cortex, and two local cortical seizures may have a subcortical intermediary. This accounts for those seizures which appear to arise ectopically at distant points. The role of the corpus callosum in the transmission of the seizure discharge to the opposite hemisphere has been studied by Erickson (1940) and Kopeloff et al. (1950), whereas the propagation of electrical after-discharge, which has many similarities to the epileptic discharge, has been investigated by Walker and his colleagues (Poggio et al., 1956) with particular reference to the subcortical distribution of suc? discharges. It thus appears that the spread of the seizure to other parts of the brain occurs over conventional pathways which

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interconnect these parts. It would also appear that certain circuits appear to be preferentially involved (Poggio ef al., 1956). This progressive involvement of additional circuits presumably accounts for the development of the generalized convulsion. The fact that the pattern of spread of the seizure is constant from seizure to seizure in many patients is clinical confirmation that certain patliways of spread are preferentially involved. Why this shoulcl be so is not entirely clear. This may be a property of the projections from the epileptogenic focus. It may also be that preferential spread is a “learnecl” property occurring as the result of repeated bombardments. Wada and Cornelius ( 1960) ha\xe presented experimental evidence to indicate that this may occur. They also reported that many of the subcortical structures they investigated developed, after a time, spontaneous paroxysmal abnormalities as well as hypersensitivity to various activating agents. The independence of such subcortical foci was also shown by their persistence after surgical removal of the cortical epileptogenic lesion. Thus “mirror foci” can apparently occur subcortically in much the same manner as in the contralateral cerebral cortex (Pope et al., 1947). However the development of cortical mirror foci in the monkey has been an inconstant finding in our laboratory, and it would appear to be relatively infrequent in the human. Nevertheless the fact that it occurs at all is of major importance to our understanding of the epileptic process. Few specific studies of this phenomenon have been undertaken, and much more controlled data is needed before it can be determined whether this plienomenon plays a significant role in human epilepsy. Summary. The spread of a seizure discharge from the epileptogenic focus can be postulated to occur in two fashions. The first of these consists of local spread through the cortical feltwork and may involve mechanisms in common with spreading depression. The second consists of propagation of the seizure discharge to other portions of the cortex as well as wide subcortical spread over conventional neuronal projections. It appears that the area in which such a disturbance has been generated has the same ability to provoke seizures elsewhere in the same manner in which its own seizure was provoked. In this way, the seizure can potentially be relayed throughout the entire brain.

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V.

181

Precipitation of Seizures

Little controlled data is available regarding the mechanisms in Jolved in the precipitation of a “spontaneous” seizure. Many have otlserved that, in the epileptic monkcy, sudden environmental stress \v 11 precipitate a seizure and evamples of such precipitation in the human are common. It is not known, however, how such stress precipitates the fit. Presinnably seizures are precipitated either by all eration of local mechnnisin~in the epileptogenic focus or by all eration of the properties of the circuits which permit the seizure to spread. I t is known that both EEG spikes and actual clinical seizures may occur more frequently during sleep. Even in the epileptic mlmkey with a focus in sensorimotor cortex, it has been oiir expc rience that many more spontaneous seizures occur during the night than during the day. \TWe and associates (1960a) have shown that the direct cortical response is augmented during sleep. In Section 11, B, the p o s d d c role of augmented dendritic activity in the generation of epileptogenesis was discussed. Thus the demonstrated increased responsiveness of postsynaptic membrane during sleep may account for the increased epileptic activity at such times. Variable alterations of metabolic factors may play a role in determining when certain seimres will occur. Although increasing metabolic rates by induced hyperthermia appears to have no effect ori cortical epileptogenic foci (Schmidt et a]., 1956), fever appears to precipitate seizures of suhcortical origin and such “centrencephalic” seizures also tend to be precipitated by alterations of pH induced by hyperventilation. Until more is known regarding the fundamental mechanisms involved in the production of the epileptic process in a cluster of cortical neurons, it is discouraging to try and study mechanism^ by which such activity can be precipitated into an overt seizure. VI. Concluding Remarks

It should again be emphasized that this entire discussion has been directed at seizures of focal cortical origin. Until we have a ckar understanding of the mechanisms involved in this relatively

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uncomplicated type of epileptic process, it would seem pointless to speculate on other varieties of epilepsy where our knowledge is even more restricted. It should be evident from this discussion that even the body of data dealing with seizures of focal cortical onset is discouragingly small. Thus, in the attempt to form hypotheses, much of the discussion has, of necessity, been speculative. The limited data has had to be reinforced by arguments based on analogy. In this fashion, an attempt has been made to construct a model of the epileptic neuron as well as models of mechanisms which might account for the precipitation of seizures and their spread. There should be no apology for the construction of models of this kind. The intention and the result of scientific inquiry is to obtain an understanding and a control of some part of the universe. Certainly no substantial part of the biological universe, such as epilepsy, is so simple that it can be grasped and controlled without abstraction. Abstraction consists in replacing the part of the universe under consideration by a model of similar but simpler structure. Models are thus a central necessity of scientific procedure. Scientific knowledge consists of a sequence of abstract models, preferably formal, occasionally material in nature. Material models start by being rough approximations, surrogates for the real facts studied. Let the model approach asymptotically the complexity of the original situation. It will tend to become identical with that original system. As a limit, it will become that system itself. That is, in a specific example, the best model for a cat is another, or preferably the same cat. In other words, should a material model thoroughly realize its purpose, the original situation could be grasped in its entirety and a model would be unnecessary. Quite clearly, in the field of epilepsy, we are at the stage of rough approximations. Concepts and hypotheses regarding epileptic mechanisms have been presented in this discussion in a somewhat dogmatic form, not in the effort to substantiate their possible validity, but to focus future observations which will either confirm or disprove them. It should also be apparent from this discussion that insight into a phenomenon such as epilepsy requires a complete and intimate knowledge of normal cortical physiology. At present, neither the function of the individual cortical elements nor their intimate morphological interrelationships are well understood. Thus we must be satisfied with relatively crude models of a pathological process such

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as epilepsy-models which will become more precise with increasing information. Thus the fascination of epilepsy is the search for a complete knowledge of the functioning of the brain-the organ of the mindand the most complex communication network ever to be devised. REFERENCES

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Erickson, T. C . (1940). A.M.A. Arch. Nerirol. Ps!/chiat. 43, 429. Eyzapirre, C., and Kufflcr, S. W. (1955a). J . Gen. Plzysiol. 39, 87. Eyzaguirre, C., antl Kiiffler, S. IV. (1955b). J . Gen. Ph!/,siol. 39, 121. Fatt, I-’. (1954). P/i!/siol. Rcos. 34, 674. Fntt, P. ( 1957). J . Weuropliysiol. 20, 27. I’orster, F. hl. (1951). J . Ncuroputhol. Expt/. Ncurol. 1, 98. Gastaut, H.,and Fisher-Williams, hl. ( 19.59). I n “IIandbook of Physiology,” Ncnropliysiology I ( J . Field, ecl.), 11. 329. \Villiams & \%‘ilkins, Baltimore, hlarylantl. Ccrard, R. W., ant1 Lihet, B. ( 1940). A i t ~J. . Psychicit. 96, 1125. Goldring, S., and O’Leary, J. ( 10.51 ) . J . Nerrrophysiol. 14, 273. Gowers, W. R. ( 1001 j . “Epilepsy and Other Chronic Convulsive Diseases: Their Causrs, Syinptoins and Tre;ltment.” J. ;md A. Chmchill. London. Gray, E. G . ( 1959). J . i h i t . 93, 420. Green, J. D. ( 1958 1. “Temporal Lolic Epilcpsy” ( hl. Baldvin antl P. Bailey, eds.), p. 581. C. C Thomas, Springfield, Illinois. Grundfcst, 11. ( 1957) . Physiol. Rcos. 37, 337. Jackson, J. 13. ( 1870). Troris. S t . Anrlrctcs. Afed. Grad. Assoc. 3, 1. Jnng, R. ( 1939). Arch. Psychiut. Neroenkronkh. 183, 206. Jung, R. ( 1953 j. Elcctroenccphulog. ond Clin. h’europ/i!/sio/. S u p p l . 4, 57. Kennarcl, h l . A. ( 1957). Neurolog!/ 7, 404. Kolmotlin, G . hl., and Skoglnncl, C. H. (1955). Actn Physiol. ScmtZ. 44, 11. Kopcloff, L. XI., Harrera, S. E., and Kopcloff, N. (1942). Am. J . Psyclzbt. 98, 881. Kopeloff, A’., Kennartl, hf. A,, Pacella, B. L., Koprloff, L. Lf., and Chiisid, J. G. ( 19.50). 4.Al.d. Arch. Ncarol. Ps!/chicit. 63, 719. Kristimsen, K., a n d Corirtois, C . ( 1949). E2ectroencrl)ltnlog. a r i d C h . Neurophysiol. 1, 265. Leao, A. A. 1’. ( 1 9 4 3 ) . J . Neurophysiol. 7, 358. Li, C. L. (19Fj9a). J. Neurophy~iol.22, 436. Li, C. L. ( l 9 5 9 b ) . Science 129,753. Li, C. L., and Chou, S. N. ( 1960). “Inhibitions of the Nervous System and y-Aminohutyric acid,” 1’. 34. I’ergainon, New York. Li, C . L., antl Jasper, H. (1953). J . Physiol. (Londolz) 121, 117. Libet, B., und Gerard, R. (1941 ). J . Netrrophysiol. 4, 438. Lissmun, H. W. ( 1058). J. Exptl. R i d . 35, 156. Loewenstcin, W.H. ( 1939) . Aiiu. N.Y. Acc~l.Sci. 81, 367. hlahnke, J. H., and Ward, A. A,, Jr. ( 1960). E x p t l . Weurol. 2, 31 I. hlarshall, W.H. ( 1 ) . Pli!/siol. Hetic. 39, 238. hlartin, A. K., and Branch, C. L. (1958). J. R i e ~ r ~ p / t y ~21, ~ i368. ~~l. Alatthews, B. I-I. C. ( 1 ~ 7 ) .Proc. no!/.Soc. B123, 416. .\Iorit:i, H. (1DFjO). J . Cellidor Cottip. Ph!/sioZ. 54, 189. ). A.Jl.A. Arch. Neurol. 1, 141. hlorrell, F., antl Florenz, A. ( 1!138). Electroenccphalog. and Cliii. Neurop/iy,sio[. 10, 187. hlonntcastle, V. B., Davics, 1’. \V., :ind Bcrman, A . L. (1 9 5 7 ). J . Neurophysiol. 20, 374.

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Nins, L. F., hlarshall, C., and Nielsen, A. (1941). Y a k J. B i d . Aled. 13, 477. Ntrnbcrger, J. I., and Gordon, hl. W. (1057). Progr. in Neurohiol. 2, 100. O’:;eary, J. L., and Coldring, S. (1960). Epilepsiu 1, 561. Pa ay, S. L. (1956). J . Biophys. Biochmn. Clytol. 2, 103. Penfield, W., and Jasper, 1%. 11. ( 1054). “Epilepsy and the Functional Anatomy of the Human Brain.” Little, Rrown, Boston, Xlassachusetts. Phillips, C. G. (1956). Quart. J. Exptl. Pliysiol. 41, 58. Poqgio, G. F., Walter, A. E., and Antly, 0. J. (1956). A.M.A. Arch. Neurol. Psychiut. 75, 350. Pope, A., hlorris, A. A., Jasper, II. H., Elliot, K. A. C., and Penfield, W. ( 1947). Research Pubk. Assoc. Heseurc/a Nerooiis Afentcil Diseuse 21, 218. Purpura, D. P. (1959). Intern. Rcu. Ncurdliol. 1, 47. Rall, W. (1959). Exptl. Nerrrol. 1, 491. Rose, J. E., and hlountcastle, V. 13. ( 1954). Bull. Johns Nopkins Hosp. 94, 238. Rosenbluetli, A,, Bond, D. D., ant1 Cannon, W. R . ( 1942). Ani. J. Physiol. 137, 681. SClkl, hf. ( 1952). J
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Wall, P. D. ( 1959). J. Neurophyyiol. 22, 305. Ward, A. A., Jr. (1952). Epilepsia 1, I. Ward, A. A,, Jr. (1960a). Epilepsia 1, 600. W’nrd, A. A,, Jr. ( 1961). Epilepsia 2, 70. \Yard, A. A , , Jr., and hlalinke, J. H. ( 1960). Trans. A n t . Nwrol. As.voc. 85, 93. Ward, A. A., Jr., and hlorlock, N. L. (1‘959). Neurology 9, 187. W-ard, A. A . , Jr., and Thomas, L. B. (1955). Electroenccphmlog. anti Clin. Neuroph!/siol. 7, 135. L\arcl, A. A,, Jr., hlcCulloch, W. S., and Kopeloff, N. (1948). J . Yeurophysiol. 11; 377. Ward, A. A , , Jr., Thtm~as,L. B., and Schmidt, R. 1’. ( 195.5). Fetlcratiun Proc. 14, 158. Ward, A. A , , Jr., Thomas, 11. P., and Schmidt, R. P. ( 1956). l’ran9. Am. N e r d . A.ssoc. 81, 41. White, J. C., l
FUNCTIONAL ORGANIZATION OF SOMATIC AREAS OF THE CEREBRAL CORTEX By

Hiroshi Nakahama

Department of Physiology, School of Medicine, Keio University, Shinjuku-ku, Tokyo, and Neurophysiology Section, Seishin-lgaku Institute, Itabashi-ku, Tokyo, Japan

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

11.

111.

1V.

1'.

VI. 1111.

Afferent Projections A. Ipsilateral Projection\ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Medial Lemniscal arid Spinothalamic ABcrcnts . . . . . . . . . . C. Functional 0rganiz;lt ioii . . . . , . . . . . . . . . . . Efferent Projections 4. Cortical Projcctioiis t o tlic Diencephalon . B. Cortical Projections to the Cranial Motor Lateral Tegmentuiii . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Cortical Projections to thc Solitary Nuclei D. Cortical Projections to the Gracile and Cuneate Nuclei and to the Dorsal Columir Nnclci , . , . . . . , . . . . . . . . ..... E. Corticoapinnl Pathways . . . . . . . . . . . . . . , , . . . . . . . . . . . . . . . 12. Movements of Cortical Stimulation and Ablation . , , , . , , . , Regulatory Mechanisms A. 'rhc Rcticular Formation . . . . . . . . . . , . , . , . . . . . , . . . . . . . . I3. The Unspccific Tha1;nnocortical Projection Systcm , . , . . . . . C. Sensorimotor Integraiion at Cortical Level . . . . . , , . , . . . . . Corticocortical Projections A. Cerelxoccrebellar 1ntc.rrelationsliips . . . , , . . . . . , , , , , . . . , . B. Callosal Intcrhemisph(~ricConnections , , . , . . . . . . , . , . . . . . C. Ipsilatcral Intercortical Connections Behavioral Conditioning Stiidics . . . . . . . . . . . . , . . . . . . . . , . . . . . Conclusions . , . . . . . , . . . . . . . . . . . , , . . . . . . . , , . . . . , , . , . . . . . , References . . . . . . . . . . . . . . . . . . . . . . . . . . , , , , . . . , . . , , , . . , . ,

I.

187 191 193 196

203 203 206 206 208 213

215 218 223 "25 230 232 236 240

2442

Introduction

A double sensory representation for the contralateral limbs in the cerebral cortex of cat was described by Adrian (1940, 1941). They were represented in tlic one area, which was located in the 187

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posterior sigmoid and coronal gyri from above downward: the hind legs, the trunk, the forelegs, aiid the face. In addition to this classic sensory area lie found another (t he second somatic sensory area), lying next to the former and located at tlie rostral tip of tlic anterior ectosylvian gyrus, in w7liich only the forelegs and tlie liind legs were represented. Tlic area of the hind legs was caiidal to that of the forelegs. W'oolsey (1933, 19-14), in a study of cat, dog, aiid monkey, found this second somatic sensory area present in all t h e e species, and, in addition foiind that face, proxiinal portion of limbs, ancl trunk were also represented. Further ( electropliysiological ) researches on rabbit ( Woolsey and Ff'ang, 1915) slieep a n d guinea pig ( \f7001sey and Fairman, 1946), inan (Penficld and Rasmussen, 1'350) cat (Knighton, 1950), marsupial ( Adey and Kerr, 1934), dog ( Pinto Hamiiy et nl., 195G), porcupine (Lende ancl Woolsey, 1956). aiid raccoon ( Welker and Seidenstein, 1959) proved the existence in these various forms of a second soinatic sensory area with sornatotopic arrangement. Since tlie anatomical and functional significmvx of these areas \vas not known, Woolsey (1937a) siiggestctl the terms somatic arcti I ( SI ) and somatic iireii I1 ( SII ) . Hc refcrred, i n cat, to the posterior siginoicl gynis as SI (limb) and tci the rostral region o f the anterior cctosylvian gyrus ;is SII ( liml) ) . J-Iowever, both tlie anterior and the posterior sigmoid gyri \vert' ternicd SI (limb) by Nnkahama and Saito (l956), since tliese gyri produce movements of tlic contralateral limbs ( AIcKibben and LVlicclis, 1932; Ward and Clark, 1935; Garol, 1942; Nakaliama cf (/l., 1956) and receivc impulses froin tlie afferent nerves of tlie contralateral limbs ( Nukahania and Saito, N56). Tlic posterior sigmoitl ~ y r i i s was referred to a s the posterior region of SI (limb) and the anterior sigmoid gyrus as the anterior region of SI (limb), since SI potentials evoked by ipsilatcxil SIT stimulation were divided into two categories of wave forms ( Nakahamn and Saito, 1956; Nakahama, 1960). The niesial surfncc of tlic frontal lobe anterior to tlie classic motor area (area 1) lias been suggested to elFect motor activity by Foerster ( 1936), wlio described synergistic niovcments produced by stimulation of this area. Penfield and \Yelch (1939) identified a similar area in inan and called it the siippleinentary motor area. This area has been further investigated in man ( Erickson and Woolsey, 1951; Penfield and Welch, 1951), chimpanzee ( Woolsey et al., 1952a), macaque (Penfield and U'elch,

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189

1951; Woolsey et nl., 1952b), a n d squirrel monkey (Welker et nl., 1957 ) . In these four forms, there is a topographical localization of motor function on the mesial cortex. \Voolsey (1955) has shown diagrams of the cortex of rat, rabbit, cat, and monkey, sliowiiig locations and general arrangements of the “precentral” ( XI1 ) and supplementary ( MII ) motor areas, “postcentral” sensory ( SI ) and second somatic ( S I I ) sensory areas, a n d visual (VI and VII) and auditory areas ( A 1 and M I ) . MII, however, has not yet been studied in rat and cat (Fig. 1). Kennard and McCulloch ( 1943) studied the electrical excitability of the postcentral gyrus some time after removal of Brotlmann’s areas 4 and 6 from infant monkeys, and found that it was still possible to intliice focal movements similar to those normally elicitable from t l i c prcccntral gyriis. Tf‘oolsey ct 01. ( 1953) demonstrated conclnsively the existence of a well-organized postcentral motor outflow aftcr complete degeneration of the motor pathways from both the precentral and supplementary motor areas of adult monkeys. Cliusitl et al. (1955) demonstrated that focal contralateral motor convulsions occurred in chronic monkeys in which the postcentral cortex hat1 been injectcd with alumina cream. These responses persisted after ablation of the ipsilateral precentral motor cortex, again indicating that the postcentral region has its own efferent connections to the contralateral musculature. In none of thcse studies, lio\vcver, had the second somatic area ( SII ) been removed. The topographical pattern in the postcentral area is a mirror image of that of the precentral area (Lilly, 1953; \l’oolsey ef- ul., 1953; \Velkt.r cf nl., 1957). This suggests that two topographically distinct motor syst e m exist in this region ( Welker et nl., 1957). It has been indicated in SII of cat (Woolsey, 1947a,h) and of monkey (Benjamin and Welker, 1957; Welker et nl., 1957) that the sensory and the motor patterns arc laid clown together and coincide somatotopically. The evidence that somatic afferents distribute to the precentral as well ;IS to the postcentral region has been demonstrated anatomically ( TT’alker, 1914 ) and by recording cortical potentials evoked by electrical stimulation of cutaneous 1947). nerves (Malis et nl., 1953) and dorsal roots (TVoolsey ct d., I n man, sensations persist on stimulation of the precentral gyms after postcentral resection ( Peiifield and Rasmussen, 1950; Penfield and Jasper, 1954) and frequently involve a “desire to move.” The

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precentral afferent fibers arrange topographically in a similar pattern to the precentral efferent fibers in the mediolateral direction ( Woolsey et d.,1947; Malis et nZ., 1953). The supplementary motor

RAT

RABBIT

MONKEY

FIG. 1. hfotor and sensory representations in the cerebral cortex of rat, rabbit, cat, and monkey. Each diagram shows locations 'and general arrangements of the postcentral ( S I ) and second ( S I I ) somatic sensory areas, precentral (MI) and supplementary ( h l I 1 ) motor areas, and t h e visunl ( V I and VII) and auditory ;areas ( A 1 and A I I ) . However, hlII has not yet been established in rat and cat. In the diagrani of monkey cortex, SII lies largely on the upper Imnk of the sylvian fissure adjacent to the insula and the auditory area on the lower bank (not illustrated). The anterolateral boundary of VI is illustrated by the thin line, with an asterisk placed at the center of the macular projcction area. ( From Woolsey, 195s.)

area ( M I I ) may also be involved in sensory function, since electrical stimulation in conscious patients has been found to induce sensations referred somewhat diffusely to various parts of the

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body, sometimes to both sides (Penfield and Rasmussen, 1950; Penfield and Jasper, 1954). From the date mentioned above, Woolsey (1958) proposed the following designations: somatic sensory-motor area I ( SmI) for the postcentral gyrus and its homologs in nonprimate forms; somatic sensory-motor area I1 (SmII) for the second sensory area; somatic motor-sensory area I ( M s I ) for the precentral motor area; and somatic motor-sensory area I1 ( MsII) for the supplementary motor area. The present review uses the nomenclature mentioned above as much as possible. Therckore the anterior region of SI corresponds to MsI; the posterior region of SI, to SmI; MII, to MsII; and SII, to SmII. SI includes both MsI and SmI. In various old papers quoted in this review, the words “motor area’’ and “sensory area” are often ambiguous giving no exact sites of the area referred to. In such cases, for expediency, the motor area is understood as MsI and the somatic sensory area as SmI in the present review.

II.

Afferent Projections

A. IPSILATERAL PROJECTIONS Nakahama ( 1958) discovered in cat the ipsilateral projection to SmI. This had never been found from tactile stimulation alone. HE carried out experiments which excluded transfer from the opposite cerebrum, from the cerebellum, and from the unstimulated side of the body through reflex mechanisms of the spinal cord. He emphasized that both Sin1 a n d SrnII have bilateral connections, which are stronger in SmII than in SmI, although good ipsilateral responses in SmII are well known since Woolsey and Fairman’s paper (1946). He also brought out some observations on the matter of blocking interaction between evoked responses, depending on whether or not they are evoked in areas of overlapping projection of the nerves concerned both with SmI and SmII and also depending on the laterality of the conditioning and testing shocks. These observations might be interesting for sensory discrimination. Employing the method of double simultaneous stimulation it has been found that man may give one of three types of responses.

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EIIROSHI RTAKAHAhlA

Depending on various factors in tlie test situation, tlie subject may (1) perceive and correctly localize both stimuli (expected normal) ; ( 2 ) perceive and correctly localize one stimulus but not perceive tlie other, even though the latter is perceptil )le to the patient on single stimulation (phenomenon of extinction ); and ( 3 ) perceive and correctly localize one stimulus and perceive but mislocalize the otlier ( phcnomenon of displacement) ( Render, 1945, 1948, 1951; Bender and Furlow, 1945; Bender and Teuber, 1946; Bender ef ul., 1949a, b, 1931; Jaffe and Bender, 1952; Fink et al., 19S3). Tlie occurrence of the phenomenon of extinction or displaccment definitely indicates that a sensation evoked by one stimulus is constantly influenced by other stimuli ( Bender, 1951). These phenomena might be explained by bilateral cortical representation in Sin1 and SmII, their overlap, and their mutual interference. Increased study of such phenomen:t, with both obscrviitions on man and experiments on animals, may lead to the discovery of sensory function from the vienrpoint of neural mechanism. Sites of blocking action to the cortical responses in Sin1 and SmII are considered at or/and be lo^ the level of the cerebral cortex. Amassian ( 195%) has shown that interaction at the thalamic level accounts for most of the cortical blocking and inhibition from peripheral stimulation; this finding supports Head’s concept ( 1920) of tlic: thalamus a s a great sensory integrating center, and this is true of even the relay nuclei. Recently Mountcastlc and Powell ( 19591)) have sliown by the physiological stimulation of the skiii of monkey that a few nciirons in SmI may be activated b y stimiilation of the ipsilatcral side of the body, and that they may be inliibitecl hy stimiilation of the contralateral side wlwn so driven by ipsilateral stimuli. These obscrvations arc quite in accorclance with those of Nitkahama ( 195s). Xlountcastle 11) have further indicated that these neurons have huge peripheral receptive fields; that the inhibitory receptive field may also be very large, and correspondingly located with respect to tlie ipsilateral excitatory receptive field; that these cells may be excited by stimuli which are clearly injurious to the skin; and that they may be inhibited by light mechanical stimuli delivered to exactly the same receptive field. These properties are strikingly different from all the other neurons of SmI (klountcastle and Powell, 1959b), and resemble in almost every way those of neurons

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of the posterior thalamic group in cat (Poggio and Mountcastle, 19,SS). This is interpreted to indicate a projection iipon SmI of tw70 quite different ( lemniscal and spinothalamic ) components of the somatic afferent system ( hlountcastle and Powell, 1959b).

B.

MEDIAL

LEMNISCAL AND

SPIKOTIIALAhlIC fiFFERENTS

Anatomically it is well known that the spinal component of the medial lemniscal system is formed by axons emanating from the cells of the spinal ganglia. These ascend on the ipsilateral side of the cord in the posterior column and synapse on cells in grlcile and cuneate nuclei. Thcw are also axons originating from thf3 cells of these nuclei, crossing ( a s far a s is known, entirely) to the opposite side and ascending in the medial lemniscus to end upon the cells of ventroposterolateral nucleus, ;IS well as axons that originate from tlie cells of the latter elcment and project upon SmI. The trigcminal component of the lemniscal system arises in the main sensory nucleus of the fifth nerve. The m.ain outflow of this nuclcus consists of axons crossing to the opposite side. The pathway adjoins mediodorsally tlie medial lenniscus, forms an integral part o l it, and terminates in ventropcisteromedial nucleus. l h e cells of the latter project, a s do the cells of ventroposterolateral, iipon Srnl ( see Rose and Mountcastle, 1S859). The spinothalamic system is considered as consisting of tv7o components. The first is thc spinothalarnic tract arising in the posterior liorns of tlic, spinal cord and the second is tlie spiiiothnlaniic tract originating in the spinal nucleus of the fifth nerve (the bulbothalamic tract) (see Rose and Mountcastle, 1959). While tlie lemniscal systcni pertains to tactile antl kinesthetic activity, the spinothalamic system transmits impulses provoked by painful and thermal stimuli h i t there is adequate evidence as well that some tactile impulses are also relayed through it (see R x e and Mountcastle, 1059). It is customary to distinguish within tke spinothalamic system of the spinal cord, although not in the bulbothalamic tract, a vcntral antl a lateral spinothalamic pathway. The first is assumed to conduct tactile impulses, the second is known to be important for arousal of painful and thermal sensations. In the dorsal portion of pulvinar-posterior system designated b y Rose and Woolsey (1958), the lateral sector is the pulvinar,

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IIIROSIII NAKAI-IAMA

while the medial sector is often referred to as nucleus lateralis posterior. They referred to the ventral portion as the posterior group of nuclei. It includes the suprageniculate nucleus and at least part of the region which some workers call the anterior portion of the medial geniculate body itself. A portion of the posterior thalaiiiic group receives a bilateral cutaneous afl'erent projection thought to be rclayed by the spinothalamic tract ( Whitlock and Perl, 1959; Poggio and Mountcastle, 1959). This projccts upon tile cerebral cortex, and may be the thalamic relay for SmII (Rose and Woolsey, 1958). It has been generally held that all the fibers of the spinothalamic tract originating in the cells of the posterior horn cross to the opposite side and ascend within the anterolateral column. However, evidence is accumulating that, in contrast to the medial lemniscal system, the spinothalamic system possesses an ipsilateral component from the body surface. This may terminate partly or wholly in the segment of the posterior thalamic group which adjoins the ventrobasal complex posteriorly. Rose and Woolsey (1958) showed that, if renioval of the auditory region was augmented by removal of SmII, the anterior portion of' the posterior group degenerated most severely; and that an isolated removal of SmII was without major effect on this group, whereas the preservation of SmlI, when the auditory region was removed, assured its relative intactness. It appcars then that the R X O I ~ S of this thalamic region have connections (probably of collateral nature ) with SmII. Observations made upon SmI cells by Xlountcastle and Powcll (1959b) and upon SmIl cells by Carreras and Levitt ( 1959) suggest that the posterior nuclear groiip projects upon both SmI and SmII, \vhich is relevant with its sustaining nature. The ventral thalamic nuclear group is divided by Rose and Mountcastle ( 1952) into three complexes: the ventrolateral, the ventromedial, and the ventrobasal. The ventrolateral complex comprises the ventroanterior and ventrolateral nuclei with their subdivisions, and is known to receive the main thalamic inflow of the superior cerebellar peduncle and to possess connections with the cortical motor system. The ventromedial complex is made up of the ventromedial, the interventral, and submedial nuclei. Its connections and its projections to the cortex are not so well known. The ventrobasal complex consists of the external ( ventropostero-

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lateral) and the arcuate (ventroposteromedial) nuclei, and has been known for a long time as the posterior portion of the ventral nuclear group in particular, which is the thalamic synaptic region for the medial lemniscus, spinothalamic, and quintothalamic tracts (Rose and Mountcastle, 1952). It is well establishcd that the medial lemniscus passes upward through the brain stem to the ventrobasal complex of the dorsal thalamus ( Ranson and Ingram, 1932). Recently it has been found that the ventrobasal complex niiclei receive their afferent inflow predominantly, if not exclusively, from the contralateral cutaneous nerves, ascending through ventrolateral funiculi of the spinal cord, which also send axons to the posterior thalamic region ( Whitlock and Perl, 1959). Recently, hlacchi et nl. (1959) havc used the method of the retrograde ch~generationafter ablations of SmI and SmII in cat. They found ( 1) that the ventrobasal complex projects to both SmI and SmII covering the posterior sigmoid gyrus, the posterior lip of the coronal sulcus, and the antcrior ectosylvian gyrus, and ( 2 ) that certain numbers of cells belonging to the ventrobasal complex depend essentially on Sin1 for their thalamocortical connections, arid other neurons in this complex depend likewise on SmII. In addition to a focal retrograde degeneration in this complex, a pxt i a l overlapping of the thalamic degenerated areas was seen after cortical removal which involved different portions of SmI arid SmII. These overlapping areas indicate that discrete parts of the ventrobasal complex project to SmI as well as to SmII. Their results are not in agreement with Knighton’s (1950) investigations in which he concluded, from direct stimulation of the ventrobasal complex, that only the caudal tip of the ventroposteromedial nucleus represents the thalamic relay for SmII, as far as can be determined from his drawings. Rose and Woolsey ( 1958) pointed out, however, that Knighton was probably activating SmII quite often by stimulation of the anterior portion of the posterior group. Another disagreement was presented by Rose and Woolsey (1958) in that the isolated removal of SmII did not cause a significant degeneration of neurons in the ventrobasal complex. A reinvestigation of these discrepancies is necessary. These findings, however, seem to support Woolsey’s (1947a, b ) view that the afferent volleys to SmI and SmII arise over different pathways. I t was also found that SmII receives projections from

196

HIROSHI NAKAHAMA

the anterior portion of the posterior group of the pulvinar-posterior system (Butler et al., 1957; Rose and Woolsey, 1958). A topical distribution of the projection fibers from the ventrobasal complex to SmI was confirmed by Knighton ( 1 9 3 ) and Macchi ct al. (1959). The anterolateral portion of the thalamic relay area was linked with the more medial sector of the posterior siginoid gyrus and the posterometlial part of the ventrobasal complex with the more lateral sector of tile sigmoiclal gyrus and the postcrior lip of the coronal S U ~ C U S ( XIacchi ct al., 1959). These anatomical data are in agreement with the findings of Rose and Mountcastle (1952) who observed that the anterolateral part of the vcntrobasal complex was activated by tactile stiinulation of the contralateral leg whereas the medioposterior portion of the ventrobasal complex was activated by tactile stimuli in the cephalic region of the body. Physiological investigations on the cortical ;ireas by means of evoked potentials indicate that the legs arc represented in the posterior sigmoicl gyrus whereas the head is represented chiefly in a postcoronal c:ortical area (Adrinn, 1940; Marshall cf al., 1942). In Sin11 it is conceivablc that there exists also a topical organization of the thalamocortical connections, since ablntions of different portions of SmII produce retrograde degeneration in distinct areas of the ventrobasal complex ( M x c h i ct al., 1959). I t is believed that a topographically organized projection s!.stem constitutes the substrate for spatial discrimination. A small portion of the ventrobasal arciiate niicleus has been shown to project to MsI (face area), which receives a tactile projection from the mouth, and which possesses n distinct granular layer ( Krugcr and Porter, 1958 ) .

C. FUNCTIONAL ORGANIZATION Mountcastle and Powell (1959b) made a study of macaque SmI neiirons activated by stimulation of the skin using the method of single unit analysis. They present evidence that the excitatory peripheral receptive ficld for a cortical neuron is surrounded by a belt of skin within which stimulation will inhibit the cell 1 inder study (Fig. 2 ) ; that inhibition w a s not seen under deep anestliesia; that inhibition appeared with increasing incidence as the anestlietic state lightened, shon7ing that the central relays subserving in-

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hi bition are qualitatively tliiferent from those concerned with excitation; and that cells closely adjacent to one another in the ccrtex are reciprocally re1atc.d to appropriately located cutaneous st:.muli. The evidence places particular emphasis upon the role surrounding inhibition p h y s in sensory discrimination. The clisposition of peripheral inhibitory fields for cortical cells surrounding their excitatory fields has also been observed in cat SmI by M.ountcastle ( 1957). His interpretation of this phenomenon was

FIG. 2. Spntinl relation of tlic, c,\citatory and inliil,itory cutaneous rccept:ve fields playing np(m six I I V I I Y O I ~ S of monkey SmI. In four ctiscs tlic. inhibitory fic.1~1 s1irroumtls conrpl(,tt.ly; in tlic other tww the inhihitory ficlcl surr ~ u n d spartially. ( From hIoiiiitca\tle airtl l’owell, 19591).)

that a single stimulated point wjll inhibit the surrounding cortical c.ells, and that the reciprocal ovcrlap of their central inhibitory z.ones will sharpen and limit their discharge zones as the two stimulated peripheral points approach each other, contributing certainly to the double peak of cortical activity, which is assumed to be necessary for two-point discrimation. I t has been shown that the response patterns of the first order afferents activated by joint rotation are divisible into two groups, those which adapt rapidly, and those which adapt slowly (Boyd

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IIIROSHI iVAKAHAMA

and Roberts, 1953, Andrew and Dodt, 1953; Skoglund, 1956). The quickly adapting elements occur very rarely and have becn tentatively correlated with the Vater-Pacini corpuscles ( Boyd and Roberts, 1953). In monkey SmI quickly adapting cortical neurons sensitive to joint rotation have been similarly observed by Mountcastle and Powell (1959a), again much less commonly than have slowly adapting ones. They also showed that the latter discharge rapid onset transients, with decline to steady adapted rates diuing steady joint positioning, just as slowly adapting receptors do; that the level of that steady rate is determined by joint angle, and is independent of the speed or direction of the movement bringing the joint to that angle; and that movement of a joint away from the excitatory direction causes a decrease in the discharge rate of such a cortical cell, and closely adjacent cells respond in a reciprocal manner to a given movement. They support the hypothesis that this class of cells of SmI, together with the relevant peripheral receptors, afferent nerve fibers, and relay neurons, constitutes the essential neural substratum of proprioceptive and kinesthetic sensation. This is contrary to the widely held belief that kinesthesis depends as well upon afferent input from muscle stretch receptors. Direct stretch of muscle evoked no detectable response in SmI (Alountcastle et al., 1952). Nor have single unit analysis studies reve'iled any cells at thalamic or cortical levels responsive to muscle stretch ( Mountcastle, 1957; Mountcastle and Powell, 1959a). Afferent volleys confined to group I afferent fibers from muscle (which innervate the annulospiral endings and the Golgi tendon organs) evoked no response in SmI (,l/lountcastle et al., 1952). Some differences of opinion exist as to whether group I1 afferents (which innervate the flower spray stretch receptors) and group I11 afferents or only groiip I11 myelinated afferents from muscle project upon the somatic sensory cortcx ( Mountcastle et al., 1952; McIntyre, 1953). These differences are probably due to the different muscle nerves used, for it is known that some fibers from joint receptors, periosteum, and deep fascia, may travel in some muscle nerves and are of group I1 size (Gardner, 1944; Skoglund, 1956; Stilwell, 1957~1, b, c ) . The peripheral endings of group I11 afferents are thought to be bare ncrve terminals and there is no evidence that they are sensitive to mechanical changes in muscle. It seems safe to assume that these endings

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are of no significance for position sense (Rose and Mountcastle, 1959). It is clear that miiscle stretch afferents are unlikely to play a role in position scnse for they are under control of the gamma efferent loop and may discharge over their full frequency range at any muscle length (Rose and Mountcastle, 1959). For further details of the function of the gamma efferent-spindle afferent loop, readers are refcrred to Granit (1955). Mountcastle et nl. (1952) showed with the method of gross electrode recording that receptors in and about joints project to SmI and SmII of the contralateral cerebral cortex and to SmII of the ipsilateral hemisphere through the ventrobasal nuclear complex of the thalamus. They also indicated that the afferents from bones and joints form together with afferents from cutaneous receptors a common topographical pattern. It is quite possible, as pointed out by Mountcastlc and Powell (1959a), that the receptcr organs of the joint capsules and pericapsular tissue play a major role in position sense, together with their afferent projections into the dorsal columns, their related medial lemniscal and tl ialamic ventrobasal relay neurons, and that class of nerve cells of SmI upon which neural activity set up by joint rotation finally impinges. The method of single unit analysis clarified that at each successive relay and in SmI itself neurons activated by these joint receptors are arranged in topographical patterns mutually interlocked with those representing the cutaneous sensory sheet ( \lountcastle, 1957; Mountcastle and Powell, 19594. Mountcastle and Powell ( 1959a) indicated that cortical neurons activated bv joint rotation are inhibited by stimulation of the skin, although the physiological significance of this interaction is obscure. Clark and Powell (1953) have indicated that each of the three subdivisions of the monkey SmI, described by Brodmann ( 1905), possesses differential afferent connections from the ventrobasal complex of the thalamiis. They studied the retrograde cellular degeneration and atrophy in this thalamic nucleus resulting from cortical lesions confined to either area 3, 1, or 2 of Brodmann. They indicated that area 3 receives connections from cells over the entire rostrocaudal extent of the nucleus, area 1 receives the exclusive projection from the cells of the caudal half, and area 2 receives the eiclusive projection of no thalamic cells. Area 2 seems to receive only a collateral outflow from areas 3 and 1. Electrophysiological

200

HIFiOSHI NAKAHAMA

experiments of Powell and Mountcastle (1959b) showed that the architectonic subdivisions of SmI also have a functional significance. The cells of area 2 are almost exclusively related to tlie deep tissues of tlie body, but more anteriorly, in areas 1 and 3, progressively increasing proportions of' cells are related to the skin. Their finding that this change is a graclual one without abrupt steps fits well with that of Powell and Mountcastle (195%) who indicated that the divisions between three cytoarchitectural areas are regions of gradients of morphological change, rather than sharp lines. Tliis is one functional expression of the very clear cytoarchitectural differences of SmI. Mountcastle (1957), in an elegant study using the method of single unit analysis of cat SmI, found that the neurons encountered in a perpendicular traverse of the cortex were sensitive to stimulation of either the skin or the deep tissues, and that elements of the two classes were not indiscriminately mixed, supporting the hypothesis that the neurons lying in narrow vertical columns extending from layer 2 through layer 6 make up an elementary unit for function, and that those vertical columns related to various submodalities of sensation are arranged in a mosaic which in sum makes up the cortical projection pattern. This hypothesis was fully supported by Powell and Mountcastle (l959b) for areas 1 and 2 of monkey SniI. For area 3 the collection of wholly convincing data was prevented by technical difficulties, but they believe that it holds for that region also. They cited the observations of Sperry (1947) and Sperry et al. (1955) who showed that cats with subpial vertical dicing of the cortex on a l-mm grid are still capable of making very fine visual discriminations, and that monkeys in which the sensory and motor cortices have been treated similarly are still capable of what appeared to be normal motor activity; emphasizing that these observations show very clearly that rather small vertical columns of cortex are capable of integrated activity of a rather complex order, which is independent of lateral intragriseal spread of activity. Splanchnic afferent representation was demonstrated in the trunk region of contralateral SmI and SmII of rabbit, cat, dog, and monkey and an ipsilateral representation mas in SmII of cat (Amassian, 1951; Ruch et nl. 1958). Within the area of SmII a third auditory area was found to exist in dog (Tunturi, 1945; Pinto

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Hiimuy et nl., 1956), in addition to the areas homologous to auditory area I and I1 of Woolsey and Walzl (1942). Since the studies of Allen (1945) on dog and of Meyer and Woolsey (1952) on cat are in substantial agreement, Rose a i d Woolsey ( 1955) concluded that SrnII can b e shown to possess auditory functions. Mickle and Ades ( 1952) demonstrated that projection of the auditory, vestibular, arid tactile system overlaps within a region which partly includes SrnII, suggesting that their projection is direct from the thalamic relay nuclei, and that this area plays a part in the maintenance of posture and in spatial orientation. It is apparent that information cclncerning the orientation of the body in space and of the spatial relations between its parts depends upon afferent inputs from both scmatic sensory and vestibular receptors as well as from the visual arid auditory apparatus. Bremer ( 1958), Bremer et nl. ( 1954), Per1 and Casby (1954), and Buser and Heinze (1954) reported that potentials evokecl by auditory and tactile stimuli in SmII or in the cortex adjacent to it can be shown to interact in such a manner that it must be concluded that some auditory potentials in SmII indeed originate within this field (Rose and Woolsey, 1958). Rose and Woolsey's ( 1958) demonstration that SmII receives sustaining projections from the anterior portion of the posterior thalamic nuclear group is in harmony with all these findings. This portion of the group is intercalated between the ventrobasal complex and the medial geniculate, which makes it an ideal place for tactile and a iditory interconnections ( Hose and Woolsey, 1958). Thompson and Sindberg (1960) mapped cortical response fields of cat under c'hloralose anesthesia to click auditory stimulation. The fields were not only on the middle suprasylvian and anterior lateral gyri but on NsI. Although some degree of response was in SmI (leg) , the fociis of activity was in hlsl ant1 the bulk of SmI w7as silent. Indirect evidence regarding the subcortical pathways for these res-3onse systems was provided by thc chronic ablation data (Thompson and Sindberg, 1960). ,411 focal responses were present and normal after ablation of aiiditory area I, auditory area 11, postlxior ectosylvian area, SmII, and the insulartemporal regions b'ilaterally. Retrograde degeneration studies with the Nissl method indicated that direct projection from the medial geniculate body and nucleus posterior was completely excluded. However, the significance of these fields for the functioning of the nervous system is mknomn.

202

HIROSHI NAKAHAMA

Electrical stimulation of the chorda tympani in cat evoked a potential within tlie facc somatic area ( a localized area on the orbital surface of the cerebrum just rostra1 to the tip of thc anterior ectosylvian gyrus ) ( Patton and Amassian, 1952). Bilateral destruction of this region in rabbit and rat caused impairment of taste (Brcmer, 1923; Bciijamin and Pfaffmann, 1955). Removal of thc face som‘itic area in monkey and chimpanzee had no rffcct on quinine preference thresholds ( Bornstein, 1940b; Patton ct al., 1946). Studies on monkey showed that the parainsular and insular cortex must bc included to obtain severe deficits in quinine preference thresholds (Ruch and Patton, 1946; Bagshaw and Pribram, 1953). In man with bullet wounds of the inferior SmI there was reduced gustatory and tactual sensibility of the tongue ( Bdrnstein, 1 9 4 0 4 . Gustatory sensations were elicited in conscious human patients by electrical stimulation of the lower end of SniI ( Penfield and Boldrey, 1937). hlicroelectrode studies showed that cortical cells in the tactile tongue area of cat respond only to tactile, thermal, or gustatory stimuli applied to the tongue (Cohen et nl., 1957; Landgren, 1957a, b ) . Other cells in this area showed convergence of tactile, thermal, and gustatory impulses ( Landgren, 195% ) . The taste cells appeared to be less chemically specific than the single afferent fibers for they responded to almost all types of gustJtory stimulation ( Cohen et nl., 1957). Landgren ( 1960a) showed in enckphale isoli. cat that the thalamic relays in the tactile path from the tongue are grouped together within ventroposteromedial nucleus and that they were not interspersed in the neighboring facial relay nucleus. It was also demonstrated in similar preparations that tlie majority of “cold cells” in the thalamic ventroposteromedial nucleus did not respond to any other physiological stimulus than cooling of the tongue ( Lantlgren, 1960b). For stimulation and ablation studies on cerebral somatic x e a s conccrned with pain, readers are referred to a recent review ( Sweet, 1959). Ill.

Efferent Projections

The recent remarkable development of the Nauta and Gygax stain ( 1954 ) for specifically impregnating degenerating axons allows satisfactory identification of degenerating axons including their end-ramifications. The Nauta-Gygax method apparently has

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an advantage over other silver techniques, in that it will stain the degenerating axoplasm of large and small, myelinated and unmyelinated fibers. Many excellent investigations have been made by means of this method during the past several years. The studies are mainly concentrated in the output distribution from the cerebral cortex, although the input projections must also be traced by this method. Further studies with the Nauta-Gygax method combined with electrophysiological techniques will lead to a much more coinplete understanding of the output and input organization of the central nervous system.

A. CORTICAL PROJECTIONS

TO THE

DIENCEPHALON

Auer (1956) studied the cortical efferents to the diencephalon with the Nauta-Gygax technique for degenerating fibers of passage an3 terminals, 5 to 6 days after the ablation of both MsI and the gyms proreus of cat. The degenerating fibers were directed toward the ventrolateral nucleus and the ventrobasal complex through the lateral part of reticular nucleus. The head of reticular nucleus had the greatest concentration of terminal degeneration. The lateral part of the dorsomedial nucleus as well as the adjacent intralaminar nuclei presented degenerating terminals. The center median nucleus and the lateral part of the central nucleus received degenerating terminals. The subthalamus had terminal degeneration in the zona incerta and the subthalamic nucleus, appearing to be a thoroughfaie for fibers of passage to the midbrain reticular formation and probably also to the center median nucleus. The hypothalamus was characterized by terminal degeneration and degenerating fibers of passage in the medial forebrain bundle. Little degeneration was observed in the ventromedial, the mammillary and the posterior hypothalamic nuclei.

B. CORTICAL PROJECTIONS TO THE CRANIAL MOTORNUCLEI AND LATERAL TEGblENTUhl

THE

The existence of cortical fibers to the spinal trigeminal complex and the lateral tegmentum were reported in monkey by h4ettler (1935), Nisino (1940), and Kuypers (1958b), in cat by Brodal et nl. (1956) and Kuypers (1956, 1958a), and in rat by Torvik ( 1956). Recently, Kuypers ( 195%) presented very interesting

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IIIROSIII NAKAHAMA

findings with the Nauta-Gygax silver technique. I n monkel, the cortical fibers to the spinal trigeminal complex proper are priinarily SmI projection, while MsI projects primarily to the lateral tegmentum and the motor nuclei; and the projection to tlie motor nuclei arises predominantly in the caudal parts of MsI (face area), adjacent to the central sulcus, while the fibers to tlie lateral tegmentum have a more diffuse hIsI origin. In chimpanzee, i n contrast to monkey, the caudal parts of MsI (face area; approximately arca 4 ) project almost exclusively to the motor nuclei, \vhile the fibers to the lateral tegmcntum originate from rostrally adjacent cortical areas, together with additional fibers to the motor niiclei. Furthermore the volume of the direct corticonuclear connections was noticeably l q e r in chimpanzee than in monkey ( Kiiyiers, 195%). In man a similar distribution of degenerating fibers to the trigeminal complex ancl tlie adjacent lateral tegmcntiim was found, and a slightly more massive, direct corticonnclcar projectioii appears to exist (Kuypcrs, 1 9 5 8 ~ )Conversely, . hlsI projection to the lateral tegmentum decreases in volume in chimpanzee as coml)~ircd with monkey (Kuypers, 1958b). In cat cortical connections to the motor nuclei were almost completely lacking, and it is siispcacted that the corticifugal impulses reach the cranial motor niic1c.i indirectly through one or more synapses in the spinal trigeniinal complex or the lateral tegmentum, or both ( Kuypers, 1 9 5 8 ). ~ These observations indicate that during phylogenetic clevelopment the almost exclusively indirect corticonuclear connections of lower animals were augmented in primates by direct corticonuclear projections ( Fig. 3 A ) , which apparently began to arise especially in the caudal part of tlie field of origin of tlac indirect connections ( Kuypers, 1958b ) . These direct connections subsequently incrcmed in number at tlie expense of thc indircct projections ancl begnn to concentrate heavily in the more caudal part of MsI (Fig. 3 B ) . The existence of direct and indirect corticonuclear connections is substantiated by electropliysiological findings of a similar nature in regard to the spinal cord, reported by Uernliard and Hohm ( 195421, b, c ) . On the other hand, these authors also expected a phylogenetic decrease of the ipsilateral direct corticomotoneuronal connections. However, in the brain stem the opposite appears true, since from monkey to man more and more direct projections to the ipsilateral trigeminal motor and facial nuclei were found. This,

205

SOhlATIC AREAS OF TIIE CEREBRAL CORTEX

however, could conceivably be a characteristic of only the cranial components of the segmental inotor apparatus (Kuypers, 1958b ). Kliypers (195Sb, c, d ) suggcstcd that SmI fibers to the trigeminal co nplex, which arise from the somatosensory cortex proper, subseive primarily a “sensory” function by influencing the threshold

FIG.3. A. Diagram showing the difl‘erenccs in corticifugal clonnections in cat and man. In man thc iiidircct corticonuelear connections are augmented by a massive system of direct corticonuclcar projcctions, while in cat only thc indirect connections are denionstratecl. ( From Kuypers, 1958c. ) B. Diagram illustrating tlic cortical origin of thc projections in cat, monkey, and chimpanzee. Direct connections conccntrntc in the caudal part of MsI; 0 : projec tions to the lateral tegmentiiin of pons a n d mcdnlla oblongata, projection to the motor nuclei, x: projection to the spinal trigeminal complex. (From KLypers, 195%. )

m:

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HIROSHI SAK.\IIA1\IA

of primary sensory synapses (Hagbarth and Kerr, 1954), and that MsI projections to the motor nuclei and the lateral tegmentiim, on the other hand, are likely to subserve more truly “motor” functions. The indircct corticonuclear pathways are consequcntly most likely reprcsented by the fibers to the lateral tegmentnm rather than by the cortical fibers to the trigeminal complex. This is consistcmt with the observation that the lateral tegmcntuni constitutes the uninterrupted rostral continuation of the zona intermedia and the basal parts of the posterior horn in the spinal cord, which contaiii the majority of the spinal internuncial elements ( Lloyd, 1941) . C. CORTICALPROJECTIONS TO

THE

SOLITARYNUCLEI

Cortical projections to rostral parts of the solitary nuclcxis and their vicinity bilaterally was found in rat by Torvik ( 1956), in cat by Broclal et 01. ( 195G), in monkey by Kuypers ( 1958b), a i d in . experiments ( Kuythe human brain by Kuypers ( 1 9 5 8 ~ )Monkey pers, 1958b) suggested that cortico-solitary fibers ( a t least in part) originate in the most ventral part of MsI, in the vicinity 01 the inferior precentral dimple, possibly including parts of the adjacent opercular cortex. In view of their termination these fibers can be classified in one category with Sin1 projections to the spinal tri5eminal complex and the posterior horn (nucleus proprius) of the spinal cord. Since the rostral part of the solitary niicleus seeins to receive primary sensory taste fibers (Lewis and Dandy, 1930; Torvik, 1956), it could be inferred that the area from whicli the cortico-solitary fibers originate subserves specifically the perception of taste (Kuypers, 1958b). This view is supported by the observation of taste deficits in monkey following ablation of the most ventral part of MsI together with the opercular cortex and a part of the insula (Patton and Ruch, 1946; Bagshaw and Pribram, 1953). D. CORTICALPROJECTIONS TO THE GRACILEAKD CUNEATENUCLEI AND TO THE D O R S A L COLUhfN NUCLEI The degeneration of a cortical pathway to the gracile and cuneate nuclei v7as first described by Redlich ( 1897) in cat following extensive cortical damage. Walberg (1957), using Glees’ ( 1‘346) silver method, described terminal dc>generation in these nuclei in cat after removals of both Msl ancl SmI. However, the cJxternal

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nucleus was free from degeneration. The corticifugal fibers to the dorsal column nuclei descend with those of the pyramidal tract, some of them leaving the pyramid at the medullary level and entering the medial lemniscns from which they finally reach the nuclei, while other fibers appear to pass through the reticular formation of the medulla on their way from the pyramidal tract to the nuclei ( Walberg, 1957). With the Nauta-Gygax method Chambers and Liu (1957) observed after the ablation of either both MsI and SmI or only of SmII that the corticospinal tract te minates mainly in the contralateral gracile and cuneate nuclei. Following subtotal lesions of both hlsI and SmI in cat it was found that the leg area appeared to be the prime source of the projection to the area of gracile nncleus, ~ h i l ethe arm area appeared to project to the region of cuneate nucleus (Chambers and Liu, 1937; Kuypers, 195Sa). An analogous arrangement was found in monkey MsI, although cortical fibers from the arm area of MsI to the region of cuneate nucleus are less iiiiiiieroiis than those from the leg area to the region of gracile nucleus (Kuypers, 1958b). Monkey SmI appears also to send numerous fibers to the region of these nuclei ( Kuypers, 195Sb). The fact that fibers from different cortical areas (MsI, SmI, frclntoparietal, occipital, temporal, medial, and basal regions of the cerebral cortex) converge on the dorsal column nuclei points to these nuclei as being structures subserving a certain integration of va:*ious kinds of impulses (Walberg, 1957). This assumption may also be applied to all divisions of the spinal and principal trigeminal sensory nucleus and the niiclcus of the solitary tract, since corticif iigal fibers to these nuclei, like those to the dorsal column nuclei, we're derived from various parts of the cerebral cortex (Broclal ct d., 1956). The anatomical fintlings indicate that the cerebral cortex, particularly SI, must be able to influence the central transmission of impulses via the dorsal column nuclei, the trigeminal nucleus, and thc: nucleus of the solitary tract. This effect must be exerted on serisory messages coming from a wide variety of receptors (Walberg, 1957 ) . Some electrophysiological studies in curarized cat are in agreement with this assumption. Hernhndez-Pkon and Hagbarth (1955) observed a depression of an afferent test volley in the sersory trigeminal nucleus following repetitive SI stimulation. HernBndez-Pebn et al. (1956) described depression or abolition of

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the postsynaptic potentials in the gracile niicleus evoked by single shocks supplied to the corresponding dorsal column following SI stimulation. It appears entirely possible that these effects m a y be mediated via the anatomically demonstrated cortjcifugd fillers even if a transmission Jlong corticoretieulonucle~~r patlnva>;s cmnut be cxcludecl.

E. CORTICOSPINAL PATHWAYS It was found in cat with the Nauta-Gygax method that only two pathways originate from both MsI and SmI, a large crossed lateral corticospinal tract and a small uncrossed ventral corticospiiial tract, and that the latter is confined to the upper cervical segments, crossing in the ventral white coinmissure before entering the spinal gray matter ( Chambers and Liu, 1957). Data obtained by tlie hlarchi method (Boyce, 1595; Swank, 1934; Tower, 1935) and hy silver techniques ( FVa11m-g and Brodal, 1953 ) makc it highly probable that an uncrossed lateral corticospinal tract exists. Since it is very small, it is difficult to see this pathway with the iise of the Marclii (Chiarugi of crl., 1955) and of a silver technique (SzentiigothaiSchimert, 1941) . \Valberg and Rrodal ( 1953) dcscribed four spinal pathways for the corticospinal tract of cat, a crossed and uncrossed ventral corticospinal and a crossed and uncrossed lateral corticospinal tract. However, four similar pathways were not found following both MsI and SmI lesions or the pyramidal section, wliereas they were found following SmII removal in cat ( Cham1,ers and Liu, 1957). All four tracts from cat Sin11 contain few fibers, h i t the crossed lateral and uncrossed ventral contain the most ( Chambers and Liii, 1957). Chambers and Liu ( 1957 ) s h o ~ e dthat unilutcral Smll removal resulted in mild degeneration in the spinal cord of both sides, definitely more 011 the contralateral side. The degeneration was found in a11 segments from C1 to S2. They also indicated that thc corticospinal system originated from cells in the sigmoid, coronal, and anterior ectosylvian gyri, and that no evidence was found for an origin from the parietal, occipital, and temporal areas. It seems definitely reasonable tliat few if any corticospinal fibers originate in the cortex other than from the sigmoid area, coronal gyrtis, and anterior ectosylvian gyrns. These findings are in close agreement

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wit li the results of the electrophysiological investigations which indicated that largest cortical potcntials, responding to antidromic stimulation of medullary pyriiniid, occurred in hlsI, SmI, and SmII of monkey, cat, and rabbit (\Voolsey and Chang, 1948; Lance and Manning, 1954; Porter, 1955). However, Landau ( 1956) observed that antidromic potentials evoked by the bulbar pyramid stimulation could bc recorded only from the pericruciate area, chiefly from Ms.!, containing the Retz cclls. He suggested that the wider site of origin of the pyramidal fibers, reported by others, is cluc to a diffuse recording of the antidromic potentials and to the recording of dromic potentials from the inadvertently stimulated medial lemniscus. He also expressed tlie opinion that the antidromic method is 1:mitetl to the detection of the cortical origin of only the large fibers (cells) of the pyramid tlnc to the limitation of recording in a vohime conductor. The histological method of Nauta-Gygax is not subject to the above limitations, and is superior to the antidromic method for determining the eoiinections of the system. Topographical organization in corticospinal tracts of monkey and rat was stutlietl by Barnartl and Woolscy ( 1956) with the Marchi method following carefully controlled discrete cortical lesions. SmI fibers s h o m d a lateral disposition in relation to hls1 fibers throughout the bra n stem, but below the p\mmidal dccussatioii no differenccs could be found. Within h,IsI projection there were soinatotopic 1occ.lizatioii in the internal capsule and cerebral peduncle. Leg fibers \irere situated most caiitlally in the posterior limb of the caps&, with fibers for distal ;irm and proximal arm located more rostrally in that order. At pontiiic levels localization became less distinct so that in the medulla 110 separation was fouiid except that facc: area fibers were concciitrnted a bit medially and dorsally, doubtless because the face fibers were leaving the pyramid. No IocJization could be seen in the spinal cord although there was evidence of variation from onc animal to another. Chambers and Liu (1957) were the first to visualize clearly the distribution of fibers to the apex of the posterior horn and the degeneration in the medial portion of the base of the dorsal horn by the Nauta-Gygax method following removal of both hlsI and SmI or SmII. Their hist'slogical findings of the collateral distribution and the pericell ~ l a arborizations r in the basolateral area, around the large cells of the posterior horn (nucleus proprius) and the cells of the inter-

210

I1IROSIII N.\KAIIA;\Z:\

mediate region, agree with the physiological observations of Lloyd (1941) as to thc spinal connections of the pyramidal tract i n cat. His experiments were performed on the lumbar region. The corticospinal activation of motoneurons in the cervical region was also found to be of internuncial order in this animal (Bernhard et al., 1953a). In monkey, Kuypers (1958~1)showed that MsI lesions produced degeneration of corticifugal fibers which were primarily distributed to the intermediate region and the anteiior horn of the spinal cord, and that Sin1 lesions, on the other hand, produced degeneration of corticifugal fibers distributcd primarily to the posterior horn of the spinal cord. Ho\wver, some overlap bctween h1sI and SmI projections seems to exist, eslxxially in the leg area. Thc SmI projections to the posterior horn of the spinal cord and the spinal trigeminal complex probably constitutes a “sensory” feedback mechanism capable of influencing secondary cell groups, from which SmI ultimately receives at least part of its information (Kuypers, 19581). cl ) . Sucli a cortical influence upon secondary sensory cell groups in the spinal cord was demonstratcd physiologically in curarized cat by Hagbnrth and Kerr (1954). This influence was the depression of the afferent response evoked by a dorsal root stimulus during the stimulation of R;IsI, SmI, or SmII. Electrical responses of the corticospinal tract were studied in cat and monkcy by Patton and Amassian (1954a). Responses recorded through an electrode inserted into the bulbar pyramid or lateral column of the cervical spinal cord following orthodromic stimulation of MsI, Sml, or SmII consisted of a stable early positive deflection ( D wave) followed by a series of later positive deflections ( I wave) of variable latcncy and configuration. They concluded that the D wave results from direct excitation of pjmmidal neurons whereas the I waves result from indirect or relayed excitation of pyramidal neurons via cortical interneurons which are arranged in closed chains permitting recirculation of impulses and repetitive bombardment of pyramidal neurons. In cat, Patton and Roscoe (unpublished observations, cited by Patton and Amassian, 1960) found that stimulation of the leg and arm subdivisions of MsI, SmI, and Sin11 (limb), respectively, produced the D wave which indicates the existence of corticospinal projections. Direct pyramidal responses from the face area are presumably excluded by bulbar recording. In cat (Patton and Amassian, 195421) and

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monkey (Patton and Amassian, 1954b), the major projection zone is MsI; SmI stimulation evoked a pyramidal response which is doininated by the I waves. For a more extensive discussion on the pyramidal tract, readers are referred to Lassek et nl. (1957) and to Patton and Amassian ( 1960). Bernhard and Bohm (l954b) referred to the fraction of the co1ticospinal fibers which activates the motoneurons directly ( i.e., monosynaptically) as the coi ticomotoneuronal system. When the existence of this system was found electrophysiologically in monkey, they thought that there might be a relationship between the existence of the corticomotoneuronal system and the special pattern of the muscular activity which monkeys, primates, and humans develop when moving their hands and fingers. The difference in the movement pattern between an animal with paws and an animal with hands concerns not only the activity of the muscles serving the digits but also that of all the muscles of the extremities. A monosynaptic system would be more apt to serve movement patterns in which there is a great demand on the regulation of the aclivity in restricted muscles or muscle groups, as is the case in an extremity equipped with a hand, since in such a system there are a priori fewer possibilities of functional divergence than in a polysynaptic system (Bernhard and Bohm, 195413). The pyramidal system is much more developed in monkey than in cat, and inclL,des both a ventral and a lateral pyramidal tract. In this connection Bernhard et al. (1953a, b ) made experiments on monkey, in which MsI was stimulated and the action potentials were recorded from the peripheral nerves in the extremities and from veqtral roots in the lumbar region. They concluded that spinal motoneurons in the lumbar region can be activated monosynaptically by descending volleys in corticospinal fibers. The monosynaptic motoneuron response to MsI stimulation was also shown to be distributed to nerves both to proximal and distal muscles of the fore and hind limbs. The monosynaptic activation of the spinal motoneurons could only be produced by stimulation of the cont r a lateral subdivisions of MsI which correspond to the innervation fields, and no monosynaptic response from peripheral nerves was obtained by stimulation of premotor, SmI, and MsII (Bernhard et al., 1953b). These authors showed in monkey that monosynaptic activation of spinal motoneurons could be obtained by

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ipsilateral MsI stimulation only after transection of the lateral pyramidal tract (in the cervical region) contralateral to the stimulated cortical area, indicating that pyramidal or extrapyramidal inhibitory influences were eliminated by the transection. In this connection they draw attention to some classic investigations ( Wertheimer and Lepage, 1897; Hering, 1899; Probst, 1905) whiclz indicate that, in monkey, movements produced by ipsilateral cortical stimulation appear more easily when the contralateral movements are prevented from occuring. This ipsilateral monosynaptic activation of the motoneurons may be mediated by uncrossed corticospinal fibers either in the anterior column or in tlie lateral column of the spinal cord. In monkey MsI, Bernhard and Bohm (195413, c ) sliowed that single cortical shocks do not elicit any monosynaptic response in the spinal Inotimeuxons. Repetitive stimulation liad to be used, and during the course of stimulation the monosynaptic responses were built up in a regular way. It was shown by them that the repetitive cortical stimulation builds up a tonic asynchronoiis discharge and a long-lasting facilitatory action which successively raises the excitability of motoneurons; that when this facilitatory effect reaches a certain level, a monosynaptic response to the descending volley in the corticomotoneuronal fibers breaks tlirough; and further, that the cortical field from which facilitation of a certain group of niotoneurons could be evokcd has a greater extension in the anterior direction than the cortical field from which a monosynaptic response in the samc group of motoneurons could be elicited. I t appears that monosynaptic discharge of motoneurons hy pyramidal volleys occurs only on a background of facilitation provided by bombardment through more complex pathways. Among the different frequencies tested, those between 20 and 25 per second were found by them to be most effective for the building up of the monosynaptic response. Therefore they used these frequencies for the mapping experiments, indicating that the cortical representation of tlie corticomotoncuronal system for one muscle is more restricted than that of tlie system which is responsible for the late responses (probably polysynaptic), and that the 1.‘1 t e responses are evoked in tlie same and other motoneurons with a wider peripheral distribution. The observation in monkey, that stimulation at one cortical point activates a much more restricted

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group of motoneurons by direct action, is different from the indication in cat that a descending volley set up by stimulation at a cortical point is bound to evoke widespread muscular activity, owing to the divergence in polysynaptic rclay systems (Bernhard and Bohm, 1954b). This difkrence might be of great importance in the organization of the spinal integration mechanism of the cxticospinal system. A reciprocal arrangement of the monosynaptic responses in the nerves to tlie two antagonistic muscles of curarized monkey could l)c elicitcd by cortical stimulation in the absence of influence from the muscle receptors (Bernhard and E’ohm, 1954b, c ) . This indicates that a reciprocal arrangement to antagonistic muscles from hIsI exists without afferent back responses from the muscles tliuing tlie contraction. Such a peripheral influence had been excliidecl by Sherrington (1893) on the cortical activation of the eye' muscles. Excluding the proprioceptive inflow is necessary wlien attacking this problem, since Gellhorn and Hyde (1953) found that the initial position in which an extremity is placed or held has a large influence on the pattern cf response to stimulation of any cortical site and also on the map cf motor cortex responses.

1’.

MOVEMENTS OF CORTICAL STlhlULATION AND

ABLATION

Movements obtained by stimulation of cortical somatic areas have already been discussed. More detailed descriptions of MsI stimulation and ablation are available in a recent review (Terzuolo and Adey, 1960). Unilateral excision of hlsII in man yiclded no permanent impairment of posture or movement ( Penfield and Welch, 1949, 1951), hut only a transient and moderate hypertonia (Penfield and IVelch, 1.951). Penfield and Welch (1951) showed that the grasp reflex is a sign of MsII injury in unilateral lesioiis in monkey. Erickson and Woolsey ( 1951) reported a weak and short-enduring grasp reflex after unilateral involvement of MsII in man. Travis (1955) confirmed these results showing in the contralateral limbs of monkey after a unilateral lesion of MsII a moderate bilateral hypertonia at the shoulders h i t no noticeable paresis. He also clemon5trated that MsII is a bilateral system, since unilateral lesions produced minor deficits, whereas, simultaneous bilateral removals

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HIROSHI NAKAHAMA

yielded more prominent effects on posture and tonus. Hopping and placing reactions persistently disappeared in the affected extremities following unilateral MsI and MsII ablation ( Travis, 1955). Stimulation of monkey MsII produced movements of the contralateral somatic musculature which were of different type from the ones obtained by MsI excitation; they were slower, more tonic, more “postural.” These movements could be elicited even after excision of MsI in monkey and, in man, they were obtained after JlsI and SmI ablation (Penfield and Jasper, 1954). Moreover, Bertrand ( 1956) showed that monopolar stimulation with single shocks in monkey MsII evoked a response of the pyramidal type in both ipsilateral and contralateral corticospinal tract. The presence of the D waves in the pyramidal tracts, on both sides of the cord, after one MsII stimulation suggests that MsII projects directly into the pyramidal tracts in a manner similar to MsI. As in the case of MsI, the crossed group of fibers is the most important, but MsII seems to contribute a larger proportion of direct fibers to the ipsilateral corticospinal tract than MsI does, since the higher amplitude of the D waves and the wider distribution of the responses were recorded in the lateral column of the spinal cord (Bertrand, 1956). However, anatomical studies ( Nauta-Gygax method) have not disclosed any degenerating fibers in the medullary pyramids or the spinal cord following ablation of a large part of monkey MsII ( D e Vito and Smith, 1958, 1959). MsII projection fibers passed down the internal capsule and terminated in the reticular formation of the upper pons and in the pontine nuclei at the lower border of the pons. These projections were ipsilateral. The thalamic projections were relatively minor and consisted mainly of fibers with small diameters, which were found in the ipsilateral center median and intralaminar nuclei ( D e Vito and Smith, 1959). These anatomical data suggest that MsII contributes to regulation of movement through a multisynaptic pathway rather than through a direct corticospinal system. In the light of these anatomical findings it has been shown that the D wave recorded from the pyramidal tract disappeared before the I waves when MsII was stimulated, while the I waves disappeared first when MsI was stimulated (Smith et d.,1958). This suggested that no element of the corticospinal tract arose from MsII. Spread of stimulating current to MsI is probably responsible for the D

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waves after MsII stimulation ( Smith et al., 1958). No neurological deficits were found in monkcy after bilateral SmII lesions and no impairment of performance on peanut slide, problem boxes, or life-saver bar was detected (Pinto Hamuy, 1956).

IV.

Regulatory Mechanisms

A. THERETICULAR FORMATION The brain stem reticular formation was identified as the primary agent in the maintenance of the waking state (Moruzzi and Magoun, 1949; Lindsley et ul., 1949, 1950; French and Magoun, 19551; Magoun, 1953, 1954). The arousal function, discovered by Moruzzi and Magoun (1949), is best known, and is concerned witk the transition from sleep to waking. The area implicated in conveying such “arousal” information to the brain was occupied principally by the reticular formation and related thalamic nuclei; hence these structures became kiio\vn as the “reticular activating system.” Recently, Rose and hIountcastle ( 1959) have presented indi -ect evidence that the reticular activating system is activated mainly or solely through the aiiterolateral columns and the tracts arising from them rather than through the posterior column and the medial lemniscal system. Reticular neurons are considered to be intricately linked togetlicr by midtisynaptic relays (French et al., 1952, 1953a, b; Starzl et al., 1951a, b ) . Frontal corticoreticular fibcrs, more particularly alleged to come from MsI, have been describcd in human material with Marchi method by Hoche ( 1898), D6jerine ( 1901), Sand ( 1903), and some other authors. Among the first expcrimental studies relevant to this subject are those of Simpson and Jolly (1907) in monkey. In Marchi preparations these workers found degenerating fibers leaving the pyramid and entering the contralateral bulbar reticular formation after crossing in the raplie. The fibers are given off along the entire extent of the medullary pyramid. Mettler (1935, 1945) found many fibers from monkey MsI passing through the reticular formation to reach tlic facial, ambiguous, and hypoglossal nuclei. Levin (1936) reported similar findings and emphasized the bilateral distribution, with predominance contralaterally. Verhaart and Kennard (1940) also described these fibers, but maintain that

216

HIROSHI N A I U I i A M A

the corticobulbar fibers are very scarce. Minckler et al. (1944) commented upoil the reduction in the number of descending degenerating fibers at levels between the midbrain and the spinal cord in a study of human and monkey material with tlie Marchi and Weigert methods. In R4archi and Weil sections from hemidecorticated rats Combs ( 1949) describing degenerating fibers passing from the pyramid to the bulbar and pontine tcgmentum, and Escolar (1950) found, in cat, degenerating fibers leaving the pyramid and passing to the medial reticular formation of both sides. Krieg ( 1954b) distinguished different contingents of fibers, passing from MsI to the reticular formation of the lower brain stem. With the Nauta-Gypx technique Kuypers ( 1956) indicated in cat MsI that corticotegiiiental connections, largely diffuse and contralateral in the medulla, are especially abundant with the magnocellular part of tlie lateral reticular nucleus, while corticotegmental projections are largely ipsilateral in the pons. With the Glees method Rossi and Brodal (1956) showed in cat that corticoreticular fibers originate from widespread areas of the cerebral cortex, but the majority come from SI, particiilarly A M . Smaller contributions arise from the medial and basal surfaces and only very few come from the temporal and occipital regions. The connections are crossed and uncrossed, with some prepontlerance of the former. They further indicated that the terminal regions of fibers from difl‘ercnt cortical areas are identical, and that scattered fibers terminate throughout the length of the reticular formation, but there are two regions which clearly stand out as major terminal stations: one was located in the Iateral pontine tegmentnm, and tlie other resided in the medulla near Olszewski’s nucleus reticularis gigantocellularis ( Olszewski, 1954). With the evoked potential method, French et 01. (1955) identified and studied tlie extent of the several cortical arcas which project to the brain stem reticular formation of monkey. These loci were found to be situated in sensorimotor cortex (SmI, MsI, and probably SmII, MsII, in the writer’s judgment of their figure), frontal oculomotor fields, paraoccipital region, anterior cingulate gyrus, and superior temporal lobe. Stimuli applied to each of these zones could be recorded throughout the extent of tlie reticular ascending activating system, including the intralaminar and midline tlialamic nuclei. The area of projection of each locus was

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generally coextensive with that exhibited by other cortical zones, and no pattern of response could be elicited which characterized individual cortex-brain stem relationships. French et al. ( 1955) further indicated that it has been possible to record, at a single electrode placement in the reticular formation, potentials evoked by stimulation of multiple cortical loci and many sensory pathways. Such potentials of cortical and peripheral origin were found

FIG. 4. Lateral view of monkey brain which denionstrates corticifugal projections and collaterals of classic afferent projections converging on reticular formation of lower brain stem. (From French et al., 1955.)

to interact, generally occlusively, in the brain stem. These data suggest that sensorimotor (SmI, MsI, and probably SmII, MsII) projection to the reticular formation converges with the outputs from other various cortical areas as well as with the afferent inflows from peripheral receptors within the reticular activating system. Figure 4 illustrates that both corticipetal and corticifugal connections interrelate the reticular formation of the brain stem and the cerebral cortex, and each of these parts of the brain can markedly influence the activity of the other. Recent studies indi-

catc that tlic fiinctional interrelations of t h e two portions ot the brain iria\~lw significantly involved in conditioiicd learning ( see \~lagoiiii,1958). I t is of grcxt iniportance that scmsorimotor cortex ( SmI, and proliably Smll, \Is11 ) , a s well a s other cortical areas descrllxd above, concerii mechanisms throrigh which the cciitral ncrvoi~s system is able to inotlrilatc or control its own scnsory input. StimuIntion of the reticular forination cstcwsivcly modificd the discharge cc:ntrifugal impulses directly rate of miiscle spiiicllc,s, ant1 Iic~ncc~ iirfluenccd proprioccption ( Grauit and K a a d a , 1952). Stiiii~dation of not only the rcticiilar formatioii h u t SI in1iil)itetl the conduction within the tlorsal coliinin o f thc spinal cord ( H-Iigbarth antl Kerr, 1954 ) and witliin tlic glacile iriicleiis ( Sclicrrcr antl Herniindezl’ebn, 1958) . Excitation applied to tlich cortical loci tlescribetl iiiclutling tlie rl~iriencc~plialoi~, which are known to project to the reticular activating systcin, coiiltl eithcr inliiliit or facilitate ceplialically dirc,ctcd conduction within the reticular formation itself ( Adry et d,, 19.57). T h e r c h r e thc cortcs plays an important role in the control of seirsory concltiction witlrin the ncm’oiis s!,steni. It scenx prol)alile that in this way tlic cortcs is able to alter tlie state of coiisciousnws antl plays ;i rolc in tlic proc’sscs of slcep : ~ n dawakening and in tlic state of alertiicss. S\-s.rE:.\[ B. THEUNsrw:im: TIIAI,Ah10~:01il.I(:AL I’IIOJEC:~.IOS The unspecific tlialamocortical projrction system, which is sometimes called the thalamic rrticular systcwi ( 1aspcr, 1954 ) , constitutes the rostra1 portion of the ascending reticular acti\rating system of tlir 1)rain strm ( Alagoun, lLS0, 1953). The. anatomy and tlistinct pliysiologicd propcities of the thalamic rcticular s>.steni were first described Iiy Ilenipscy and \lorison ( 1942, 1943), nnd Xlorison and Llempsev ( I942 ) . l m v freqtiency stimulation of a thalamic system c-vokod widely tlistributcd, high voltage, cortical nxves, characterized b y long latcncics a r i d initial progrcssi\,e voltage incrt:meiit. Attcntioii \vas tlra\vn to the closc resernblancc of these recruiting responses to spindle Imrsts, occurring spontaneously i n lxirbiturate anesthesia. I n contrast. stimulation of specific ponsm in only known thalamic nuclei produced short latency local cortical B ~ P ; I S which did not affect the spontaneous cortical

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rhythms. I t seemed that these two effects must be mediated by separate thalamocortical pathways: ( a ) the well-known specific projection system with a more or less point-to-point arrangement; ( b ) a secondary nonspecific system with diffuse connections. In cat, the thalamic distribution of the unspecific projection system w;is found to be the center median and intralaminar nuclei (Morison and Dempsey, 1942), as wcll as the ventralis anterior and rostra1 pole of the reticular nucleus (Starzl and Magoun, 1951 ). These component nuclei act as a functional unit, since on repetiti1.e stimulation of any one of them recruiting waves could be recorded from all (Starzl ant1 Rlagoun, 1951). These nuclei interrelate their activities at the thalaniic level (Starzl and Magoun, 1951; Starzl and Whitlock, 1952). Starzl and Magoiin (1951) showed that the principal transmission of recruitment to the cortex occurs through connectioiis with the thalainic association nuclei ( medial, anterior, lateralis complcx, and pulvinar ), although possibly some direct connections also exist between the recruiting nmlei of origin and the cortcs. Hanbery and Jasper (19Fj3) studied cortical responses to local electrical stimulation of the unspecific thalamocortical projection system in cat under fairly deep Ncmbutal anesthesia. Recruiting responses were obtained most constantly from the anterior regions, cspecially gyrus proreus and hIsI. The responses were also obtained from sensory receiving areas, more commonly from SmI. Tl e independence of the unspecific projection system from specific t l d a m i c nuclei has been pr0\7cd b y recording recruiting responses from each cortical area, following complete destruction of the thalamic nucleus known to have specific connections with a particular cortical area (Hnnbery and Jasper, 1953). Complete destruction of the somatosensory nucleus (ventroposterolateral nucleus) did not prevent Sin1 recruiting responses when the unspecific projection system ~ v a s stimulated. Recruiting responses were also obtained from hIs1 aftcx clestruction of vcntral lateral nucleus, its specific projection nucleus. Jasper ct nZ. (1955) showed that projection to sensory areas is most labile and frequently produces lower voltage recruiting responses than obtained simultaneously in nonsensory areas. This was thought to be due to competition between afferent impulses arriving over the specific and unspecific projection systems. This thinking has been supported

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by the demonstration that destruction of the sensory relay nuclei of the thalamus greatly facilitated the amplitude of recrniting responses in their respective areas of cortical projection (Jasper, 1958,1960). Li et al. (1956) showed that the surface negative recruiting response, when analyzed with microelectrodes at varioiis depths of the cortex in SmI, was inverted in many instances to become a deep positive wave, out of phase with the surface negative response. This was a gradual change and most commonly observed at a depth of about 0.6 to 1 mm beneath the surface, suggesting that the unspecific afferent terminals distribute to the more superficial cortical layers, comparable in this respect to the late surface negative wave of tlie specific evoked potential complex. The rr:cruiting response also showed the similar intracortical distribution as does the spontaneous alpha rhythm ( L i ct ul., 1956). Individual neurons which fire with short latency following a single shock to the sensory relay nucleus of tlie thalamus are not fired by stimulating the unspecific thalainic projection system. Conversely, those neurons which fire with unspecific stimulation, in the recruiting response, cannot be fired directly by a volley from the sensory relay nucleus. There is, however, interaction betwecm the two projection systems. The excitability of these specific cortical cells can be modified so that they show increased firing to the same specific volley if paired with an earlier unspecific conditioning volley. These data indicate that the aff crent terminals from the unspecific system must have a difierent distribution and effect on the matrix of the cortex than do the terminals from the specific projection system; and that interactions must occur by intcrrelations between neurons in the cortex which are separately activated (Jasper, 1958). From XlsI long latency surface negative recruiting responses were recorded. These were shown to be elaborated chiefly in the depths of the cortex itself, since an earlier short latency negative wave was found to be present when recording with a microelectrode at a depth of about 0.5 mm beneath tlie surface whcre unit discharges were also found to initiate the surface negative wave ( L i et d., 1956). However, in SmI and in other cortical areas an initial early deep negative potential was not seen. This raises the question as to whether or not the short latency responses, particularly in MsI, are due to simultaneous

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stimulation of specific and unspecific projection fibers ( L i et al., 1956). Recruiting responses, increasing in voltage with repetitive stimulation at a critical frequency, are associated with repetitive di.jcharge of certain neurons on the crest of each negative wave, while other neurons which may be spontaneously active show facilitation of discharge cluring a recruiting wave and inhibition during the interval between, suggesting a kind of regulatory action of recruiting waves (Li, ef o/., 1956). In the motor system, Jaspcr (1949) showed that cortically induced movements could be facilitated markcdly by rapid stiniulation of the intrulamiiiar system of the thalamus. Whitlock et al. ( 1953) devised the “pyramidal” cat by transecting tlic mesencephalon exclusive of tlic ccrebral peduncles at the intercollicular level. With this unanesthetizcd animal, which exhibits spontaneously the characteristic EEG and behavioral patterns of sleep, Arduini and Whitlock ( 1953) recorded spike discharges from 34.~1and from its eff’ercnt projections. They showed that the pyramidal discharges are drivcn by low frequency stimulation of tl: e unspecific projection systcin, and that corticifugal volleys occur diiring each recruiting response ant1 follow the stimulus artifact af’ter latencies of 10-12 nisw (primary discliarge) and 2 M 0 msec (secondary discharge). The recruiting responses in MsI were also correlated with intracortical spike outbursts which tended to be subdivided into primary and secondary groups. Brookhart and Znnchetti (1956) failed to show any effect of recruiting waves in AlsI upon the activation of pyramidal cells, as recorded by means of electrodes directly in the tract at the level of the decussation. These discrepancies might be due to the utilization of a response n.hich was a mixtnre of recriiitinent and augmentation. Repetitive stimulation of specific thalamic nuclei at frequencies between 6 and 12 per second gives rise to local incremental responses. These were named “augmenting r c q ~ o n s e s ”by Dempsey and Morison (1943) and Morison and Dcnipsey (1943). The necessity for using extreme care in making the cliff erentiation betw-een augmenting and recruiting responses cannot be overcmphasized. Under certain conditions it is possible to show a remarkable degree of independence between these responses in the same area of the cortex (Brookhart and Zanchetti, 1956). The augmenting responses are not as closely related to the spontaneous spindle bursts

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as are recruiting waves (Morison and Bassett, 1945). The augmenting responses are probably more closely related to the neuronal systems of the local sensory after-discharge ( Chang, 1950). When the augmenting responses are fully developed, they usually consist of a large short-latency positive deflection, followed by a large negativity, which generally results from the merging of two initially separate negative potentials. The augmenting responses evoked in MsI by stimulation of the thalamic nuclei spccifically projecting thereon (ventralis posterolateralis, ventralis posteromedialis, ventralis lateralis) were associated with synchronous pyramidal discharges, clearly related in time to the surface positive deflection of the cortical potentials (Brookhart and Zanchetti, 1956) as u7as shown also with peripheral soniatosensory stimulation (Amassian et nl., 1955). Such cortical augmenting potentials could be either controlled or even blocked by sensory and reticularly produced arousal, the late negative component of the response sliowing greater susceptibility than the positive deflection ( Gaiitliier ct al., 1956). Stimuli capable of decreasing the negative cortical waves without affecting the positive potentials did not influence the synchronous pyramidal discharges; besides, these did not follow the waxing and waning of the more labile negative auginenting potentials (Parma and Zanchetti, 1956). When, on occasion, thalamic stimiilation failed to evoke aiigmciiting responses, inducing only l u g e positivities not followed 1)y clear and well-developed negative components, these responses could he however associated with large synchronotis pyrainiclal discharges ( Parnia and %anclietti, 1956). It was suggested that activity in pyramidal neurons and in thcir associated cortical interneiirons is related to the surface positive component of the auginenting potential ( Brookhart and Zanchetti, 1956; Parrna and Zanchetti, 1956). \\'hitlock et ul. ( 1953) observed, by recording spike discharges from single units of MsI and the pyraniidnl tract, that the spontaneous pyramidal discharge of their anesthetized preparations was completely blocked during arousal prodiiccd by sensory or dienceplialic stimuli. Since their data were obtained from a limited sample of pyramidal neurons, it could hardly be applied to the population of cells of MsI. For this reason, Zanchetti and Brookhart (1958) reinvestigated this problem in cat MsI with a method which revealed changes in responsiveness in the population of cells giving origin

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to the corticospinal tract and in their associated internenrons under various conditions. It was concluded that the unknown cortical mechanisms activated by the change from the slecping to the aroused condition of the EEG did not involve an alternation in the state of corticospinal neurons or their associated cortical interneurons.

INTEGRATION AT CORTICAL LEVEL C. SENSORIMOTOR Mott and Sherrington (1895) clarified the contribution of muscular and cutaneous sensation to purposive movement of a limb in monkey, showing that a completely deafferented limb was virtually paialyzed and that tlie ability to grasp was eliminated. However, if only one dorsal root distrihting cutaneous sensation to any part of the hand or foot remained intact, little motor deficit occurred. They further indicated, by differential section of certain roots of the animal, that if cutaneous afferents to the hand alone were intlct, the muscle affereiits lwing sectioned, little impairment of function resulted. Whereas if cutaneous afferents were sectioned with muscle afferents intact, the Iiand was useless. Similar results were obtained in monkey by T\vitcliell (1954), who re-examined the effects of deafferentation to determine precise operational mechanisms of limb afferents in piirposive movements. He also pointed out that “the cortical components of movement are dominant and are directed by contactual stimuli.” This may also be suggested by Nakahama and Nakamura’s ( 1959) observation that the action potentials from tlie peripheral nerves of a forelimb cvnked by the stimulatiori of MsI, SmI, and SmII, respectively, were facilitated following a prccetling stimulus to the cutancons nerve of the forelimb. Similarity between the motor deficit resulting from MsI ablation and that from the deafferentation \\:as observed by Mott and Sherrington ( 1895) and by Twitchell ( 1953). It was also suggested that the thrcsliold of MsI to direct stimulation becomes higher following the deafferentation ( Matt and Sherrington, 1895). Wall et nl. (1953) studied MsI excitability in cats under various types of anesthesia including chloralose and semicarbazide with an index which was the height of the response recorded from the medullary pyramid after a single stimulus to MsI. They found

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that a single light flash in thc eye or Literal geniculatc electric stimulation produced a prolonged period ( about 110 mscxc ) of facilitation of hlsI. A maximuin excitability was at 40 msec after the flash. This efIect \vas not aRected by complete removal of the occipital lobe cortex. It was concluclecl that neither the primary visual receiving area nor the visual association area need be present for the facilitating effect of visual stimulation to occur in MsI. Complete removal of the cortex of the superior colliculus also did not affect the time course or size of the facilitating effect of a visual affcrcnt volley on the X I s I excitability. Lesions placed in the prctectal region abolished the facilitatory effect of visual stimulation. Their conclusion was that visual stimulation has a direct effect on the excitability of h4s1, although this docls not imply that the excitatory effect of v i s d stimiilation does not also affect many other parts of the central nervous system. It was suggested that the ventral division of the lateral geniculate is certainly the largest ancl perhaps thc most likely structurc to transmit the cxcitatory influence of the :iHerent visual stimdiis to MsI. Wall et nl. (1953) furtlier studicd thc effects on h4sI cxcit;iliility t l the of the femoral cutaneous nerve stimulation. They s h ~ ~ ethiit time course of the increase in MsI excitability might h e faster than after visual stirmilation so that thc maxiinmi escitabi1it;i was reached 3 0 3 5 mscc after a single stimulation of the cut femoral ciitaiieous nerve; and that the effect of peripheral nerve stimulation seemed to be more effective on hIsI excitability than did the visual stimulation. In adclition to these examples of excitability control of pyramitlal motor area induced by visual and cutaneous afferents, Ascher and Buscr (19S8) studied a similar control mechanism not only by visual and tactile but by auditory stimulation in cat under chloralose anesthesia. They showed that MsI actilxtion depends upon the integration of SniI, visual, and auditory areas. However, the existelice of these specific sensory areas is not rrecessary to this activation, since it occurred even after the ablation of these specific sensory areas. Long (1959) showed in m a n estlietized cats that the modification of specific sensory ( somatic and visual) potentials from SmI, SmII, and visual areas, evoked by afferent peripheral nerve stimulation, \vas obtained most easily and the effect was most prolonged when the conditioning stimuli were applied to the reticular formation of the brain stem; and

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2%

that conditioning stimuli to the unspecific thalamocortical projection system, aniygdala, putamen, globus pallidus, and lateral aspect of the head of the caudate nucleus wwe, in this order, decreasingly effective in modifying sensory impulses. H e further indicated that the changes recorded in the evoked responses were more marked at the cortical level than at thc level of the specific thalamic relay nuclei, and the modifications Lvcre generally of equal degree and duration in SmI and SmII. The high-frequency (250/sec) stimulation of the reticular formation depressed the amplitude of evoked responses in both SmI and Smll, while the lower frequeiicies (0.5 and S/sec) of stimulation to the reticular formation and its projections augmented the amplitude of the evoked potentials from MsI and SmI. This reciprocal effect of augmentation and depression of afferent responses seems to be analogous in certain respects to the facilitation and inliibition of motor responses by the reticular system, and the augmentation of afferent signals may reprcsent a mechanism which permits limited focusing of awareness or attention ( Long, 1959). Stimulation of a skin nerve m ~ a sfound to lie capable of evoking responses in neurons in the pericruciate cortcx of cat SI including the pyramidal neurons with descending axons in the medullary pyramid (Asanuma, 1959; Li, 1959). Li (1959) further showed that a preceding sensory volley can elicit facilitation and inhibition of pyramidal activity. The intracellular records of Phillips ( 195Ga, b ) indicated that cortical hIsI stimulation may produce a sustained depolarization of pyramidal neurons. Asanuma and Okamoto (1959) demonstratctl that a single shock applied to the corpus callosum produced a long lasting inhibitory effect to pyramidal neurons, and that hyl)crPolarizatiol1iriz~~ti~)ii of these neurons occurred. These observations suggest that the excitability of pyramidal neurons in SI is largely determincd by a background synaptic impingement from other cortical cclls as well as from sensory volleys. V.

A.

Corticocortical Projections

CEREBROCEREBELLAR I N I LHHI.:I,ATIONSHIPS

Hampson et al. (1952) indicated in cat under Nembutal ancsthesia that SmI stimulation cvokes responses which are confined

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largely to the contralateral anterior lobe and lobulus simplex The face subdivision projects to the simplex and upper culmen, while the arm and leg subdivisions project to the lower culmen and centralis, respectively. The projection of the trunk area overlaps part of the areas to which arm and leg project. SmII stimulation evokes responses which are largely restricted to the contralateral paramedian lobule. The localization pattern for SmII projcction to the cerebellum did not show the striking point-to-point differences seen for SinI. Nevertheless the evidence was suggestive that the face was represented in the more rostra1 folia and the arm and leg in the more caudal folia. There was a good agreement between the somatotopic distribution within the cerebellx cortex of cerebral and sensory projections. Snider and Eldrecl (1952) carried out a similar group of experiment5 on monkeys under Nembutal-chloralose anesthesia. Area-to-area localizations were found only within the contialateral part of the lobus anterior and lobulus simplex and within both paramedian lobules, whcn either hIsI or SmI were stimul~itedwith single shocks (Fig. 5). Jansen ( 1957) clenionstrated, in c'it untler Nembutal-chloralose anesthesia, that the arm subdivision of SmI [ predominantly activated the medial part of the crus I1 while the leg area activated the lateral part of the same lobule, and that, in contrast to MsI, Sin1 did not scem to activate the ansiform lobule to any significant extent. The\, also described three types of corticocerehellar potentials : shoi t IJ tency (2-5 msec), long latency (12-25 msec) and combined short- and long-latency potentials. Short-latency potentials were most readily elicited from SmII, while long-latency potentials were most typically obtained by SmI stimulation. The long-latency responses weie less resistant to asphyxia than the short-latency responses, suggesting their origin in different elements of the cerebellar cortex. When paired stimuli of varying intervals were applied to the cerebral cortex the long-latency response exhibited a long unresponsive time up to 150 msec. The short-latency response had a much shorter unresponsive time (below 20 msec). As regards interaction between the two types of responses, the short-latency deflection had 1' prolonged (up to 150 msec) depressing effect on the longlatency potential, whereas the short-latency activity was hardly influenced by a conditioning long-latency potential. The longlatency component of the cerebellar response following cortical

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stimulation was greatly inhibited by a conditioning shock to a peripheral nerve while the short-latency activity showed very little depression. Evidence that both responses were mediated by the pontine nuclei was provided by various lesions in the brain stem. The short-latency potentials were attributed to the activa-

FIG 5 . A summarizing diaqram of hlsI and SmI projections to varioiis cerebellar areas. Alacaca nu&zttcz uncler chloralosc plus Nembutal anesthesia. Somatotopic localizations wcre only found with thrcshold stimuli, there was a considerable overlap between lcg, arm, and face areas, and the response of the ipsilateral paramedian lobule w‘is most vanable. (From Snider and Eldred, 1952.)

tion of granule cells, and the long-latency potentials to the discharge of the Purkinje neurons. Henneman et nl. (1952) stiinulated the cerebellar cortex and nuclei in cat under different types of anesthesia as well as in curarized or “enc6phale isole” preparations. Stimulation of the anterior lobe and lobulus simplex of the cerebellum evoked responses restricted to contralateral MsI, SmI, and SmII, while paramedian stimulation elicited responses in the same areas of both cortices. No point-to-point relationships between cerebellar

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nuclei and cerebrum wcre found, and responses were elicited in MsI, SmI, and SmII following stimulation of any site in the cerebellar nuclei, ipsilateral or contralateral. IIenncman ct nl. ( 1952) could not find consistent cortical responses following stiinulation of crus I or crus 11. However, Combs and Saxon (1959) have shown that single shock stirnulation of crus I or crus I1 cvoked a large potential in contralateral kIsI and Sin1 in curarizccl cats as well as in cats under Nembutal anesthesia. The ipsilatcral responses were small, inconstant, and limited to RfsI. The problem of cerebrocerebellar interconnections was also provided in relation to movement. Aring arid Fulton (1936) demonstrated in monkey and baboon that the tremor following decerebellation was abolished by resection of MsI and area 6. The ablation of area 6 alone increased the tremor. On the other hand, unilateral extirpation of MsI subsequent to a contralateral cerebellar lesion temporarily abolished and permanently depressed all signs of cerebellar deficit. Rossi (1912) made experiments on dogs injected with morphine, and confirmed that during stimulation of crus I, crus 11, arid paramedian lobulus, a previously uneffected stimulation of contralateral MsI was found to be ablo to produce a cortical movement constantly. Ether anesthesia was found to prevent the cerebellar responses. His experiments were repeated and confirmed by Bremer (1935; cited by hloruzzi, 1950) and Dusser de Bareme 11937; cited by Moruzzi, 19580). Walker (1938) observed a marked increase in the amplitude and frequency of the spontaneous cortical waves recorded from R4sI during a weak repetitive stimulation (frequency: 12-25/sec) of the surface of the cerebellar hemispheres in the unariesthetized “encc5pliale isole” cat. Tlie response was mainly contralateral, although some ipsilateral effects w7ere seen. Vermal stimulation was less active and the hemispheral effect was abolished or strikingly reduced after transection of the ipsilateral brachium conjiinctivum or by local application of Novocain or ice to cerebellar surface. In this manner Walker ( 1938) confirmed Rossi’s findings suggesting that these responses might be due to facilitating cerebellorubrothalamic volleys. Moruzzi (1941a, b ) studied the responses of cat under light chloralose anesthesia to cerebellar stimulation. Weak stimulation of crus I elicited jerky movements, but not postural responses. This clonic effect was abolished by destroying MsI, and no effect was

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obtained even when the intensity of the cerebellar stimul,‘1t’ion was greatly increased. Moruzzi (1941a, b ) showed that the anterior lobe stimulation produced a marked inhibition of cortically induced movements when it was timed to occur against a background of strong activity of hIsI which w a s easily obtained by supraliminal repetitive stimiilation or by local strychninization of the sigmoid gyms in a chlolalosed cut; and that following bilateral ablation of XlsI, a background of phasic movement could still routinely be produced by electrical stimulation of the white matter underlying the sigmoid gyrus. This movement was blocked by stimulation of the vernial part of the anterior lobe, suggesting that the cerebellar iiiliibition might occur at spinal levels. This is not, however, the only csplanation. The observation of von Baumgarten ct al. ( 1954) that cortically driven bulboreticular discharges are blocked by cerebellar stimulation suggests that subcortical relays of the cxtnipyramidal pathways may also be directly inhibited by cerebellifugnl impulses. Snider et nE. ( 1949 ) reported, in cat under Dial or Neinlmtal anesthesia, that movement arising from the pericruciate area of the cerebrum were suppressed by electrical activation of the anterior cerebellar lobe and paramedian lobulus as well a s nearby folia. Frequencies of electrical stimuli of 200 to 300/sec were foiind to be most effective for cerebellar stimulation. They concliitled that the patlway mediating this effect is cerebellar cortex to nnclciis fastigii to bulbar reticular formation, thence to the spinal cord via the reticulospinal tracts. Similar experiments were performed by Snider arid Magoiin ( 1949) who obtained facilitatory effects, i n moiikey under chloralosc, Nembiital, or chloralose-Nembutal ancstlresia, with some evidence of somatotopic localization, by applying high-rate ( 200300/sec) pulses to paramedian lobulus or to anterior lobe. It is apparent from the drawing ( see, Dow antl Moruzzi, 1959) that Snider and Magoun ( 1949) presented that the facilitatory responses were obtained mostly from the intcrmediatc antl the lateral parts of the anterior lobe, i.e., from areas projecting to the interposite and dentate nuclei (Jansen and Brodal, 1940, 1942). Facilitatory effects were obtained occasionally on two cats. Nulsen et al. (1948) stimulated the same surface point of the antcrior lobe-lobulus simplex complex in cat, dog, monkey, and chimpanzee under Dial anesthesia, finding that cortically or pyramidally induced movements were

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either inhibited or facilitated, depending upon the frequency of the stimulus. In each animal, except cat, increasing the frequency of the cerebellar stimulation resulted in facilitation of the existing cortical movement, whereas inhibition resulted from low frequency stimuli. These results are contrary to those which were reported by Moruzzi (1948a, b, c, d, e ) who limited his study to stimulation effects of vermal part of the anterior lobe on the extensor hypertonus of the unanestl~etized decerebrate cat. Both anatomical and physiological data can hardly be reconciled with the results of Nulsen ef nl. (1948; see Dow and Moruzzi, 1959), who maintained that cerebellar facilitation was related to the activity of fastigial neurons whereas inhibition would be relayed by dentate nuclei. Sjoqvist and Weinstein ( 1942) demonstrated in chimpanzees, using a trained proprioceptive skill such as weight discrimination, that lesions of neither the medial leniniscus nor the superior cerebellar pcduncle alone caused permanent loss of the trained skill. However, the combined Icsions of the two systems produced permancnt loss of the skill. “It might bc infcrred that these iinpulses may carry information of a precise sensory modality. Yet, cerebellar influences seem capable of niodifying evoked potentials in the sensory cortex which probably provide the background for an appropriate motor beliavior” ( Terzuolo and Adey, 1960, p. 816). A more extensive discussion of cerebrocerebellar interrela t‘ionships will be found in tlie monograph by Dow and Moruzzi ( 1959). B. CALLOSAL INTERI3EhllSPIIERIC COKNECTIONS In tlie cortical response following electrical stimulation to isocortex, activity occurs at the corresponding point of the contralateral cerebral hemisphere. This contralateral, lioinotopi~response was first reported by Curtis and Bard (1939), and lias been analyzed by Curtis (1940a, b ) . This finding was later confirmed by McCulloch ( 1914), who observed that local strycl~ninizationof a cortical point of one hemisphere could evoke the response of the corresponding point in tlie other. A furtlier andlysis has been reported by Cl ung ( 1953). This contralateral cortical response has been named the callosal rcsponse, since it is mediated via the corpus callosum.

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Monkey MsII projections were traced by means of the Nauta (1957) method to contralateral RlsI and area 6 via the corpus callosum ( D e Vito and Smith, 1959). In contralateral R4s1, projections were mainly to the arm arca, some to the leg area, and a very few to the face area. De Vito and Smith (1959) pointed out that the callosal fibcrs to 53sI terminate in all cortical layers up to and including layer 3, while those to area 6 terminate in layers 6, 5, and 3 on both tlie lateral and tlie mesial surface. MsII projections to contralatcral SmI, area 5 , and area 7 were very few, and there were none to contralateral area 8 ( D e Vito and Smith, 1959). It was foiintl in monkey using the Marchi method that Sin1 projects callosal fibers to contralateral SmI and hIsI (Peele, 1942). Cat SmIl lras also callosal interconnection (Nakahama et nl., 1958). Each stiniulation of SmI, SinII, or Msl evoked the electrical responses in all areas of SmI, SniII, and MsI of the contralateral hemisphere ( Nukuliatna and Saito, 1956), although it has not been confirmed tl nt these responses were evoked via the corpus callosum. Extensive connections between area 6 in the two hemispheres have been tlcscribecl ( Mettlcr, 1947; Krieg, 1954a ) . Connections between a r w 5 and contralateral area 5 and SmI were described, and also a few with RlsI ( AMcli, 1932; Peele, 1942; Krieg, 1954b). Projections from arca 7 to contralateral areas 7 and 5 as ell as contralateral Sin1 (areas 2 and 1) were found in decreasing order of sigiiihince (Peele, 1942). Chang ( 1953) suggestctl a three-component hypothesis, the callosal response being tlic algebraic siimmation of the threc cffccts. One component rcprcscnts tlie antidrornic activity of tlie axons and clenclritcs of the callosal neiirons in the deep part of the cortex. The second reprtwnts the activity of the callosal afferents terminating in the superficial layer and tlic postsynaptic activity of cortical cells activated l y them. The third component reflects the all'erent volleys arriving by way of callosal afferents brancliing and terminating in the tliird cortical layer and the postsynaptic discharge of the cortical intcmi[incial ncurons located in the siiine layer. Chang's analysis was based on combined studies of the ectosylvian as well as the s u p s y l v i a n response. This fact was pointed out by Peacock (1957), and it was concluded that these combined studies led to some confusion in the final interpretation. He also pointed out that the histological studies reported by

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Nauta (1954) on ccit are for the most part in disagreemcnt with the histological picture for the mouse prcsentecl by Chang. Nauta (1954), working mainly with tlie striate and auditory I areas of rat and cat, showed that the callosal fibers terminate predominantly in cortical cell laycrs 3 to 6. He used the Nauta-Gygu method after making a contralateral cortical lesion. On the other Iiand, Chang (1953) has reported, on tlic basis of Golgi and Golgi-Cox preparations of the brains of young mice and rats, that neurons contributing a ~ o n sor collaterals to the corpus callosuni 'ire mainly those situated in the ckeper strata and especially layer 6 of tlie cortex, whereas tlie terminals of these fibers are mainly tlistribiited as free endings in the superficial three laycrs. H e was able to confirm the inversion of the sign of an initially surface-positive callosal response for SmI and SmII (Perl and Whitlock, 1955; N'ikahama et nl., 1958; Nakaliania, 1959a), as originally clemibed by Curtis ( 1940b) for the suprasylvian gyrus. Curtis (1940b) attribiitcd the initial surface-positive phase to both aiitidroinic activity iincl activity in the ascending callosal afferents, which, he maintained, ramify in laycrs 2, and 3 and end in layer 1. He 'isserted that these callosal aflerents end on intcrneurons which descend to lower strata in the cortex, their activity producing the negative phase of tlie callosal response. In tlic evpcriments of Perl and Whitlock the "sink appeared to lie at levels deeper than those indicated by Curtis. Perl and Whitlock agreed with anatomical ninterial of Nauta (1954), finding it inconsistent with that of Cliaiig (1953). Furthermore no evidence n7as obtained to support Chang's threecomponent hypothesis, although it must be noted tliat tlic sites evamined were diflercnt ( Nakah'ima et nl., 195s).

C. IPSILATERAL INTERCORTICAL Con-xEcTIons Recently D e Vito and Smith (1959) have stticlied, with the Nauta ( 1957) method, h M I projections to ipsilnteral cortical areas of monkey after MsII lesions. One of the main projection7 w a s to MsI, especially to its arm portion. There was some projection to the leg area but very little to tlie face region. Fibers of both large and small diameter were degenerated, suggesting tli'tt the small diameter fibers are tlie only ones ending in the superficial layers 1 and 3, or that the fine fibers seen there are the terminals

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of larger fibers which give ofl collaterals at lower levels (layers 3, 5, and G ) . There was a nioderate projection to the lateral surface of area 6, above and below tlie arcuate sulcus. Degenerated fibers mere found in laycrs 6, 5, antl 3. Slight projection was traced to area 8 and the cingulate gyriis. -4minor projection to Sin1 was evidenced by degenerated fibers extending into layer 2. Area 5 received a very slight projcction with degeneration extending into layer 3. Even fewer dcgeiicratecl fibers were found in area 7. Histological examination \vith tlie Rlarchi nietliod showed extensive fiber connections from Sin1 into hlsl in monkey (Peele, 1942; Cole and Glees, 1954). Cole and Glees (1954) showed that very few fibers from SmI terininated in the posterior portion of area 6, a small number in area 5, and none in area 7. Peele (1912) indicated that area 3 of Sin1 projects fibers to the adjacent areas 1 aiid 2, and to area 5 ancl 7; and that areas 1 and 2 of SmI send fibers to areas 3, 5, antl 7. Dusscr de Barenne and McCullocli ( 193s) showed with the stryclininc neuronograpliy that SmI fires into MsI but not further anterior; and that SmI connects with area 7. There are discrepancics among these observations on SmI projections to areas 6 and 7. Histological data may be regarded as inore reliable. However, the Marchi method is not sufficient to demonstrate the exact sites o f termination of degenerating fibers, since it stains only the dcgcncrating myeliiiatecl fibers. Tlie Nauta method \vhich stains the tlc.geiicrating axoplasm of myelinatcd and unmyelinated fibers will clarifr tliese discrepancies. SmI receives fibers from area 5, and area 8 and 1 of Sml does from area 7. Areas 5 and 7 are abundantly interconnected (Peele, 1942). Peele ( 1942) showed with the hlarclli nictliod tliat areas 5 and 7, and possibly area 3 of Sml, sent fibers into the anterior occipital gyri; and that some fibers from SmI and the anterior part of 7 were sent into the rostra1 part of the superior temporal gyrus by way of the external and extreme caps~ilc~s, while some fibers from the posterior part of 7 entered the posterior half of the superior teniporal gyrus directly. It was demonstrated in cat with the electrophysiological method that SmI projects direct fibers to ipsilateral Rlsl and Smll (Nakahama, 195Ya, b ) ; that Sin11 projects to Msl and Sml (Nakahama, 1960); and that Msl projects to Sml (Nakahama, unpublished observations ) , Electrical stimulation of cat SmI evoked potentials

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in ipsilateral MsI which consist typically of a sequence of four surface-positive deflections and a siirface-negative deflection followed by a prolonged positive deflection ( Nakahama, 19S9b). Sin11 stimulation evoked different cortical responses in XlsI and SmI (Fig. 6 ) . The surface-positive component of tlie hIsI response was of greater amplitude than Sin1 response. This is considered to be the same in origin as that of the postsynaptic positive wave of the primary response evoked by afferent somestlietic stimiil at’ion.

IOmsec

FIG.6. Form arid distribution pattcm of cat SmI arid h l d rcspoiisc rvoked by SmII stimulation. Iiisert tliagram of hmin sho\vs corrrsponding positions of points from which records were taken and application points of stimulating electrode. Positivity is u p v a r d . ( From Nnk:ihama, 1960.)

The surface-negative componeiit of the AlsI respoiise, h v e v e r , was of snialler ainplitiide tlian tlie Sin1 response, and is supposed to have an origin similar to tliat of the local response to cortical surface stimulation near the recording site ( Nakaham~1, 1960). The same configuration pattern of rcspoiise as tliat of SmI rc~iponse of Sin11 origin was elicited in SrnII to Sin1 stimulation (Nakaliama, 195%). In paralyzed cats with local anesthesia tlie surfacc-positive deflections of the responses mentioned above slio\ved facilitation when two identical stimiili n7erc: applied to each somatic area. This facilitation did not occur with the intravenous injection of a very

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small amount of Nembutal ( Nakahama, unpublished observations ) . The extreme sensitivity to anesthesia does not imply lack of significance but may indicate the degree of importance it has in normal cortical function. Amassian (1952a, 1954) showed in cats under chloralose anesthesia that stimulation of any one of the four limbs and cutaneous or muscle nerves evoked large responses in the anterior lateral gyrus which was termed a somesthetic association area. However, it was demonstrated that somesthetic, visual, and auditory stimuli evoked potentials not only in the anterior lateral gyrus but in the suprasylvian gyrus of cats with or without chloralose anesthesia ( Albe-Fessard and Rouged, 1958; Buser and Borenstein, 1959; Buser et nl., 1959). Therefore it seems to be preferable to limit the description “association area” to response fields on the anterior lateral ancl the suprasylvian gyri, although “association” is not meant to imply anything about the nature of the response fields. Amassian (1954) could elicit the potentials in the anterior lateral gyrus following SmI and SmII stimulation. Moreover, Nakahama ( unpublished observations ) tlcmonstrated that stimulation of XlsI, SmI, and SmII evoked responses not only on the anterior lateral gyrus but on the suprasylvian gyrus of cat. These data suggest the intimate relationship of somatic areas to the association area. Albe-Fessard and Rouged ( l95S), however, showed tliat the association area responses to sensory somatic stimuli were not evokcd through cerebral somatic areas, since the responses were not abolished following removal of ipsilateral MsI, SmI, and SmII and of contralateral MsI and SmI together. They also confirmed that the center median receives soinesthetic afferents from diverse origins ancl diff erent regions of the body and relays sensory information to the association cortex. The center median, a part of the unspecific thalamocortical projection system, also receives projections from many cortical areas and is the site of interaction of cortical output influences and somesthetic input volleys ( cf. Albe-Fessard and Gillett, 1958).

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VI.

Behavioral Conditioning Studies

After simultaneous ablations of rat SmI and SmII, roughness discrimination can apparently be relearned and the retention of form discrimination was not impaired (Zubek, 1951, 195%). In Allen’s ( 1947) investigation on clog a differential conditioning technique was employed to test the somesthetic defects. Tlic rcspoiise of the animal consisted of lifting his foreleg to a positive stimulus and not lifting it to a negative one. The postive stimulus was brushing the skin of the back with the grain once per sccond, and negative stimuli were brnshing against the grain once per second, or with the grain t h e e times per second. The bilateral removal of SniI and SmII resulted in complete abolition of tactile discrimination. There was a fairly rapid return of correct differentiations to both sets of stimuli subsequent to bilateral roinoval of SmI. On the other hand, when SmII was removed thc animals experienced considerable difficulty in relearning the tactile discriminations. The chief effect of these lesions was an inability to withhold the forelog response when negative stimuli were applied. The response to positive stimuli w a s retained n7ithout impairment. Ziibek ( 195%) carried out experiments on four cats with vuious roughness discriminatioii tcsts in an alley-type discrimination apparatus. Thc Sin1 a n d Sin11 rcmoval together resiilted in ;i pcrmanent abolition of the capacity to make both fine anti coarse roughness discriminations. The Sin1 ablation slightly iin1)aired coarse discrimination. Bilateral SmII lesions severely impairctl fine roughness discrimination. This indicates that cat SmII may be crucial for the more difficult or finer discriminations. Unfortunately, his data were based upon the behavior of a single animal. In monkey, Orbach and Chow (1959) examined the effects 01 SmI and SmII resections pre- and postoperatively upon six somcstlietic discriminations (multiple cue, size plus form, form, and tlrrce clegrees of roughness). SmI resection alone produced marked disturbances in tactile form and roughness discriminations, while SmII removal alone seemed to be without effect on pcrforinance of tactile discriminations. SmI and SmII destructions together induced no more marked effect than that following SmI destruction. These results in monkey are much in contrast to those of Zubek in cat. I t is concluded that SmII is not involved in the performance

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of tactile form and roughncss discriminations in monkey, and its integration cannot compensate for a loss of SmI. Orbach and Chow (1959) support Fulton’s suggestion (1949, p. 337) that SmII serves “the motor aspects of attention by moving the sense organ, or the part bearing sense organs into a position better to appreciate a stimulus.” The correspontling evidcnce on the effects of SmII lesions in man has yet to lie asscmbled (Evans, 1935; Penfield and Jasper, 1954). Benjamin ancl Thompson ( 1959) utilized Zubeck‘s method ( 1952a) of training ancl compued the behavioral effects of ablation of both SmI and SmII in newborn cats with results of equivalent lesions inflicted on mature animals. The degree of behavioral impairment resulting from the ablation was a function of age at the time of operation. Nearly normal ability to discriminate on the basis of rougliness cues was retained if the lesion was made at birth. Almost complete deficiency in this type of discrimination occurred from equivalent lesions inflicted at maturity. The histological analysis of the cortex and the thalamus provided no anatomical basis for the behavioral cliff erences between the adult and infant animals (Benjamin and Thompson, 1959). Small lesions in the hand region of monkey SmI were tested for their effects on discrimination by palpation, and associated effects on dexterity and motor power (Cole and Glees, 1954). In all cases, substantial or complete postoperative recovery occurred. These lesions did not cause inability to make movements but may have caused initially unawareness of movements and arrest of a limb in an unusual position. In all cases a loss of tactile sensation occurred and before recovery this defect was compensated for by the greater use of visual cues. Cole and Glees (1954) identified intimate conncctions between Sin1 and MsI and considered that these cortical areas form a unit essentially linked with the thalamus and spinal cord, but sending few fibers into areas more anterior and posterior. Kruger and Porter (1958) reported that in six immature rhesus monkeys SmI and SmII ablations together resulted in a severe deficit in tactile sensitivity when monkeys had to discriminate the form of the test objects and secure food in the absence of visual cues. The cliscrimination was permanently impaired oiily if both MsI and SmI were included in the lesion. No deficit was demonstrable if the ablation was restricted to MsI alone. Kruger and Porter’s demonstration (1958)

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that MsI must be resected in addition to SmI to produce a prolonged and severe somatosensory deficit is very informative. It is suggested that MsI and SmI somehow form a higher order functional unit. Monocularly learned visual discriminations transferred readily to the untrained eye in cat with midsaggital division of the optic chiasma (Myers, 1955). However, if the corpus callosum had also been sectioned, the animals were unable to recognize the patterns with the untrained eye (hlyers, 1956) and could only relearn the discriminations at rates similar to the original lcarning with the first eye (Sperry et nl., 1956). These findings appear to demonstrate an important role for the corpus callosum in integrating the two hemispheres in visiial learning and memory. T o study contralateral transfer from one forepaw to the other of trained tactile discriminations, Stamm and Sperry ( 1957) trained cats to press with one forepaw the correct one of two pedals which the cats could not see and which then had to distinguish on the basis of touch. Transfer to the contralateral paw of roughness, softncss, and form discriminations in uiioperated cats was compared with that obtained in cats with rnidsaggital section of the corpus callosum. I t was concluded that the corpus callosum is essential for the contralateral transfer of somesthetic discriminations from one to the other forepaw. Using this functional independence of the two hemispheres in the callosum-sectioned cat, Sperry ( 1959 ) tested the functional capacity of the surgically isolated somatic areas. He showed that somesthetic discriminations performed with the left forepaw may be retained at a high level in this preparation and additional new discriminations can be learned readily after removal from the right hemisphere of all neocortex except the small frontal area (which includes MsI, SmI, and SmII, in the author’s judgment from his figure). This cortical remnant was sufficient for high-level retention and new learning of thesc discriminations and, conversely, removal of SI (in the author’s judgment from his figure) on the left hemisphere abolished or severely impaired discriminative performance with the right paw, but lcft unaffected that with the left paw. Therefore it seems that the specific cortical reorganizations and mnemonic changes involved in the establishment and maintenance of the discrimination habits performed with a paw were localized mainly or entirely within con-

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tralateral somatic areas (Sperry, 1959). A marked facilitation of the response, in the auditory area, to a specific (sensory or associational ) thalamocortical volley was observed by Bremer ( 1958 ) in the unanestlietized cat when it is conditioned by a callosal volley. He later extended tlie callosal facilitation and interhemispheric commissural transfer to the other cortical sensory areas and also association areds (see Bremer, 1958). H e cited Myers and Sperry’s experiments mentioned above, suggesting that the facilitatory modification may rcpresent a short term memory trace, which, by its repetition, leads ultimately to the fixation of a stable mnemonic pattern in tlie cortical neuronic networks. SmII has been dernonstratcd to possess the function of auditory as well as somethetic discriminations. Allen ( 1945) demonstrated that, whereas ablation of the traditional auditory areas temporarily impaired, but failed to dcstroy permancntly, the ability of dog to discriminate widely different frcquencies, this ability was permanently lost if the third auditory area which is in SmII was also resected. Meyer and Woolscy (1952) trained cats to discriminate small increments in the frequency of auditory stimuli. Each of the twenty trials in a session began with presentation of a varying number of 1000-cycle tones, which were usually 2 sec in duration and spaced 1 sec apart. The series was followed by a 1100cycle tone reinforced with shock and buzzer. The animals learned to avoid shock by running in ‘1 rotating cage. Test of generalization began after a criterion of 90$ responses was reached, and critical tests were given at increments of 20, 40, 60, 80, and 100 cycles. Cats were retrained by the same procedure after a variety of symmetrical extirpations of the following cortical areas: auditory area I and 11, SmII, posterior cctosylvian gyrus, suprasylvian gyrus, temporal region, and tlic cerebellar tuber vermis. It was found that if auditory area I and 11, posterior ectosylvian gyrus, and SmII were completely destroyed on both sides, the animals could no longer achieve the frequency discrimination. N o other combination of ablations had this effect and if remnants of auditory area I and I1 escaped damage, frequency discrimination was maintained. Butler et al. (1957) used a basically similar plan, and carefully analyzed the retrograde tlialamic degeneration in their cats. The results in this series of experiments differ from those of Meyer and Woolsey in that after complete bilateral ablation of auditory

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area I and 11, posterior ectosylvian gyrus, and SmII, ability to discriminate changes in frequency was essentially unirnpairecl. A temporary amnesia for the learned habit occurred but all animals were able to relearn. The difference between the results of- the two experiments was suggested by Butler et nl. to be clue to following reasons. (1) The testing inethods were different. ( 2 ) In Meyer and Woolsey’s studies, in animals with discrimination loss, the lesions, although listed as including auditory area I and 11, posterior ectosylvian gyrus, and SmII, actually extended ventrally nearly to the rhinal fissure (unlike those of Butler et nl. ). ( 3 ) In the latter group, the posterior part of the medial geniculate, pars principalis, consistently escaped degeneration, although it was also noted that the more extensive the lesion was in the ventral direction, the further the degeneration extended into the posterior tip of the medial geniculate. VII.

Conclusions

The determination of the boundaries of cortical somatic areas is of value in the study of the role of these areas in iieurological and behavioral functions. Somatic areas consist of at least four distinguishable, somatotopically organized regions. Each of them appears to be concerned with both sensory and motor functions. Therefore the present review uses the following nornei~cl~iture proposed by Woolsey: somatic sensory-motor area I ( SmI) for the postcentral gyrus and its homologs in nonprimate forms; somatic sensory-motor area I1 ( SrnII) for the second sensory area; somatic motor-sensory area I ( MsI) for thc precentral motor area; somatic motor-sensory area I1 ( MsII) for the supplementary motor area. The investigation of the functional mechanisms of these somatic areas is one of the main problems in present-day neurophysiology. Researchers have proceeded on the supposition that if the patterns of neural activity entering a somatic area, the modification of these patterns occurring across intracortical synaptic rel
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silaterally and contralaterally, to each other. Thus there are feedback circuits between the various somatic areas. These circiiits could add to or substract from the total effect of the afferent volleys passing to the diflcwnt furictional areas. Somatic areas are also centripetally and centrifugally linked with subcortical structures including the rvticular formation. The reticular units are influenced by volleys froin tlie cerebellum and cerebral cortex and by sensory inputs of different modalities. The unspecific tl ialamocortical projection system also iiitcrconnects somatic areas with tlie diencephaloii and brain stem. Tliis unspecific central integrating network of neurons and the reticular formation of the brain stem incessantly modify not only the spontaneous electrical activity of somatic areas h i t rcq~oiisesto scnsory impulses. For example, somatic areas projcct fibers to tlie center median, a part of’ the unspecific tlialamocortical projectioii system, which is the site of iiiteraction of cortical descending influences and ascending somatosensory volleys. Tliis illustrates a particular aspect of the complex corticosubcortical interrelationship which might play an important role in sensorimotor integration. The fact that collaterals of pyramidal fibers from somatic areas terminate in the cuneate, gracile, and trigerniiial nuclei suggests that the activity of pyramidal neurons modifies somatosensory iiiputs at the level of these relay nuclei. The peripheral feedback system also provides ari exceedingly important measure of somatic areas control over sensory input. An experiinciit to determine the input and output patterns of cortical activity of some spatial and temporal complexity will b e of much value. It seems likely that the analysis of single units will help the solution of this problem. Even if the arlalysis is successfully carried out, a mere accumulation of such analytical studies will not produce an over-all picture of the somatic area. Each somatic area, under tlie influence of manifold sti.uctures with a feedback tem, must be coiisiderccl as a functional unit of the “reflex” circuits receiving sensory information a t d executing motor acts. Much work has been clone on SmI and klsI, but less on SmII ar,d I\/sII. The meaning of the existence of double sensory and motor projections is not known yet. Further progress in the physiology of intra- and intercortical con~iectionsof the somatic areas will contribute to the untlcrstanding of how the activity of the brain is related to the mind.

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BODY FLUID INDOLES IN MENTAL ILLNESS By R. Rodnight Department of Biochemistry, Institute of Psychiatry, Maudsley Hospital, Denmark Hill, London, England

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251 11. Normal Body Fluid Intlolc~s . . . . . . . . . . . . . . . . . . . . . . . . 254 A . Pathways of Tr!-ptoplwi hdctaholism in Tissues . . . . . . . . . . 255 13. Tryptophan Metabolism by Intestinal Flora C. Normal Urinary Intlolcs . . . . . . . . . . . . . . . D. Normal Blood Intlolcs . . . . . . . . . . . . . . . . . E. Cerebrospinal Fluitl Indolcs ..................... 111. Body Fluid Indoles in hlrntal Illness . . . A. Hartnup Disease . . . . . . . . . . . . . . . . . . . . . B. Phenylketonuria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269 C. Major Mental Illncsses . . . . . . . . . . . . . . . . IV. Concluding Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284 V. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288

I.

Introduction

The suggestion has been frequently advanced during the past sixty years that abnormalities in the bodily metabolism of indole derivatives are in some way concerned in certain forms of mental illncss, particularly in schizophrcnia. The present account aims to assess this general hypothesis in the light of evidence obtained fIom the analysis of body fluids for indole compounds. As an introduction to the subject the speculative background will be briefly indicated and this \?.ill be followed by a short outline of present knowledge of indole metabolism in man.

H!YPOTHESESCONCERNING INDOLES IN CEREBRAL FUNCTION An involvement of indoles in cerebral function has usually been proposed on one or other of the following grounds: intestinal 25 1

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autointoxication by indoles; the occurrence in the body of abnormal tissue indoles with toxic action on the brain; or an imbalance of normally occurring indoles. The latter two of these theories are not necessarily exclusive of one another. Intestinal autointoxication. The theory postulates a toxic action on the brain by excessive amounts of indoles produced by bacterial action in the gastrointestinal tract. It has also been suggested that incomplete detoxication of the indoles by the liver may be involved. The autointoxication theory was popular at the beginning of the century and was widely believed to be responsible for many physical and mental ailments. The toxic agents were not necessarily considered to be confined to indoles, but the alleged toxicity of the latter figured prominently in the theory. Thus it was recognized that indoles were typical products of bacterial putrefaction in the intestine and that they were partially absorbed into the blood stream, detoxicated by the liver, and excreted in the urine. This sequence of events was well exemplified by indole itself, by then established as the precursor of urinary indican. Clinical workers, moreover, had noted that symptoms of irritability, insomnia, and “neurasthenia” were often associated with indicanuria and this encouraged suggestions of a causal relationship between indole toxicity and mental illness (e.g. Bianchi, 19’06). Another prominent worker of the time, C. A. Herter, studied the effects of orally administered indole on himself, and noting that large doses did indeed give rise to symptoms similar to those noted above, felt justified in writing: “I am confident that indole absorption contributes in no unimportant way to the production of chronic disorders of nervous function” (Herter, 1902). One niay also recall that an even greater confidence in the autointoxication theory was shown by certain surgeons who, in order to remove the source of the supposed toxins, practiced widely the operation of colectomy ( Lane, 1905). No convincing evidence was ever obtained for intestinal autointoxication causing mental illness and when therapeutic measures designed to remove or reduce the putrefactive source failed to effect any improvement in mental state, the theory lost support. Contributing also to its eventual rejection were the excellcntly controlled studies of Otto Folin on the urinary excretion of compounds originating in the bowel in mentally ill subjects (Folin,

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253

1804). The studies showed that no reasonable correlations could be drawn between m e n t J symptoms and the degree of intestinal putrefaction. A highly inform'itive and critical review of the subject is due to Alvarez (1924). Today, although generally rejected a j untenable, autointoxication factors in mental disease are still clmsidered possible by some schools of thought, notably those led by V. M. Buscaino in I t J y and H. Baruk in France (Buscaino, 1'358, Baruk, 1959). It is also of interest to note that recent work has shown that toxic factors from the bowc.1 are concerned in the s y d r o n i e of hepatic enceplialopathy ( Sherlock, 1957; Dawson et a I., 1957). Abnormal tissue indolcs. Undcr this heading arc included theories which postulate the intra- or extracerebral production through aberrant tissue metabolism of an abnormal indole or indoles with toxic consequences to the brain ( e.g. Stafford-Clark, 1957). Theories of this nature havc followed the discovery during the past fifteen years of drugs with powerful actions on the brain, which are also in some degree indole derivatives. Prominent among these, because of the potency and persistence of their actions, are lysergic acid diethylamide a i d reserpine. However, both these compounds are complex molecules of which the indole structure only constitutes a relatively small part. Of greater revelance to the present hypothesis, since they are much more closely related to indoles occurring in human tissues, are certain simpler indoles with hallucinogenic action, namely bufotenine ( Fabing and Hawkins, 1956), the N-dimethyl and N-diethyl derivatives of tryptamine (Szara, 1957; Boszormenyi ct nl., 1959), and psilocybine (4-phosFhoryl-N-dimethyltryptainine, Delay et al., 1959; Rinkel et al., 1960). Imbalance of normally occrrrring indoles. This would occur as the result of a derangement of iridole metabolism in the brain, or possibly in the body as a whole, not necessarily involving the production of abnormal indole structures. Thus altered enzyme f iinction either of heredit,u.y ii,iture or through the occmrence of inhibitors might result in an excess or deficiency of an indole essential to normal cerebral function; similarly a maldistribution of indoles might occur as 'i result of defective transport across cell membranes. The indolcs normally present in mammalian brain include serotonin ( 5-hydroxytryptamine; Bogdanski and Uclen-

254

€7. RODNIGHT

friend, 1956), tryptamine (Hess and Udenfriend, 1959), and tryptophan (Price and West, 1960). Tryptamiiie can only be detected in brain after pretreatment of the animal by a monoamiiie osidase inhibitor. It is noteworthy that the distributions of these compounds in brain follow similar patterns; the highest concentrations are found in the hypothalamus and midbrain structures, while negligible quantities occur in the cerebral cortes. A widely investigated theory of indole imbalance postulates a cerebral abnormality in the serotonin pathway of metabolism; this is discussed in greater detail later. No specific hypothesis has been advanced concerning tryptamine, which in general is a less powerful pharmacological agent than serotonin. Tryptamine does, however, possess a depressant effect on synaptic transmission in an anesthetized cat preparation, not shown by serotonin ( Evarts, 1958). II.

Normal Body Fluid lndoles

In investigating these hypotheses by body fluid analysis it is clearly essential to know the nature and origin of the body fluid indoles occurring in normal subjects. Since there is no satisfactory evidence that the indole ring structure can be syntlietized in the body, it must be assumed for the present that the body fluid indoles arise from indole-containing precursors in the diet. By far the most. important of these from a quantitative point of view is tryptophan, and until recently this substance was generally assumed to be the only common dietary indole. It is now known, however, that certain items of diet, particularly fruits, may contain considerable quantities of serotonin and tryptamine ( Udenfriend et d.,1959); a direct dietary source for several other less iinportant indoles has been proposed by Armstrong et nl. (1958). But while interpretation of the body fluid indoles must always be made in the light of their possible occurrence in the diet, thc consumption of non-tryptophan indoles is normally only a fraction of the total tryptophan intake, which in adult man amounts to 1-2 gm per day. The normal metabolism of tryptophan is very complex. The major part of that ingested undergoes a series of reactions involving the immediate fission of the indole ring with the formation of kynurenine and eventually the vitamin nicotinamide and re-

BODY FLUID INDOLES I N XIEA-TAL ILLNESS

255

lated substances (Dalgliesh, 1955). Except in so far as they are involved in Hartnup disease these aspects do not concern the present account, which will be confined to consideration of pathways which result in conipomids retaining the indole structure. These pathways may occur either in the tissues or in the alimentary tract as a result of bacterial action; the two sources are considered separately below, but it should be noted that in certain instances the indolic compounds formed are the same. Structural formulas of some of the compounds mentioned are illustrated in Fig. 1.

A. PATHWAYS OF TRYPTOPIIAK ~IETABOLISXI IN TISSUES (FIG.2 ) The tryptamine pathway. Despite an early report (Werle and Mmniken, 1937) of the presence in mammalian kidney of an enzyme decarboxylating tryptophan to give tryptamine, unequivocal evidence that the reaction occurs in wivo was obtained only recently ( Weissbach et al., 1959). Previous studies (e.g. Erspamer, 1955) had already shown that thc further metabolism of tryptamine was oxidation by monoaniine oxitlase to indolylacetic acid. There is no evidence for the occurrence in mammalian tissues of N mttthylated derivates of tryptaininc, but the natural occurrence of 4-phosphoryl-N-dimethyltryptamine( psilocybine ) in certain mushrooms has recently been described (Hofmanii et nl., 1958). The try ptamine pathway is summarized in Fig. 2. The serotonin pathwcry. The independent discovery of this pathway in the late 1940’s by Erspamer in Italy and Rapport in the United States was an evcnt of great importance in the elucidaticn of tryptophan metabolism. I n view of the many excellent rey.iews of the subject already published (e.g. Udenfriend et nl., 1957; Page, 1953, 1958; Costa, 1960) it is unnecessary here to do more than indicate the principal reactions. These are also shown in Fig. 2. The first step consists of the hydroxylation of tryptophan to give 5-hydrox~tryptoplianby an enzyme the location of which is still uncertain ( Dalgliesh and llutton, 1957). Decarboxylation of 5-hydroxytryptopha11in then occurs by a widely distri buted enzyme to give 5-liydrosytryptamine or serotonin, which in turn is further oxidized by monoamine oxidase to 5-hydroxyindolylacetic acid. Both the decarbosylase and monoaniine oxidase occur in the brain with roughly the same distributions as that of serotonin.

257

BODY FLUID INDOLES I N MENTAL ILLNESS

The methylated derivatives of serotonin, namely bufotenine (N-dimethyl-5-hydroxytryptamine) and its betaine, bufotenidin, occur in certain amphibia, in the poisonous fungus Amanitu muppn, ? CONJUGATES

I

7

5-HYDROXYINDOLYLACETIC ACID

( 7 5-HYDROXYINDOLYLPYRUVIC ACID)

5 -HYDROXYTRYPTAMINE ? Transamination

Decarboxylation 5 -HYDROXYTRYPTOPHAN

Hydroxylation

NICOTINAMIDE KYNURENINE’

Decarboxylation T r ansamination TRYPTAMINE /

Oxidative deamination

/-

INDOLYLACETIC ACID

1

CONJUGATES

\.

INDOLYLPYRUVIC ACID)

I

INDOLYLLACTIC ACID

FIG. 2. Pathways of tryptophan metabolism in niammalian tissues. Comp o u n d s in parentheses h a v e n o t been actually identified, probably owing to instability.

and in the narcotic snuff “cahoba” [derived from Piptadenis peregrina (Stromberg, 1954)l. There is no satisfactory evidence for their occurrence in mammalian tissues or body fluids (see p. 279).

258

R. RODNIGI-IT

Transaniin&oiz. It was observed early that indolylpyrnvic acid (Berg ct al., 1929) and indolylacetic acid (Baugess and Berg, 1934 ) could replace tryptophan as a growth factor for rats, which suggested that part of tlie dietary tryptophan may undergo reversible transamination to intlolylp~-ru~ate. At that time it was uncertain to what extent this reaction occurred in the tissues as well as in tlie intestinal contents, but recently unequivocal clemonstrations of the pthn7ay have been made in liver and kiclney (Lin et al., 1958; IVcissbach ct ul., 1959). Indolylpyruvic acid is an unstable substance ~vliicli readily undergocs further reactions to give indolyllactic and indolylacetic acids, both of which are found in body fluids. Thus there are two routes by ~ h i c hindolylacetic acid may be formed by tissue metabolism: by tlie deamination of tryptnmine or by tlie transamination of tryptophan. Tliese are shown in Fig. 2. Evidence has also been obtained that some transamination of 5liydrosytryptoplian may occur in aniinal tissues ( Sandlcr ct d., 1960). Therefore in Fiz. 2 an alternative source of Fj-hyclroxyinclolylacetic acid tliroiigh this route is indicntcd, although the cxtcnt to which this actually csists in ui~ois less certain tlian in thee case of indolylacetic acid.

B.

TRYPTOPHAN

~IETABOLISMBY I'VTESIINAL FLORA(FIG.3 )

Pathways to indole and skntole. Quantitatively the most iinportant reaction undergone by tryptophan in the intestinal tract results in tlie formation of indole by tlie enzyme tryptoplimase, which is particularly active in Esclzeiiclzin coli. Thc enzyme catalyzes the direct splitting of the tryptoplmi molecule into equimolar amounts of indole, pyruvic acid, and ammonia, without the intervention of any indolic inteimecliate ( Stephenson, 1949 ). Contrary to tlie assumption often made in the older textbooks of hiocliemistry, indolylacetic acid is not an intermediate in this path~vay. Part of the indole formed in tlie bowel is absorbed into the portal circulation and detoxicated by the liver by oxidation to indoxyl and conjugation with sulfate to form indicaii. Some oxidation to indoxyl inay 'ilso occur in the intestinal wall during absorption ( Nicolai, 1942). The bacterial putrefaction of tryptophan also gives rise to

259

BODY FLUID INDOLES IN MENTAL ILLNESS

3-methylindole or skatole, often described as a normal constituent of feces. The details of this pathway, including the identity of the fecal bacteria responsible, remain to be elucidated, but it is known that some skatole may be absorbed into the blood stream INDOXYL SULFATE (INDICAN)

6-SULFATOXYSKATOLE Liver

INDOXYL

6 -HYDROXYSKATOLE Liver or intestinal mucosa

.

EINDOLE Co\/,

tryptophanase reation

SKATOLE

NICOTINAMIDE

TRYPTOPH

TRYPTAMINE

INDOLYLFORMIC ACID

INDOLYLACETIC ACID

CONJUGATES

INDOLYLLACTIC ACID.

r

CONJUGATES

FIG.3. Pathways of tryptophan metabolism originating from the intestinal flora.

and detoxicated by hydroxylation in the 6-position and then conjugated with sulfate to give a compound known as 6-sulfatoxyskatole, sometimes referred to as skatoxylsulfate (Horning et al., 1959). Other bacterial pathways. Fecal bacterial have been shown to be capable of degrading tryptophan to tryptamine and indolylacetic acid ( Weissbach et al., 1959); presumably a bacterial decarboxylase

260

R. RODNIGHT

TABLE I IKUOLE DEHIVATIVES DESCRIBED I N NORMAL URINEa Approximate excretion ( nig/24 hours)

Authors

L-Tryptophan Indoxyl sulfate (indican) 6-Sulfatoxyskatolc Indolyl-3-acetic acid Indolybcetylgliitaiiiine Indolylace tylgliicuronide Indolylacetylglyciric

20 100 0-50 5 No data NQ data No data

Frank1 and Dunn (1947) Sharlit (1932) Homing et ul. (195!3) Weissbach et (11. (1959) Jepson (1956) Jepson (1958) Ewins and Laidlaw (1913)

Indolyl-3-acetnmide 5-Hydroxyindolylacetic acid Indolyl-3-lactic acid Indolyl-3-f oniiic acid Indolylforniylglucuronide

No data 5 0.5 No data 0-60

Iiidolylglycolic acid Inrlolylacrylic acid N-Acetyltryptophan Tryptaniine 5-Hydroxytryptamine (serotonin)

No data No data No data 0.06 0.06

Jcpson (1958) Udcnfriend et (11. (1955) Armstrong et al. (1958) Armstrong et nl. ( 1958) Balahrishnan and Kodnight (1960) Armstrong et al. (1958) Armstrong et al. (1958) Armstrong et al. ( 1958) Rodnight (1956) Rodnight (1956)

Corn pound

a

See text for further comment.

and a transaminase are responsible for these reactions. Under anaerobic conditions cultures of E . coli metabolize tryptophan to indolylpropionic acid (Woods, 1935), but there is no satisfactory evidence for the occurrence of this compound in man. From cultures of Bacillus protezcs, however, a normal urinary indnleindolyllactic acid-may be isolated ( Sasaki and Otsuka, 1921).

C. NORMAL URINARY INDOLES Indoles which have been identified in normal urine with a reasonable degree of certainty are listed in Table I. Some comment on their probable derivation now follows. Iiztlicnn. With regard to the indican of urine, the suggestion has often been made (particularly in the early literature, but see Peters and Van Slyke, 1946) that some formation of indole from tryptophan might take place in the tissues as well as in the in-

BODY FLUID INDOLES IN MENTAL ILLNESS

261

TABLE I (Continued)

Probable origin

Other comnients

Diet, tissue proteins Intestinal flora Intestinal flora Tissues, intestinal flora, diet from ( 18) Conjugation of ( 4 ) Conjugation of (4) Conjugation of (4)

Microbiological determination

Degradation product of ( 6 ) Tissues, diet from (17) Tissues, intestinal flord Intestinal flora, diet Conjugation of (11)

-

Diet Intestinal flora, diet Diet Tissues Tissiies

-

Especially after feeding ( 4 ) Status uncertain: possibly confused with ( 5 )

-

See also Jepson (1958)

See also Greenberg et al. (1957) See also hlilne et al. (1959)

testinal tract. The experimcntal evidence, however, strongly favors the intestine as the sole source of indole in the body (Stoppani, 1945), and there appears no reason to question this in the light of modern knowledge. Some doubts have been expressed because sterilization of the intestinal tract in man and animals with antibiotics does not always abolish indican excretion ( Wooldridge et al., 1950; Conochie, 1953). This observation is most probably explained by the survival of drug resistant strains of organism; alternatively, continued excretion may be the result of the release of indole stored in the intestinal wall, such storage having been demonstrated in rats (Nicolai, 1941). By feeding aureomycin for 6 days Baron ct al. (1956) were able to abolish completely indican excretion in a case of Hartnup disease. Urines containing a high concentration of indican sometimes develop blue or purple colors on standing. This is due to hydrolysis of indican to indoxyl (possibly by naturally occurring arylsul-

262

R. RODXIGHT

fatases), which is readily oxidized to the dyes indigo and indirubin. The resulting colored urine may lead to a false diagnosis of porphyria (Berlin, 1957). Zndole acids. These may originate from tissue metabolism, bacterial mctabolism, or the diet. This is clcarly the case for indolylacetic acid, although the relative contributions made by the first two sources in any individual cannot be determined except by lengthy sterilization experimcnts. The major dietary source is represented by the tryptamine of tomatoes and certain species of plums (Udenfriend et nl., 1959). Indolyllactic acid is probably mainly derived from tissue metabolism (Roclnight, 1959), but an intestinal source has not been cscluded. With respect to S-hydroxyindolylacetic acid, tlie only other soiirce apart from tissue 1iic:tabolism would appear to be dietary serotonin occurring in bailanas, tomatoes, and other fruits (Utlenfrientl et al., 1959). Thore is no evidcncc that tlie enzymes of the serotonin pathwq7 are present in fecal bacteria. Of the conjugates of indolylncctic acid in urine, the glucuronicle is particularly interesting since this was s h o ~ by Jepson ( 1958) to break do\\7n under m-monical conditions to give indolylacetaniide. Thus the finding of the latter compound in urine, especially after chromatograp1;y in ammonia-containing solvents, must be interpreted in terms of indolylacetylglucuroiiidc. Minor and less constantly observed indole acids in urine arc the indolylformyl, indolylglycolic, and indolylacrylic acids, all of which probably arise principally from bacterial metabolism, but also possibly from the diet and tissue metabolism. The first two acids apparently result from the breakdown of indolylpyruvic acid formed by the transamination of tryptophan; such breakdown may take place in the body or in the urine if the latter is stored or examined under alkaline conditions ( Jepson, 1958 ) . Indolylpyruvic acid itself cannot be detected in urine even when extreme precautions are taken to preserve it, although some may he excreted after oral ingestion. The unstable indolylacrylic acid is probably the product of indolylacetic acid oxidation by fecal bacteria; its glycine conjugate has been reported in urine after feeding tryptophan (Xlilne et ( i l . , 1959) and may also be the major indole in urine in certain cases of actinodermatitis (Kimmig et al., 1958). All three acids probably occur to some extent in a normal vegetable diet (Jones and Taylor, 1957; Greenberg et d . , 1957; Armstrong et d., 195s).

BODY FLUID INDOLES I N MEXTAL ILLXESS

263

Acetyltryptophnn. The occurrence of this compound in normal urine has been reported by Armstrong et nl. (1958), by Trevartlien and Shaw (195s) after the ingestion of coffee, and by Langner and Berg (1955) after ingestion of D-tryptophan. Both acetyl-L- and acetyl-D-tryptoplian are apparently absorbed from the intestine without dcacylation and are only partially further metabolized (Price ant1 Urown, 1956). Thus it appears most likely that urinary acetyltryptoplian arises from u-tryptophan or acetylated tryptophans in thc: tlict. Zriclole bases. The trq)tamine and serotonin found in urine are almost certainly derivccl ciitirely from tissue metabolism. The tryptainine formed by the action of intestinal bacteria would be expected to undergo oxidation to indolylacctic acid before reaching tlie urine. Similarly, following ingestion of normal amounts of the fruits rich in indole :unines (Udenfriend et nl., 1959), an increase in the urinary crcrction of inclolylacetic acid and 5-hydroxyindolylacetic acid rather than the amines may be expected. It is possible that urinary tryptaniine and serotonin are derived exclusively from metabolism in the kidney. This is suggested by tlie fact that tryptainine is virtually absent from blood ( Rodnight, 1959), that blood serotonin is localized exclusively in cellular elements ( Zucker ci u!., 1!)5-2), and that trypotophan and 5-hydrosytryptophan decarl)o.iylases are both prcsent in kidney (Weissbacli et nl., 1959; Clark ct ol., 1954). D. NORMAL BLOODINDOLLS The known indoles normdly found in blood are listed in Table 11. When this list is coinp,ired with the urinary indoles in Table I, it may be seen that there ‘ire not,ible differences, indicating that many of the urinary compounds are rapidly excreted by the kidney if they are transported there by the blood stream. Particularly noteworthy in this respect i 5 tlie absence in blood of tryptamine and 5-hydroxyindolylacctic acid, neither of which can be detected by methods capable of dctccting as little as 0.02 pg/ml of the former or 0.05 pcg/ml of tlie latter ( Rodnight, 1959). This situation may b e contrasted witli t h t pertaining to serotonin and indolylacetic acid, both of which occur in blood. It is possible that serotonin remains in blood by virtue of the ability of the

BODY FLUID lNDOLES IN MENTAL ILLNESS

265

platelets to absorb it from solution (Hardisty and Stacey, 1955), but it appears that no such mechanism exists for tryptamine which cannot be detected in platelets any more than in plasma. With regard to the two indole acids, it appears that the body is well adapted to the rapid excretion of the breakdown product of serotonin, but curiously less efficient in excreting free indolylacetic acid. However, in contrdst to 5-hydroxyindolylacetic acid, a considerable proportion of the urinary indolylacetic acid is conjugated to glutamine or glucuronic acid, and it is possible that in this case conjugation is a nrw\\ary preliminary to efficient excretion.

E. CEREBROSPINAL FLUID INDOLES Probably owing to clifficulties of collection there has been relatively little research into the indoles in normal cerebrospinal fluid. Pathological studies are referred to later.

Ill.

Body Fluid lndoles in Mental Illness

In considering these aspects of the subject the comparatively rare conditions of Hartnup disease (formerly known as “H” disease) and phenylketonuria will be discussed first. This course is adopted rather than thc more obvious one of dealing first with the major mental illnessw, because only in Hartnup disease and phenylketonuria have well-defincd abnormalities in indole metabolism been found in subjccts suffering from a mental disorder. In addition, discussion of these conditions will form a useful background against which the more nebulous results obtained in the field as a whole can be considered.

A. HARTNUP DISEASE: This is a rare and recently discovered hereditary condition showing clinical resemblances to classic pellagra (Baron et nl., 1956; Rodnight and McIlwain, 1955; Hersov and Rodnight, 1960). At the time of writing thirteen cases have been described. The gene is apparently recessive. In view of the rarity of the condition and its recent discovery a brief description of the clinical symptoms is perhaps indicated. The manifestation of these is

266

R. RODNIGHT

intermittent m c l the first attacks usually occur in childhood or early adulthood; moreover they may only occur very rarely and their severity varies greatly from subject to subject. The actual symptomatology is complex and inconstant, but usually includes a light sensitive dermatitis and a variety of symptoms indicating an involvement of the central nervous system. In tlie ciises described by Baron et al. (1956) tlie latter consisted primarily of neurological signs, particularly a reversible cerebellar ataxia, wliereas tlie three cases of Hersov and Rodnight (1960) presented as a confusional psychosis, a depression, and an anxiety state; all were initially treated as psychiatric illnesses. It has been widely statcd, apparently on the basis of a very cautious statemerit in the paper of Baron ct al., that Hartnup disease is associated with progressive mental deterioration; no evidence for this was found in the three cases of Hersov and Rodnight, who included two adults of normal intelligence. For further clinical descriptions the reader is referred to papers already cited and to other references cited therein. In contrast to the variable clinical picture encountcrecl in Hartnup disease, the biochemical abnormalities are remarkahly constant from one case to another, and, so far as is known, throughout the life of the aflected individual. They consist of renal amiiioaciduria, gross indicanuria, and an excessive excretion of indolylacetic acid and its conjugate indolylacetylglutamine. The magnitude :ind nature of the amiiioaciduria has been studiecl by Evered (1956) and Hersov and Rodnight (l960), who fiiicl the pattern is unique and that tryptophan is among the many amino acids whose daily excretion is raised some 3-5 times. The amoiiiits of indican excreted are usually 3-4 times normal, ~ h i l efor indolylacetic acid values ranging from normal to 200 mg per day have been reported (Jepson, 1956). The clinical reseinblance of Hartnup disease to classic pellagra suggested some abnormality in nicotinamide metabolism, and indeed in some (though not all) cases nicotinamide therapy has proved successful in curing the symptoms of the condition (e.g. Hersov, 1955). However, no abnormalities could be detected in the excretion of nicotinamide metabolites after ingestion of the vitamin (Rodnight and McIlwain, 1955), nor is there usiially any history of a dietary deficiency. Besides the diet, another

BODY FLUID INDOLES IN MENTAL ILLNESS

267

source of nicotinamide in the body is from tryptophan through the kynurenine pathway (Fig. 2 ) , and it has been calculated that in adult man as much a s half the daily requirements of nicotinamide may be supplied by tryptophan (Goldsmith et al., 1952). I t was therefore proposcd hy Rodnight and McIlwain (1955) and Baron et al. (1956) that a nicotinamide deficiency might occur in Hartnup disease as a result of a faulty metabolism of tryptophan, whereby tryptophan is diverted from its route to nicotinamide into the nonessential patlnvays leading to the formation of indole ( and thus indican) and inclolylacetic acid. This proposal received experimental support when it was shown by Milne et al. (1959) that orally administered tryptophan is converted to kynurenine significantly less efficiently in IInrtnup subjects than in normals. Kynurenic and xanthurcnic acid excretion is also low in the condition (Hersov and Rodnight, 1960). Regarding the naturc of the abnormal tryptophan metabolism in Hartnup disease, there would apliear to be two possibilities: a deficiency in one of the enzymes responsible for the conversion of tryptophan to nicotinamide; or a distortion of tryptophan metabolism occurring a s a secondary consequence of the aminoaciduria, possibly involving tlic transport of tryptophan. The available evidence strongly supports the latter hypothesis. First, Baron et n2. (1956) showed by feeding antibiotics that the indicaniiria was intestinal in origin, tliiis indicating that the bacterial flora of the bowel were in some way implicated. Second, Milne ct al. (1959) demonstrated that when tryptophan is fed to Hartnup subjects a rise in urinary indican and indolylacetic acid occurs which persists for more than 24 hours, while in normal subjects tryptophan ingestion is followed by a rise in urinary indoles which return to normal within 10 hours. This suggests a delayed absorption of tryptophan from the intestine with a consequently greater opportunity for bacterial degradation to indole and indolylacetic acid. I t is unlikely, however, that this diversion in itself is the only cause for the low efficiency in converting tryptophan to nicotinamide, sincc it only accounts for the loss of 15-20oC,’0 of the dietary tryptophan, which, at 1.5 gm per .day, is about six times higher than the minimum requirement for normal subjects (Denko and Grundy, 1949; Leverton et nl., 1956). In all probability a defect in tryptophan transport is also present in

268

R. RODNIGHT

other cells of the body, such as those of the liver, with the result that tryptophan fails to reach in adequate amounts the enzyme sites concerned. Furthermore, since the aminoaciduria involves many other amino acids, it is probable that defects in the trailsport of these also occur in the condition. The intermittent nature of the clinical attacks of the disease, despite the constancy of the biochemical abnormalities, suggests that a precarious balance exists in these subjects between dietary supply and the demands of the tissues, where an inefficient metabolism of tryptophan results in a requirement for nicotinainide and/ or tryptophan which cannot always be met. In the normal subject a more efficient metabolism ensures a lower requirement, and deficiency only occurs under extreme conditions of deprivation. Explained in these terms the clinical manifestation of Hartnup disease may be considered as attacks of pellagra with the typically variable symptomatology often seen in the early stages of that condition (Bicknell and Prescott, 1953). However, this may well be an oversimplification of the situation. In the first place nicotinamide does not always alleviate the symptoms of Hartnup dis1956) whereas it only fails in classic pellagra ease (Baron et d., where irreversible damage has occurred. Second, it is not yet clear whether the essential deficiency in the disease is one of nicotinamide or of nicotinamide derived from tryptophan. This distinction is of iinportmce because of obscrvatioiis wliicli indicate that the nicotinamide derived from tryptophan is utilized for the synthesis of essential pyridine nncleotides in blood and liver to a greater extent than is dietary nicotinainicle (Clraloupka ct al., 1957), thus suggesting that tryptophan plays more than a supplementary role in the nicotinamide economy of the body. Finally the possibility of deficiencies involving other amino acids or their derivatives occurring in the condition must not be overlooked. Hartnup disease has been discussed at some length despite its apparent rarity, since it aifords an interesting example of a metabolic abnormality encountered in psychiatric practice involving indoles. Careful screcning of a large number of mental hospital patients suggests that the incidence of the condition is probably very low. For the wider problems of mental illness, therefore, Hartnup disease illustrates mechanisms by which a

BODY FLUID INDOLES I N MENTAL ILLXESS

269

hereditary biochemical defect may periodically become manifest as a psychiatric illness.

B. PHENYLKETONURIA The clinical, genetic, and principle biochemical aspects of phenylketonuria are too well known to merit more than a brief outline in this account (recent reviews are by Kretchmer and Etzwiler, 1958; Harris, 1959). The condition is characterized clinically by oligophrenia of carly onset and biochemically by a deficiency in the enzyme phenylalanine hydroxylase, which is responsible for the introduction of a hydroxy group into phenylalanine to form tyrosine. In cases of phenylketonuria the gene is recessive, and homozygotes constitute between 0.002 to 0.006% of the general population in the United Kingdom ( Munro, 1947). Associated with the enzyme defect is a diversion of phenylalanine metabolism into abnormal pathways and excessive amounts of unusual degradation products of the amino acid are excreted in the urine. The most important of these is phenylpyruvic acid, but phenyllactic, phenylacetic, and O-hydroxyphenylacetic acids also occur. The excretion of these phenolic acids may be reduced to near normal levels by drastically restricting the phenylalanine content of the diet (Woolf et al., 1955, 1958; Armstrong and Tyler, 1955). The expedient of a low phenylalanine diet may also result in a clinical improvement in the condition (Coates et al., 1957). Abnormalities in indole mctabolism in the condition were unsuspected until recently. They are quantitatively very much less impressive than the phenylalanine abnormality, but are of interest in view of the potential significance of indoles in cerebral function. The indolic abnormalities involve two different pathways of tryptophan metabolism: the route leading to indolyllactic and indolylacetic acids and the 5-hydroxyindole pathway. Indolyllactic and indolylacetic acids. Armstrong and Robinson (1954) were the first workers to observe an increased excretion of indole acids in phenylkctonuria. The excretion of indolyllactic acid was found to be greatest, the output ranging from 20 to 150 mg/gm creatinine. Assuming a daily excretion of 1 gm of creatinine, this suggests the excess excretion in phenylketonuria is about 100 times the normal value (Table I ) . Figures for indolylacetic acid

270

R. RODNIGIIT

were not quoted, but the excretion was stated to be usually increased in this disease. 5-Hyd~oxyintZoZepatlitcay. It was found by Pare et al. (1957, 1959) that the serum levels of serotonin and the urinary excretion of 5-hydroxyindolylacetic acid were significantly lower in children with plienylketonuria than in other mentally defective children, or in a group of children awaiting tonsillectomy in a general hospital. The means of the serum serotonin levels for tlie three categories of subject were: children with phenylketonuria: 57 m g / m l ; other mentally defective children: 270 m\rg/ml; mentally normal children: 124 ml,ig/ml. A similar gradation of results was seen in the urinary output of S-hydroxyindolylacetic acid Some of the mentally defective children not sufkring from plicnylketonuria showcd abnormally high serum serotonin levels, hut this observation has not, as yet, been followed up. During treatment with a low plienylalanine diet the excasive excretion of indole acids disappears and serum serotonin and urinary 5-liydroxyindoly1acetic acid levels return to normal ( Armstrong and Tyler, 1955; Pare et nl., 19S8). Since tlie abnormal phenolic acid excretion is also corrected by the diet, it appears possible that the indolic abnormalities are the result of a distortion of tryptophan metabolism by an accumulation of plien~ l~ il~ uiine metabolites. In support of this are observations that phenolic acids inhibit the enzyme 5-hydroxytryptophan clecnrboxylase in tiitro (Davison and Sander, 1959). It is also relevant to note tliat a deficiency of blood catcchol aniincs has been found in phenylketonuria ( Weil-Malherbe, 1955) and that dihydroxyphenylalanii~e decarboxylasc, an enzyme concerned in tlie biosyntliesis of the catechol amines, is also inhibited by phenolic acids ( Fellman, 1956). However, it is possible that 5-hydroxyindole meta1)olism is affected at other sites besides the decarboxylation of 5-hydroxytryptophan; for example, a reduced activity of tryptophan hyclroxylase might also result in a serotonin deficiency. Finally tlie possibility must be considered that a defect in the transport of tryptophan is the cause of its distorted metabolism. Here again tlie high levels of plienylalanine and its metabolites might conceivably be responsible, but at tlie moment there is no esperimental evidence to support tlie hypothesis. If the indole abnormalities in phenylketonuria are proliably

BODY FLUID INDOLES I N MENTAL ILLNESS

271

secondary to the defect in plienylalanine metabolism, the question still arises as to whether they are in any way related to tlie mental deficiency of the condition. I n Hartnup disease, for instance, the indole abnormalities arc in a sense secondary phenomena, but nevertheless of great importance in its clinical manifestation. The evidence regarding plienylketonuria suggests a very much more complex situation. Thus Pare et al. (1959) found no correlation between the magnitude of the serotonin deficiency and intelligence in the disease, and Kirman et ul. ( 1959) were unable to increase intelligence by raising serum serotonin levels through administration of 5-hydroxytryptoplian.

c.

M A J O R h'lEiVTAL I L L N E S S 3

No consistent and ~ l l - d c f i l ~ eabnormalities d in indole metabolism comparable to those describcd above in Hartnup disease or phenylketonuria have yet been discovered as characteristic of any of the major mental illnesses siich as schizophrenia or endogenous depression. The subject will tlierefore be considered under different aspects of indolc metabolism, rather than under clinical diagnosis. This will enablc the ininor abnormalities, which have been found in mentally ill patients as a group of subjects to be considered in perspective; however, it will be pointed out in the text where a teiideiicy toward abnormality appears to be confined to a single diagnostic category. 1. Urinary Indoles of Excliisioe Iiitestinnl Origin

Indican. A moderate degree of indicanuria has often been reported in mental hospitd patients ( e.g., Townsend, 1905, G d lotta, 1929; Sano, 1954). This is clearly differentiated by both its variability and quantity from tlie persistent liigli excretion of indican found in Hartnup disedsc and is most probably related to the gastrointestinal disturbances which are common in mental patients (Henry, 1928; Altscliule, 1953). This conclusion is borne out by other workers who find a greater than normal variation of indican excretion among mentally ill pitients but no consistent or reproducible trends (Ijorden, 1906; Hodnight and Aves, 1958; Goldenberg et al., 1960). Sulfntoxyskntole (skatoxylsulfate). In a study of urinary in-

T dole excretion in mental illness, Rodnight and Aves ( 1958), using paper chromatography, found that while there were no niajor abnormalities evident, there did occur a tendency for more strong spots of an unknown indole (“unknown 2”), since identified as 6-sulfatoxyskatole, to appear on the chromatograms of specimens from the mental patients. The tendency was most marked in new admissions to a psychiatric observation ward and in patients suffering from depression, but also occurred to some extent in a group of schizophrenic subjects. Occasional strong spots of the substance were also found among the normal population. This finding is supported by other workers, also using paper chromatography, who report an abnormal incidence in schizophrenia of a urinary indole with the characteristics of sulfatoxyskatole (Leyton, 1958: spot Q; Feldstein et al., 1958: spot 32; Riegelhaupt, 1958: spot QFB; Forrest, 1959: spot Q.F.B.; Buscaino and Stefanchi, 1958; Sprince et al., 1960). In these studies groups of schizophrenic subjects were compared with normal subjects, and in most instances other classes of mentally ill patients were not examined. This is important because there is no evidence that the excretion of the substance is characteristic of schizophrenia. Thus in addition to the study of Rodnight and Aves referred to above, Curzon (1958) was unable to detect differences in its excretion between patients with schizophrenia and other diagnoses, and Armstrong et al. (1958) found that what is almost certainly the same substance (compound 13) frequently occurs in excessive amounts in urine from mentally defective childrcn in institutions. Moreover, a very high excretion of sulfatoxyskatole occurs in the malabsorption syndrome, certain anemias, and in spontaneous Heniz body formation (Homing and Dalgliesh, 1958). These authors suggest the precursor of sulfatoxyskatole, 6-hydroxyskatole, may play a role in the pathology of these conditions by damaging cellular structures. Studies are at present in progress to determine whether a high sulfatoxyskatole excretion in mental illness is akin to the moderate indicanuria often found in patients or whether it is indicative of a more fundamental abnormality as is the gross indicanuria of Hartnup disease. In the latter case the excretion in patients would be expected, first, to be very much higher than the maximum found in normals, and, second, to be a persistent reproduci-

273

BODY FLUID INDOLES IN MENTAL ILLNESS

ble feature of their excrctory pattern of indoles. Daily excretions of sulfatoxyskatole have therefore been determined in groups of normal and mentally ill subjects by an ion exchange and paper chromatographic method which will be described elsewhere. (Quantitative information of- this nature was not available in the studies quoted above, since these were essentially qualitative surveys of single samples of urine.) Preliminary results of this study show that, as previous work had indicated, the normal range of sulfatoxyskatole excretion is very wide and that the frequency distribution curve of indivitlu,il values is highly positively skewed, more than half the values occurring in the range <S-15 mg per day (Table 111). The mean cwretion of the 65 patients studied TABLE 111 DAILY EXCRETION OF SULFATOXYSKATOLE IN NORMAL AND MENTALLYILL SUBTECTS

Excrrtion ( r n g / U Iroiirs) Per ccnt subjccts with values in range:

Subjects Normals ( 4 3 ) Patients ( 65)

\lean

Ihijie

< 16

24

<5-130 <5-240

58 46

37

1 6 4 8 48-80 80-160 26 30

11 12

5 10.5

> 160 1.5

so far is about 50% higher than that of a comparable group of 43 normals and all the indications are that this is due to a less positive skew on the distribution curve, rather than to a distinct group of high excretors. Regarding persistence of excretion in both normal and mentally ill subjects the level of excretion, even if high, remains reproducible over a period of weeks, rarely varying by more than i-50c/o of the mean in a series of determinations (Table I V ) . In these results no trend was observed and the fluctuations which did occur were randomly distributed and were not correlated with mental state (the writer is grateful to Dr. I. Lodge Patch for this information). Further, no correlations were noted with indican output or with the excretion of indole acids. In one patient a high lcvel of excretion was abolished by administering antibiotics for 3 days, but the excretion was back to its normal level 3 days after withdrawal of the drug. A high level of sulfatoxyskatole excretion does not appear to be related to constipation when the occurrence of this complaint was assessed by the statement of the patient or the nurses’ reports. As is well known, however, minor disturbances of bowel function

TABLE IV PERSISTENCE OF SULFATOXYSKATOLE EXCRETION IN MENTALLY ILL SUBJECTS Subject and sex

Age (years )

Diagnosis

1M 2M 3F 4F 5F 6F

54 30 44 30 41 15

Depression Schizophrenia Schizophrenia Schizophrenia Depression Schizophrenia

Duration of experiment

Number of

Output (mg/% hours)

a

( weeks )

determinations

Mean

Range

rn

7

8 5 9 5 8 5

61 60 90 56 70 70

32-110 32-96 48-160 48-80 24-96 48-80

2 6 4

7 7

88 3

BODY FLUID Ih-DOLES I S hfENTAL ILLNESS

275

are difficult to judge by subjective report, particularly in mental patients, and efforts are being made to apply more objective measures. From these results some provisional conclusions may be drawn. I n the first place, while cscessive sulfatoxyskatole excretion is distinguished by its persistence from the moderate indicanuria often found in mental illness, no support has emerged so far for the suggestion that the high excretors of the substance in the mentally ill population constitute a distinct group of subjects, as do the high indican excretors who are Hartnup subjects. Second, the characteristically constant nature of the excretion in any one individual shows that the factors which condition it are relatively constant features of tlie individual’s environment. Among these are presumably diet and internal factors concerned with intestinal function, such as motility, mucous secretion, gastric acidity and absorption. Diet is probably the least important since minor variations (such as omitting meat protein) do not alter excretion, and both low and high excretors may be found among individuals on the same diet. The relative contribution of the other factors is unknown, but related to them is the question as to whether urinary sulfatoxyskatole is derived from skatole produced in the small or large bowel. Despite the enormously greater numbers of bacteria in the colon, an origin in the small bowel is suggested by the absence of a correlation between excretion of the substance, indican output, and large bowel function. Moreover, although in health the greater part of the small intestine is sterile, it is possible that some bacterial growth may occur in the terminal portion. This is suggested because recent work by Dixon has shown that sterility in the small bowel is not maintained by a bacteriocidal action of the intestinal contents but by mechanical removal by peristalsis of tlie bacteria introduced by the diet (Dixon, 1960). Clearly then, differences in motility of the small intestine [which commonly occur in mental illness, Henry (192S)I would influence the opportunity for bacterial growth in the ileum; and since skatole as a lipid-soluble substance would be expected to be absorbed more rapidly from the small intestine than from the colon, it is conceivable that urinary sulfatoxyskatole excretion is an indication of the number of skatole-producing bacteria surviving in the small intestine. This viewpoint is taken by Horning and

276

R. RODNIGHT

Dalgliesh (1958) with respect to the biochemical picture in the malabsorption syndrome, where a high excretion of skatole metabolites is associated with extensive infection of the small intestine ( Black, 1959 ) .

2. Various Urinnr!j Inclolc Acids ( Excliuling 5-Hydroryindolykcetic A c i d )

Inclolykicetic cicirl. A high excretion of indolylacetic acid in schizophrenia was claimed by Sherwood ( 1957 ) , However, the present writer, using a highly specific paper chromatographic method (Rodnight, 1959), was unable to find any consistent diffr*rences from normal in several mental illnesses, including schizophrenia (Tablc V ) . The same negativc conclusion was reached by Leyton TABLE V INDOLYLACETKACID EXCRETION IN MENTAL ILLNESS Mean age of subjects ( years )

Mean output ( mg/gni crratinine,

Normal (18)

29.2

1.25 i.0.02

hlentally ill subjccts Schizophrenic ( 20) Schizophrenica ( 1 ) Depressive (15) Dcpressivc ( 1) Hypomania ( 3 ) Dcinentia 1

33.8 15 48.4 52 28

1.95 t 0.38 16 2.58 iz 0.49 13 1.51 0.35

Diagnosis and number of subjects

a

60

t standard error)

Subject 6F in Table IV.

(1958) and Weissbach et 01. (1959). In the author’s study, however, high outputs were observed in two patients, one a schizophrenic and the other a depressive. Excretion in the schizophrenic subject, who also showed a high excretion of sulfatoxyskatole (subject 6F, Table I V ) , was observed to be persistently high over a period of some weeks. The high excretion apparently originated in the intestinal tract since treatment with antibiotics reduced it to normal levels, without, ho\vever, affecting the subject’s mental state. Here again there must be special reasons to explain why the intestinal flora produce an excess of this particular degradation product of tryptophan.

277

BODY FLUID INDOLES IN MENTAL ILLNESS

Indolyllactic acid. In the study referred to above (Rodnight, 1959) no abnormalities were found in the excretion of indolyllactic acid in schizophrenia or depression. Indolylformic acid (indolylcarboxylic acid ) and conjugates. Free indolylformic acid (often named indolylcarboxylic acid) has not been widely observed on urinary chromatograms, possibly because it reacts with the usual detection reagent ( p-dimethylarninobenzaldehyde, or Ehrlich’s reagent) very slowly. In the course of developing the method for determining sulfatoxyskatole in urine, however, a fast reacting unknown indole was frequently observed on chromatograms of urine extracts. This has been provisionally identified as indolylformylglucuronide ( Balakrishnan and Rodnight, 1960). Excretion of this substance shows some correlation with sulfatoxyskatole excretion and also occurs more frequently in mentally ill subjects (Table V I ) ; it may possibly be identical TABLE VI DAILYEXCRETION OF INDOLYLFORMYLGLUCURONIDE IN NORMAL AND MENTALLY ILL SUBJECTS Range (mg/24 hours )

37 Nonnal subjects

48 hlentally ill subjectsa

(%)

(%)

< 20

70 19 11

42 30 18 10

20-40 40-60 > 60 a

None

Seventy-three determinations in all were made.

with Leyton’s spot S (Leyton, 1958). Since its origin is apparently intestinal, the remarks already made concerning sulfatoxyskatole excretion are presumably also applicable in this case.

3. Indole Amine 2lletabolism This has been studied in mental patients in two ways: by determination of the body fluid levels of the amines themselves and by following the urinary excretion of the indole acids derived from the amines. The advantages and disadvantages of these two approaches are discussed later. Urinary indole aniines. Serotonin and tryptamine are the only indole amines detectable in urine by a sensitive ion exchange and paper chromatographic method ( Rodnight, 1956); tryptamine has

278

R . RODNIGHT

also been determined in urine by a fluorometric method, with similar results (Weissbach et nl., 1959). Values for the daily excretion of these substances in some cases of mental illness are given in Table VII; further details of the methods used are given by Rodnight DAILYEXCRETION

OF

SEROTONIN

Diagnosis, sex, and num1)er of subjects

TABLE v11 AX’D THYP TA~I IN I N ENORMAL ILL SVUJECTS Output (pg/24 hours) hlcan

Range

AND .\[ENTALLY

Combined means for inales and females

Serotonin Normal, male ( 2 0 ) Normal, female ( 6 ) Schizophrenic, nialc ( 1 2 ) Scliiiophrenic, fenialc> ( 10 ) Depressive, male ( 8 ) Depressive, feinale ( 1 0 )

72

55 54 47 40 54

45-110 10-83 15-90 10-120 30-68 15-80

68 51 47

Tryptamine Normal, inale ( 1 3 ) Normal, fem‘ile ( 6 ) Schizophrenic., male ( 9 ) Schizophrenic., female ( 8 ) Depressive, male ( 8 ) Depressive, female ( 10)

64 56 52 72 39 30

20-120 40-72 10-110 30-170 10-60 10-80

61 62 34

(1959). The results of this study indicate no major abnormalities in indole amine excretion in any of the subjects esainined, all of whom were acutely ill patients, hospitalized for a mavinium of S weeks. There was, ho\vever, a tendency for both thc schizophrenic and depressive subjects to excrete less than the normal mean output of serotonin and for the depressives to excrete rather less tryptaniine than normal. These differences can be shown to be statistically significant ( P < O.OS), but it is doubtful if they are of importance in view of the following points: (1) the chomatographic method used for the determinations, while being highly specific, possesses an error of t 205; ; ( 2 ) difficulties were encountered in collecting the full 24-hour volume of urine from some of the patients; ( 3 ) the metabolism of serotonin may lie affected by the level of tryptophan in the diet (Zbinden ef al., 1958) : faulty feeding habits are common in mental patients and if prolonged might conceivably be reflected in

BODY FLUID INDOLES IN MENTAL ILLNESS

279

serotonin output; ( 4 ) the fact that several low excretion values for both amines were found in the normal population suggests that with a larger sample the differcnces might be less. Absence of ntethy luted intlole nrnines in urine. The hallucinogenic properties of N-dimethyltryptamine and N-dimethylserotonin (bufotenine) have already lwen alluded to; it was therefore of interest to examine the possibility that they occur in the urine of mentally ill subjects. In the method used for determining serotonin and tryptamine the recovery experiments showed that the niethylated compounds would have been detectable if their 24-hour excretion had been of the order to 20 pg or greater. Although the relevant areas of the chromatograms were always carefully examined, no trace of these compounds IIU ever detected. A more sensitive procedure was therefore devised, whereby an excretion of 5 pg per day could be measured. This mcthod was based on elution of the relevant areas of preliminary clrromatograms and re-chromatography of the eluates in a higher equivalent volume of urine; details will be published elesewhere. E x h determination was controlled by recovery experiments. In eight specimens of urine from mentally ill patients, including six from schizophrenic patients, no excretion of bufotenine was detectable; methyltryptamines were absent from five of these specimens, but in the remaining three the presence of interfering material made it impossible to exclude an excretion of less than 15 pg per day. Serotonin levels in blood. Reference has already been made to the fact that serotonin is the only indole amine detectable in the blood of normal subjects, this observation was also found to apply to a series of ten mentally ill patients (including three with schizophrenia). The limits of detection of tryptamine or bufotenine by the method used was of the order of 0.10 pg/ml. Other blood indolcs ( Table I1 ) were also nornicil. Serotonin levels in blood have been compared in normal subjects and mental illnesses by Felclstein et nl. (1959) and by Jus et 01. ( 1958) using fluorometric and bioassay techniques, respectively. Their results are summari7etl in Table VIII. In Feldstein’s study there was no difference in the mean or range of results found in 17 normal subjects and 22 chronically ill schizophrenic subjects, but a significantly low mean ( P 0.02) was found in 13 acutely ill newly admitted psychotic patients. JLISel nl. also report at low mean value

280

R. RODNIGHT

in 44 schizophrenic patients, but here no division into acute and chronic patients was made; in addition the upper and Iowcr limits in the mentally ill subjects are much wider than in the study of E’eldstein et al. Both groups of workers are righly hesitant in ascribing any significance to their results in relation to mental illness in view of the wide scatter of the values. It is also possible that the nutritional state of some of the patients contributed to the tendenc!. for low levels to occur (see Zbinden et nl., 1958). TABLE VIII BLOODSEROTONIN LEVELS I N MER’TAL

ILLNESS

Conccntration ( nipg/ml) Alcan Authors and subjects Feldstein et al. (1959) Normal males ( 15) Chronic male schizophrenics (22 ) Acute male psychotic subject3 ( 13) Jus et al. (1958) Normal subjects (10) Schizophrenic subjects ( 44)

( +- standard deviation)

R,inge

190 -c 80 170 5 60 130 k 70

60-390 60-3 10 50-290

135 67

5-187 17500

Effect of reserpine and electroshock on blood serotonin. The administration of reserpine in therapeutic doses has been found to cause a rapid fall in the blood serotonin to very low levels (Green et nl., 1957; JUSet al., 1958; Rodnight, 1959). Typical results from the author’s own work, where a bioassay method was used for the determinations, are given in Table IX. The rate of fall is seen to be very rapid and in fact the low values are reached some days before any clinical response to the drug occurs; after withdrawal of the drug from one subject (subject 4 in the table) the l~looclserotonin level remained low for at least 2 weeks. No correlations betv7een the rate of the fall or its magnitude and the therapeutic result of the drug have been observed. Since the very high levels of blood serotonin which occur in malignant argentafinoma ( carcinoidosis ) ( Stacey, 1957) are not associated with psychiatric disturbance, it appears that wide variations in blood serotonin in either direction do not affect mental status. In contrast to the striking effect of reserpine, electroshock treat-

281

BODY FLUID INDOLES IN hIENTAL ILLSESS

ment to patients (Green et al., 1957) or to rats (Bertaccini 1959) does not affect the blood levels of serotonin. Serotonin levels in cerebrospinal and ventricular fluids. These have been determined in a variety of neuropsychiatric conditions by Turner and Mauss ( 1959), using a bioassay technique (see also Sachs, 1957). Serotonin, when present, was found to be very low, generally of the order of 0.03 irg/ml or less; it could not be detected in 77% of the samples of spinal fluid and 47% of the ventricular TABLE IX EFFECTOF RESERPINEO N BLOODSEROTONINLEVELS IN ;\IENTALLY ILL SUBJECTS

Subject

Age ( ycars)

1. Psychotic illness

40

2.

3.

Neurotic disorder

Schizophrenia

4. Senile dementia

36

Reserpine dosage (mg/day) None 5 5 None 1.*5 1.5 1.5

18

None 10 5 to 7

G8

None 1 1 None

Period of Serotonin administration concentration (days) (mpg/ml)

1

7 7 14 63 1

7

1 5 14

160 15 5 170

< 15

< 15 < 15 375

< 15 < 1.5 350 320 About 15 40

fluid. No correlations were found with diagnosis, but in one catatonic schizophrenic patient the ventricular fluid serotonin level was surprisingly high-0.2 Pgiml. Further details of this interesting case are given in the original paper. Urinary indole acids, particularly 5-hydroxyinclolylncetic acid. Both tryptamine and serotonin are broken down in the tissues by the enzyme monoamine oxidase to give indolylacetic acid and 5-hydroxyindolylacetic acid, respectively. Reference to the excretion of indolylacetic acid in mental illness has already been made; these measurements of urinary indolylacetic acid, however, are of no value in assessing tryptamine metabolism since the acid is also the end

282

R. RODNICHT

product of another pathway of tryptophan metabolism (Figs. 2 and 3 ) , which occurs, moreover, in the alimentdry tract as W C ~ Ias in the tissues. 5-Hydroxyindolylacetic acid is certainly derived entirely from tissue metabolism, but even here the extent to which transamination of 5-hydroxytryptophan rather than oxidative deamination of serotonin is responsible for the urinary product is uncertain (Sandler et al., 1960). There have, however, been many studies of urinary 5-hydroxyindolylacetic acid in mental illness and some of these will now be discussed. Determinations of endogenous excretion rates for 5-hyclroxyindolylacetic acid in normal and schizophrenic subjects have been made by Buscaino and Stefanchi (1958), Ranerjee and Agarwal (1958), Sano et d.(1957), Feldstein et nZ. (1959), Curzon (1958), Lauer et al. (1958), and Kopin (1959). In all these studies the method of Udenfriend et nl. (1955) was used. Except in Kopin’s work the diet of the subjects was not strictly controlled, ant1 in the interpretation of the results this is of particular importance in view of the known influence of the ingestion of serotonin-containing fruits on 5-hydroxyindolylacetic acid excretion ( Udenfriend et nl., 1959, Barbeau and Witkofl, 1959). It is also necessary to point out that only in the studies of Lauer et nl. and Kopin is it certain that the patients were not receiving drug therapy during the period of urine collection; drug control is of importance because Ross et ul. (1958) showed that phenothiazines, both when added to urine and given by mouth, seriously interfere with the Udenfriend method for determining 5-hydroxyindolylacetic acid by inhibiting the color tlevelopment. In view of these uncontrolled interfering factors, it is perhaps surprising to find that the results of the studies as a whole are clearly negative. Only in one study, that of Banerjee and Agarwal, were markedly higher than normal excretion rates obtained in ten schizophrenic subjects, and these are so much at variance Xvith all the other results that it must be assumed that a methodological error or inadequate sampling was responsible for their results. Buscaino and Stefanchi claimed a slightly raised excretion for catatonic patients as compared with other types of psychotic illness, but this was not confirmed by Sano et nl. or Feldstein et al. Low values for some patients with schizophrenia were found by Sano et al., but this most probably was due to an interference in the determination through chlorpromazine medication. Kopin, however, found

BODY FLUID INDOLES IN MENTAL ILLNESS

283

that low values were often obtained in schizophrenic subjects in poor nutritional state and that treatment of these patients with pyridoxine tended to bring the excretion closer to normality. An excellent discussion of these and other results in mental illness is given in the paper of Feldstein et al. Among the points stressed by these authors is the variability of 5-hydroxyindolylacetic acid excretion even under controlled conditions : variability, moreover, may be greater in schizophrenic subjects than in normals, and consequently, in order to obtain a reliable estimate of mean excretion rates, long collection periods should be used. Effect of tryptophan ancl serotonin administration on urinary 5-hydroxyindolylacetic acid. E. A. Zeller and associates, in a careful study (Lauer et d., 1958) found that seventeen normal control subjects approximately doubled their output of 5-hydroxylindolylacetic acid during the 5-hour period after oral administration of L-tryptophan (0.1 mmole per kilogram body weight, equal to about 1-1.5 gm per subject). In a group of twenty-six schizophrenics the same dose of tryptophan produced no rise in 5hydroxyindolylacetic acid; pretreatment levels of excretion were the same in both groups and the differences in the response were statistically significant ( P < 0.01 ). Kopin ( 1959), however, studying sixteen schizophrenic subjects and six normals under controlled conditions failed to reproduce this interesting result, finding that both groups responded eqiially. Yet another result was obtained by Banerjee and Agarwal ( 1958), who reported that schizophrenic rather than normal subjects responded to oral tryptophan with a rise in urinary 5-hydroxyinclolylacetic acid. These two latter studies have led Feldstein et a1. (1959) and Kety (1959) to clismiss the results of Zeller’s group as of doubtful significance. In the author’s opinion, however, the subject might justify further investigation. In the first place the results of Banerjee and Agarwal are by no means comparable, since the rise in urinary 5-hydroxyindolylacetic acid they reported apparently occurred during two successive 24-hour periods after tryptophan administration, whereas in the other studies observations were confined to a period of 5 hours after administration. Furthermore, as has already been mentioned, the basal levels of 5-hydroxyindolylacetic acid excretion quoted by Banerjee ancl Agarwal are inexplicably some four times higher than those reported by numerous other workers in

284

R. RODNICIIT

schizophrenia. In Kopin's study the conditions would appear to be very similar to Zeller's, except that he used a dose of tryptophan ( 5 gm.) some three to four times higher than did Zeller. This in itself would not be likely to obscure a major deficiency in the serotonin pathway, but might conceivably fail to uiicovcr a minor defect of interest. At all events more work is required to clear up the discrepancy between the rcsults. Buscaino and Stefanchi ( 1958) gave serotonin by intramuscular injection to a group of fifteen normal controls and nonpsyehotic hospital patients on the one hand and thirty-six schizophrenic subjects on the other, and then measured the precentage recovery of serotonin in the urine as 5-hydroxyindolylacetic acid. This they claimed was greater in the schizophrenic group ( 42% ) than in the controls (25% ) . There was, however, considerable overlap in the figures and the result should perhaps be considered as demonstrating that no major differences wcre prescnt in the oxidative deaminatioii of serotonin in these subjects. Etfect of reserpine on urinary 5-hy~lroryindolylncetic acid. Reserpine in therapeutic dosage h a s been found often to efiect a small but definite rise in urinary 5-hydroxyindolylacetic acid excretion during a period of 24 hours after administration, but particularly in the first 8 hours (Todrick et d.,1958; Vnlcourt, 1959). Some of the patients in Todrick's study failed to respond with a rise in excretion, but no correlations between this i 1'11 ure and psychiatric diagnosis were observcd. The rise, when it occurs, is presumably related to the action possesscd by reserpine of releasing serotonin from its stores in the body and exposing it 1955). to the action of monoamine oxidase (Pletscher et d.,

IV.

Concluding Discussion

The findings pertaining to Hartnup disease and phenylketonuria have already been discussed under these headings. It remains therefore to consider, in relation to the hypotheses set out in the introduction, the mainly negative results which have been obtained in the studies of the major mental illnesses. Autointoxication. This, it will be recalled, postulated a toxic effect on the brain of indoles derived from the action of bacteria

BODY FLUID INDOLES IN MENTAL ILLNESS

285

in the intestinal tract. Present studies have reinforced the earlier convictions that the theory is groundless, for although modern analytical methods have shown that a considerable number of the body fluid indoles do, in fact, originate in the intestine, their occurrence cannot be corrclated with mental abnormality. These indoles are, moreover, qiialitatively similar in both normal and mentally ill subjects, an observcition ~ h i ~ lends l l no support to the special autointoxication thcory of V. M. Buscaino [which proposes that a characteristic intestinal flora results in the production of abnormal toxins in sclrizophrenia ( Bnscaino, 19SS)l. Nor is there any reason to bclicve that an impaired detoxication of intestinal indoles by the livcr occurs in mental illness since most of them are found in urinc from patients, as from normal subjects, in the conjugated form. Abnormal tissue indolcs. Evperience suggests that the term “abnormal” should be regurded as relative, for it is doubtful if any metabolic disorder has yet been discovered which results in a strictly abnormal product in the sense of a compouiid for which the normal organism has n o potentiality. Either the product is metabolized normally ( a s is the homogenistic acid found in alkaptonuria), or traces of it arc fouiid in normal body fluids when sufficiently sensitive metliods are used ( e.g. the phenylpyruvic acid of phenylketonuria), or it is built up from smaller normally occurring units ( a s are the abnormal hemoglobins). This situation is presumably a reflection of the fact that the known hereditary metabolic disorders are the results of either enzyme deficiencies or of defects in cellular transport, which distort normal metabolism rather than create ncw pathways. As far as the present subject is concerned, in the studies reported here no indole has been found which is in any way characteristic of mental illness. I t is important to recognize, however, that sensitive though they are, the chromatographic methods which have been so far applied to indoles are in general not capable of detecting less than about 0.5 pg indolc,/ml of blood or a daily excretion of less than 10100 pg, depending upon the indole. Concentrations of this order could be of significance, in view of the potency of some indoles for the brain: for example, an intravenous dose of 100 pg of lysergic acid diethylamide in an average adult results in a maximum blood concentration of 0.02 pg/ml, and this no doubt rapidly

286

R. RODNIGHT

declines through diffusion to other tissues, metabolism, and excretion. A further point to bear in mind is that the metabolism of an abnormal indole may convert it to a normally occurring indole (or a close derivative) before it appears in the body fluids. Such a possibility is illustrated by the fate of the hallucinogenic indole, N-dimethyltryptamine, which Szara and Axelrod ( 1959) find partly undergoes demethylation to give tryptamine (and therefore indolylacetic acid) and is partly hydroxylated to give 6-hydroxy-N-dimethyltryptamine; the latter appears in tlie urine as 6-hydroxyindolylacetic acid. Thus while the present negative findings clearly exclude the occurrence in mental illness of a major indole abnormality in the body as a whole, they by no means indicate that further study should not be undertaken. linbalance of normally occurring indoles. In the case of this hypothesis also, the significance of mainly negative results must be considered in relation to the limitations of the methods and approaches used. For example, cerebral serotonin has been estimated as comprising 3% of tlie total serotonin in the body. Clearly, then, if abnormalities in this pathway are confined to the brain there will be very little hope of detecting them in the body fluids. However, with the one exception of the controvcmial result of Lauer ct al. (1958) regarding the conversion of orally administered tryptophan to urinary 5-liydroxyindolylacetic acid, tlie results may be said to csclude a general bodily imbalance in basal indole metabolism in mental illness. For the indole amine pathways, moreover, this conclusion is especially securc since here it is based on measurements of several metabolites in both bloocl and urine; results considered separately woiild hc more difficult to interpret. Thus the urinary inclolc amines may only reflect renal metabolism, blood indole amines may be influcnced by homeostatic mechanisms and in the case of urinary 5-hydroxyiiidolylacctic acid it is uncertain to what extent this substance is derived from oxidative deamination of serotonin or the transamination of 5-hydrol;ytryptophan ( sec Section 11, A ). Geiiernl remarks. Indoles are a group of compounds which illustrate very well the many problems facing the biochemist in studying the biology of mental illness by analysis of body fluids, and it may legitimately be asked whether such studies arc worth conducting if control of diet, intestinal bacteria, and other relevant

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variables cannot be fully achieved. In the author’s opinion this is justified if the limitations of such partly controlled investigations are clearly stated and recognized, if they are regarded a s preliminary, and if the results, puticulurly positive results, are interpreted with caution. However, although the discovery of many metabolic abnormalities has not been prevented by the fact that ideal experimental conditions are rarely attainable in clinical research, poorly controlled work h a s no doubt obscured others. In the present field it appears that the point has been reached when partly controlled investigations of the kind described in the review are not likely to contribute further. klore sensitive methods, a wider use of drugs and 1irectirsors, and above all the application of stringent controls are needed to elucidate more clearly the metabolism of indoles in the major mental illnesses. V.

Summary

The review describes the occurrence of indole derivatives in the body fluids of mentally ill subjects. Studies are considered in relation to modern knowledge of the indoles in normal body fluids and to the fact that t h e indoles are derived from three sources: the diet, bacterial degradation of tryptophan in the intestinal tract, and tissue mctabolism. Only in two rare coiitlitioiis with psychiatric manifestation, namely Hartnnp disease kind jihrnylketonuria, have well defined abnormalities in indole rncta1)olism been found. In Hartnup disease the abnormalities arc’ most probably due to a defect in the transport of tryptophan across cell membranes; the resulting distortion of tryptophan mctabolism leads to a greatly increased bacterial degradation of tryptophan in the intestine and gross indicanuria. The defect is associated with an abnormally low conversion of tryptophan to nicotiilarnide in affected subjects; this is believed to be concerncd in the iiitermittcnt clinical symptomatology of the condition. In phenylketonurin an increased excretion of indole acids and low values of serotonin in blood are found; these are probably secondary consequences of the main defect in phenylalanine metabolism present in the disease, and there is now no evidence to suggest that they are related to the oligophrenia of the condition.

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In the major mental illnesses such as schi~ophrenia ancl endogenous depression an increased excretion is often obscri ed of indoles derived from the intestinal tract; in particulcu the excretion of sulfatovyskatole and the glucuronide of inclolylcarltoxylic acid and, less frequently, indolylacetic acid, may be raised. These changes are not considered to be in an) way causally related to mental illness, but are likely secondary consequences of an altered intestinal climate. No abnormal indoles have been found in body fluids from mentally ill subjects. The many investigations which have been made in the indole aniine pathuciy in mentcil illness are also reviewed, these are negative insofar as they give no indication of a general bodily disturbance in the pathway, but do not exclude the presence of local abnormalities in the brain. ACKNOWLEDGMENTS

The author is very gratefiil to Profcssor If. hIcIlwain for his liclp and encouragement and to the Board of (;ovcrnors of the Betlilcni Royal and the hlaudsley Hospitals for gencroiis finnncial support. Sonic of the author’s own investigations described in the rcvicw contributed to a thesis prwented to the University of London for the degree of P1i.D. hls’EHENCES

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SOME ASPECTS OF LIPID METABOLISM IN NERVOUS TISSUE By G. R.

Webster

Department of Chemical Pathology, Guy's Hospital Medical School, London, England

I.

Introduction

B. Ccrcbrosides . . . . . . . . . . . . . . . . . . . . . . . C. Phospholipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111. The Breakdown of Lipids . . . . . . . . . . . . . . .

299

Summary and Conclusion\ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . ...

313 314

17.

I.

Introduction

A considerable body of knowledge coiicerning the metabolism of lipids in the nervous system has been built up in the last two decades, largely as a result of tlic use of radioactive isotopes. Several reviews covering much of this work have been published; for example Sloane-Stanley ( 1952), Webster (1956), Kossiter (1957), LeBaron (1959), and Thompson and Webster (1960). It is the purpose of this article to consider some aspects of this work particularly in relation to the metabolism of lipids in the myelin sheath. In the first section, studies on the formation and turnover of lipids in nervoiis tissue, using isotopically labeled precursors, will be discussed; secondly, in view of the importance in human medicine of the demyelinating diseases and of our lack of knowledge concerning the biochemical mechanisms involved in the breakdown of lipids in the nervous system in these pathological states, an account will be given of some recent investigat'ions bearing on this latter aspect of lipid metabolism. I t would be beyond the scope of this article to discuss in detail the molecular structure and composition of myelin, but the 293

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following brief account is included to serve as a background to the subsequent discussion of metabolic aspects. From a comparison of the lipid composition of cerebral gray and white matter and from observed differences in cornposition between human infant and adult brain (Johnson et nl., 1049a) sphingomyelin, cerebroside, and free cholcsterol were considered to be the lipids most characteristic of the myelin sheath. A detailed study of the chemical events accompanying myelination in the mouse has shown that marked increases in these lipids are closely associated with the formation of myelin (Folch, 1955). Phosphatidylserine is also considered by some authors to be present in myelin (Brante, 1949; Robins et al., 1956). Brante (1949) also states that some ethanolamine phosphatide and perhaps lecithin are also present, and it is of interest that recent studies support his view that all or nearly all of the ethanolamine phosphatide of the myelin sheath is in the plasmalogen form (Webster, 1960). Proteolipids, described originally by Folch and Lees (1951), are present most abundantly in white matter and constitute important structural elements of central myelin (Folch et al., 1958); the finding by these latter authors and by Finean and associates (1957) that sciatic nerve is essentially free of proteolipid indicates important structural differences between central and peripheral myelin. The extensive studies by electron microscopy and X-ray diffraction techniques that have led to the current concepts concerning the arrangement of the lipid and protein components in the structure of the myelin sheath have been summarized by Finean (1957) and Finean and Robertson (1958).

II.

Lipid Turnover in Nervous Tissue

The view that at least some of the lipids of the nervous system are constantly being broken down and resynthesized has gained wide acceptance (Dawson and Richter, 1950; Dawson, I953b, 1954a; Streicher and Gerard, 19%; McAlpine et al., 1955; Ansell and Dohmen, 1957). Evidence suggesting that this process of turnover might occur at a relatively rapid rate has been described by several authors from studies of the rates of incorporation of isotopically labeled precursors into certain classes of lipids. For

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example, Dawson and Richter (1950) calculated that the rate at which the whole phospholipid fraction of mouse brain became labeled following administration of P3.)in vim corresponded to the complete turnover of this fraction in 70 hours, and Gidez and Karnovsky ( 1954) reported that C14-labeled glycerol was incorporated into the triglycerides and phospholipids of rat brain at a rate exceeded only by that found for liver. A considerable proportion of the lipids of nervous tissue are known to be present in nuclei, mitochondria, and other cellular structures (Tyrrell and Richter, 1951; Petersen and Schou, 1955; McIlwain, 1955). As Davison et al. (1959a) have recently emphasized, one of the outstanding questions of particular interest medically to further understanding of the pathogenesis of the demyelinating diseases in man is whether the lipids of the myelin sheath, as distinct from other lipid-containing structures of nervous tissues, are constantly broken down and resynthesized, or whether they remain comparatively inert metabolically once they have been formed during myelination in the growing animal. A. CHOLESTEROL In early experiments on the metabolism of cholesterol in nervous tissue it was shown by Waelsch and associates (1940a, 1941) that deuterium can be rapidly incorporated in vivo into the unsaponifiable lipids of brain in young rats, but not in adult animals (Waelsch et al., 1940b). Bloch and associates ( 1943) fed deuterium-labeled cholesterol to adult dogs and studied its incorporation into various organs for the succeeding week; although the labeled lipid was found in various non-nervous organs none was recovered from the spinal cords or brains of these animals. Negligible incorporation of radioactive carbon of CI4-acetate into adult rat brain cholesterol has been observed in vivo (Van Bruggen et al., 1953; Nicholas and Thomas, 1959), in vitro (Srere et al., 1950), or following perfusion of isolated cat brain with l-C14-octanoate (Sperry et al., 1953). On the other hand, other workers have reported that slight but definite labeling of cholesterol was found in adult mouse brain in vivo following injection of C14-labeled hexose (Moser and Karnovsky, 1959), and in adult rat brain and cat sciatic nerve in uitro after incubation with C14-acetate (Rossiter, 1957). Kline et al.

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(1958) also found a slight degree of incorporation of C'1 from acetate into the cholesterol of segments of cat nerve. Other studies have also been carried out recently with the particular view of obtaining more information concerning tlie metabolic activity of cholesterol and other lipids of the myelin sheath. The design of these experiments has been to administer isotopically labeled lipids or precursor substances to young animals during the stage of active myelin formation and then to determine the degree of persistence of radioactivity in various lipids for long periods into adult life. Davison et nl. ( 1958) injected 4-C' k-cliolesterol into the yolk sacs of ne\t7ly hatched chickens, which were thcn killed at intervals of up to 200 days later, and determined the radioactivity present in tlie cholesterol isolated from the brain and other organs. Activity disappeared from the plasma and liver rapidly and only a very small fraction of that present originally was found in these sites 8 weeks after hatching. I n contrast, the level of radioactivity in the brain cholesterol fell only slowly and a considerable proportion of the original activity was still present 220 days after hatching. The presence of radioactivity in cholesterol in rat brain has also been found as long as a year following administration of C'+-acetate to young animals ( McMillaii et d., 1957; Nicholas and Thomas, 1959) and also in rabbit brain for up to 200 days after injecting 3-C' 4-serine into 11-day-old animals (Davison et al., 1959b). Davison and his co-workers suggested that persistence of radioactive cholesterol might be more marked in the myelin sheaths than in the cells of the nervous system. In later experiments ( Davison et al., 1959a) 4-C14-cholesterol was injected intraperitoneally into 17-day-old rabbits, and the activity of the lipid present in whole brain, separated cerebral gray and white matter, spinal cord, sciatic nerve, heart, liver, and kidney was studied for periods of up to 1 year. In the non-nervous tissues the activity fell to very low levels in 4 to 6 months. In brain, however, a maximal activity was found at 26 days which then declined slowly over the next 4 to 5 months, followed by a slower decline to about 25% of the peak value at a year. Cerebral gray matter also showed an initial phase of relatively more rapid decline in the amount of labeled cholesterol present, which was succeeded by a more stable phase. In spinal cord and cerebral white matter, on tlie other hand,

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hardly any turnover of cholesterol was found. The results with these tissues throughout the whole experimental period of 12 months thus resembled that of the later stable phase in gray matter. These findings seem to imply that cliolesterol in the adult nervous system can be considered as being in two compartments of differing metabolic activity; a metabolically active one being present particularly in gray matter and indicating therefore turnover of cholesterol in this tissue, and a stable compartment present in both gray and white matter sliowing little or no metabolic activity. Davison et 01. (1959a) suggest that the cliolesterol of the stable compartment may represent tliat present in the nervc sheaths. Chemical degradation of tlic cliolcsterol isolated from brain 18 months after injection of 4-C1'-cliolesterol into young animals showed that the isotopic r.ar11011 was still present in the 4 position and, furthermore, all the radioactivity present in the extracted lipids could be accounted for as cliolesterol (Davison and Wajda, 1959). These findings would appear to exclude the theoretical possibility that apparent persistence of radioactivity in brain cholesterol in adult animals could have rcmlted from breakdown of the molecule followed by a high degree of reincorporation of the labeled fragment into brain lipids, since if this had occurred some redistribution of the isotope within thc brain lipids m7ould b e expected.

B. CEREBROSIDES The metabolism of cerebrosicles in the nervous system has been, until recently, less extensively investigated than that of cholesterol and the phospholipids. Riuton ef nl. (1958) found that C1'labeled hexose w a s incorporated into brain cerebrosides in young rats, but were able to find only slight incorporation in adult animals. They also reported that Luidine diphosphogalactose was an intermediate in the biosyntliesis of brain cerebroside from C14galactose, and that the enzymes concerned in this process were present in brain microsomal preparations. Radin et al. (1937) administered C14-galactose and S";-sulfatc to young rats and found that while labelcd sulfatide appeared to undergo no metabolic breakdown, cerebroside showed a gradual decline in specific activity over the 10 days succeeding injection of the labeled

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precursor. From their results these authors estimated that the half-life period of brain cerebrosides corresponded to 13 days. Moser and Karnovsky (1959) used C'4-labeled glucose and galactose in their studies of lipid metabolism in mouse brain; these substances were given by intramuscular injection, and the animals were killed 1 hour later. These workers reported that glucose was incorporated as a unit into the galactose moiety of brain cerebroside and that labeling from glucose was more effective than that from galactose. Incorporation of hexose into brain cerebroside was markedly more active in young animals than in adults, but, although the specific activity of the lipid in adult brain was low, the amount of radioactivity present was significant. It is of interest in this connection that Sadhu (1953) reported that after feeding glucose to young pigeons, one-third of the total brain cerebrosides were glucocerebrosides (only galactocerebrosides are present normally in the brains of these animals). Platt (1955) speculated whether differences in diet in infancy might influence the composition of myelin cerebroside and further whether variations in this might be related to the occurrance of demyelinating diseases in later life. Varma and Schwarz (1960), however, recently carried out experiments in which two groups of kittens were reared from birth on diets containing maltose and lactose, respectively, as sole sources of carbohydrate. Development was normal in both groups and no differences were found in the weights, histological appearance, galactose or glucose contents of the brains. Davison et a2. (1959b), in their experiments in which 3-C14serine was administered to young rabbits, reported that rapid labeling of brain cerebrosides occurred and that little change in activity was discernible during the subsequent period of nearly 200 days. These findings were interpreted as indicating that a proportion of the brain cerebrosides, like cholesterol, is relatively inert metabolically. These latter authors comment that the apparent discrepancy between their findings and those of Radin et al. (1957) is probably explicable in terms of the shorter experimental period used by Radin and his co-workers, which might have been too short to reveal any long term persistence of labeling of brain cerebrosides.

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C. PHOSPHOLIPIDS Early investigations of phospholipid metabolism in vivo using P32, administered orally or intravenously, indicated that this was less active in nervous tissues than in other organs in adult animals (Artom et al., 1937; Changus et nl., 1938; Chargaff et al., 1940). Similar observations were also reported from experiments with NI5labeled choline ( Stetten, 1941) and C"-labeled choline (Tolbert and Okey, 1952). Brain lipid metabolism was most active in young animals, and progressively declining rates of istope incorporation were found with increasing age. A more rapid penetration of P32 into brain occurred if the isotope was injected intracisternally (Bakay and Lindberg, 1949; Sachs and Culbreth, 1951; Strickland, 1952), and it was considered that the slower rates of phospholipid labeling reported previously were due to slow penetration of the precursors across the blood-brain barrier. Sperry et al. (1953) observed that brain phospholipids could be labeled from C14-octanoate, when this substance was perfused through isolated cat brain. Considerable incorporation of isotopes into lipids in nervous tissue has also been obtained by the use of in vitro methods (Fries et al., 1942), which have in recent years provided much detailed information concerning lipid metabolism. Dawson (1953a, 1954b) showed that the rates at which the individual phospholipids of a guinea pig brain homogenate became labeled when incubated in the presence of P32 differed widely. The specific activity of diphosphoinositide was over nine times that of the total phospholipid fraction, while lecithin and sphingomyelin were less than 10% and phosphatidylethanolamine and phosphatidylserine less than 5% as active as the total fraction. Ansell and Dohmen (1957) found a similar pattern of results in vivo in adult rats. Schachner et al. (1942) showed that phospholipid labeling with P32 in vitro was enhanced by the presence of hexose in the incubation medium and that it was reduced by anaerobiosis. Dawson (1953b) found that in a brain dispersion the incorporation of the isotope was dependent upon an active oxidative phosphorylation system. Strickland ( 1954) made similar observations with cat brain slices. More recently McMurray et al. (1957a, b ) have described the presence in brain preparations of two systems capable of supporting P32-labeling of brain phospholipids. They found a glycolytic system demonstrable in water dispersions of

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brain, and also an oxidative system in mitochondria prepared from suspensions of the tissue in isotonic sucrose. Further detailed investigations into the biosyntlietic mechanisms of brain phospholipids (McMurray et nl., 1957c; Rossiter ct nl., 1957b) have shown that incorporation of P3?into lecithin is brought about by a sequence of reactions similar to that described by Kennedy and Weiss (1956) in liver; choline is phosphorylated from ATP to form pliosphorylcholine which is then attached as a unit to an a$-diglyceride by a trmsfer reaction involving cytidine nucleotide as a co-factor. In experiments in which rat brain homogcnates and mitocliondrial preparations were incubated wit1I P32labeled cytidine diphosphate choline ( Rossiter ef nl., 1957a) the radioactivity was incorporated into the phospholipids, and the incorporation was increased by the addition of a,P-diglycerides to the system. Most of the activity of the phospholipids was found in the lecithin fraction and only to a slight extent in phosphatidylethanolaminc or phosphatidylserine; some activity was pi esent also in sphingomyelin. The phospholipids of sciatic nerve homogenates were also labeled from cytidine diphosphate choline. Majno and Karnovsky (1958) have made an extensive study of the incorporation in zjitro of C'4-labeled acetate, glycerol, choline, glucose, and into the lipids of rat brain corteu, spinal cord white matter, and peripheral nerve. Cortical lipids were found to have greater specific activities than those of spinal cord white matter or peripheral nerve with all substrates except acetate, this latter substrate was utilized by nerve to a greater extent than by either of the tissues of the central nervous system. With most substrates used thc white matter preparations showed the least activity as regards incorporation into lipids. The ability of a number of Cl4-1abeled substrates, acetate, glycine, glycerol, serine, ethanolamine, and choline, to be incorporated into various lipids in rat brain slices has also been investigated by Pritchard (1958). H e found that the last four of these substances were more effective than acetate or glycine for labeling brain phospholipids; liver lipids, by comparison, showed greater activity with acetate, glycine, and serine than with choline or glycerol. I t was evident both in brain and liver that only acetate labeled the fatty acid portions of the phosphatides. Examination of the glycerylphosphoryl diester moieties, after removal of the

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fatty acids by hydrolysis, showed that with choline and ethanolamine as substrates labeling was confined to the nitrogenous bases of the corresponding phospholipids; most of the radioactivity incorporated from serine was present in the diester moiety of phosphatidylserine, although some was found in glycerylphosphorylcholine and glycerylphosphorylethanolamine. All the phosphatides were effectively labeled from glycerol. A similar pattern of isotope incorporation from various precursors in preparations of cat peripheral nerve has been described (Pritchard and Rossiter, 1959). These findings indicate that it is possible in uitro to label each of the glycerophosphatides of nervous tissue in various parts of their molecules by suitable choice of radioactive precursors (Pritchard, 1958). Numerous investigations, of which some have been briefly outlined above, have shown some degree of active phospholipid metabolism in nervous tissue. Most of these investigations, however, both in vivo and in uitro, have generally involved experiments of comparatively short duration lasting periods of hours only, and, furthermore, have mostly been carried out with tissues from adult animals. In view of the conclusion that at least a proportion of the cholesterol and cerebrosides of nervous tissue is comparatively inert metabolically in adult animals, the question arises whether a part of the phospholipid fraction also shows similar evidence of stability. Davison and his co-workers have studied the long-term persistence of radioactivity incorporated into phospholipids in the nervous system in young myelinating animals. After the intraperitoneal injection of PS2into 16-day-old rats (Davison and Dobbing, 1958, 1959, 1960b) the radioactivity present in the whole brain phospholipids was followed for periods of nearly 200 days. A steady decline in activity was observed during the first 100 days but subsequently the activity in brain phospholipids remained virtually constant. A similar pattern of events was seen also in the phospholipids of spinal cord and peripheral nerve. The radioactivity present in the acid-soluble phosphorus of the blood reached negligible levels by 50 days. When P32 was administered to adult rats the activity of the brain phospholipids reached a maximum value later than in the young group and thence diminished gradually during the subsequent experimental period of 6 months at a rate similar to that

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seen in the early part of the experiments with rats injected at 16 days of age. In the adult rats, however, radioactivity was still detectable in the blood for more than 100 days following administration of the isotope. It was concluded that the similarity in the rates of decline in brain phospholipid radioactivity in the two groups might be attributed to this continued availability of precursor in the blood and that there was no evidence of persistence of phospholipid labeling in brain after injection of P32 into fully grown animals. In other experiments (Davison and Dobbing, 1960a, b ) C14labeled glycerol was given to two groups of rats, one 13 days old and the other fully grown animals. The radioactivity in the blood dropped in both groups to negligible levels by the end of the first week; the radioactivity present in the brain phospholipid fraction declined during the next 110 days at a much faster rate in the latter group. Subsequently Davison and Dobbing ( 1960c) have found that, in rabbits after injection of PS2 at 16 days postnatally, persistence of the isotope in phospholipids was more marked in tissue preparations rich in white matter than in preparations of predominantly gray matter. These recent studies on the long-term fate in vivo of radioactive precursors incorporated into lipids during the stage of myelination have indicated that in the central nervous system, and possibly also in the peripheral nervous system, a proportion of the cholesterol, cerebrosides, and phospholipids most probably remain relatively inert metabolically in the adult for long periods of time (Davison et al., 1959a, b; Davison and Dobbing, 1960b). It has been pointed out that the apparent metabolic stability of various lipids in these experiments may be related more to their presence in some stable anatomical unit than to their chemical structure and properties ( Davison and Dobbing, 1960a). Since persistence of radioactivity incorporated into lipids during myelination has been observed in those lipids believed to be associated particularly with the myelin sheath and has been especially evident in tissue preparations rich in white matter in contrast to gray matter, Davison and his co-workers consider their findings consistent with the view that these lipids, having been incorporated into the myelin sheaths during development, are

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relatively stable and normally undergo little, if any, breakdown and renewal in the adult nervous system. HI.

The Breakdown of Lipids

Numerous attempts have been made to detect the presence in nervous tissues of enzymes capable of hydrolyzing intact lipids. Thannhauser and Reichel (1936) claimed that brain possessed a weak cerebrosidase and sphingomyelinase. Rossi ( 1935), Goebel and Seckfort (1948), and King (1931) also studied the hydrolysis of phospholipids by brain. The latter author reported that extracts of brain and other tissues incubated for several days with lecithin preparations slowly released acid-soluble phosphorus. The enzyme thought to be responsible was relatively stable in neutral systems and maximal activity was observed at 37°C and pH 7.5. Sperry (1947) reported a decrease in the lipid phosphorus content of brain homogenates incubated in saline-bicarbonate buffers, and he interpreted this as evidence of cleavage of phospholipid by a mechanism present in brain tissue; he considered that this process was slow or inoperative in the living animal. These observations were confirmed and extended by Tyrrell (1950) who further claimed that the loss of phospholipid involved the lecithins and cephalins, and that no change was discernible in sphingomyelin. Johnson et al. (1949b), on the other hand, observed decreases in total phospholipid, sphingomyelin, and cephalin fractions, but not in lecithin, during prolonged incubation of cat brain slices. In both these last two mentioned investigations precautions were taken to maintain the sterility of the tissue preparations. SloaneStanley (1952) has reviewed this early work and has compared the apparent rates of lipid breakdown reported by the various authors mentioned above. Many of the early investigations in this field must, however, be considered of doubtful value as regards the presence and level of activity of intrinsic lipid-hydrolyzing enzymes in nervous tissue. Little or no attempts were made to characterize the enzymes thought to be concerned, and in view of the long periods of incubation which were frequently employed the possible failure to maintain sterility cannot be excluded; active lipidhydrolyzing enzymes are known to be present in certain microorganisms ( Macfarlane and Knight, 1941).

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More recently Abood and Geiger (1955) observed a significant decrease in lipid in cat brain following perfusion for 60 minutes with a glucose-free medium, and in experiments in whicli cat cerebral cortex was electrically stimulated for 20 seconds there was also a significant loss of lipid nitrogen (Geiger et al., 1956). The most dctailed account yet reported concerning eivymic hydrolysis of lipids by nervous tissue is that of Sloane-Stanley (1953) on the breakdown of inositol phosphatide by guinea pig brain under anaerobic conditions in Zjitro. He found that this lipid was hydrolyzed at a high rate initially, equivalent to 66 prnoles per gram fresh tissue per hour, which, however, declined gradually during the course of the next few hours. Hydrolysis of the lipid was abolished by heating the brain homogenates before use. Analysis of the reaction products indicated that these were mainly inositol monophosphate and inorganic phosphate in approximately equimolar proportions, and it was suggested that at least two enzymes were concerned in the reaction of inositol phosphatide with brain. In a later extension of this work Rodnight (1956) showed that Caf + activated thc release of inositol monophosphate but slightly inhibited the production of inorganic phosphate, as also did Mg'+ and Ba 1' . The enzyme system was present also in rat, rabbit, monkey, sheep, and human brain. No evidence was found in either of these investigations, however, of significant hydrolysis by brain of other cephalins or of lecithin. It is generally considered likely that the complete degradation of phospholipids, for example lecithin, by animal tissues involves the removal of fatty acids by the successive actions of phospholipases A and B, followed by further hydrolysis of the resulting water-soluble gly cerylphosphorylclioline by a phosphodiesterase. The possible presence of phospholipase A activity in nervous tissue, which could give rise to the production of lysophosphatides by removal of a single fatty acid from an intact phospholipid, has gained in interest recently from the finding that lysolecithin, and possibly other lysophosphatides, can themselves cause damaging effects in nervous tissues (see Section IV). Up to the present time, however, there has been no certain demonstration of the presence in the nervous system of intrinsic enzymes of the phospholipase A type, although the presence of such enzymes in other tissues is well established ( Francoli, 1934; Gronchi, 1936; Hanahan, 1952;

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Shapiro, 1953). Phospholipase B was first recognized in rice bran by Contardi and Ercoli (1933) and was later found in certain molds (Fairbairn, 1948) and bacteria ( Hayaishi and Kornberg, 1954). Considerable activity was also reported in rabbit pancreas and lung (Noguchi, 1944), ox pancreas (Sliapiro, 1953), and rat and sheep liver (Dawson, 1 9 5 6 ~ ) . Slight but significant phospholipase B activity has reccntly been demonstrated in rat and human nervous tissues by Rllarples and Thompson (1960); these workers incubated fresh homogenates of nervous tissues with a purified preparation of lysolecithin as substrate and assessed enzymic activity by the determination of both free fatty acid release and glycerylphosphorylcholine production. Rat brain and spinal cord showed activities equivalent to 1.7 and 0.8 pmoles substrate hydrolyzed per gram fresh tissue per hour, respectively. The activity in human brain ranged from 5.0 pmoles per gram per hour for the caudate nucleus to 0.7 pmoles per gram per hour for centrum ovale. It was evident that phospliolipase B in nervous tissue was associated more particularly with those regions rich in gray matter. THEHYDROLYSIS OF LIPIDPHOSPHATE ESTERS IN NERVOUS TISSUE The presence in brain of various mono- and diesters of phosphoric acid containing cholinc, ethanolamine, and serine that occur in the lecithins, cephalins, and sphingomyelin has been demonstrated by numerous workers [see Porcellati (1958) and Baker and Porcellati (1959) for refercnccs]. In an extensive quantitative investigation of these compounds in rat and lien brain and in hen spinal cord and sciatic nerve by paper chromatographic methods Porcellati (1958) found mean levels of 9.2, 13.8, and 4.3 mg/100 gm for phosphorylcholine, phosphorylethanolamine, and phosphorylserine, respectively, in fresh hen brain; values for rat brain were closely similar. In hen spinal cord and sciatic nerve the amounts of phosphorylserinc were similar to those in brain (5.5 and 3.9 mg/100 gm, respectively). The amounts of phosphorylethanolamine in these two tissues were, however, rather less than in brain (6.8 and 2.7 mg/100 gm, respectively). The mean level of glycerylphosphorylcholine in hen brain was 0.69 mg/100 gm. Baker and Porcellati (1959) have also studied the separation of

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nitrogen-containing phosphate esters on ion exchange columns; by this method they found 17.1 and 13.1 mg of glycerylphospliorylethanolamine per 100 gm in hen brain and spinal corcl, respectively. As already mentioned glycerylpliosplioryl diesters of clioline, ethanolamine, and serine are probable products of phospholipid catabolism. The presence of a diesterase hydrolyzing compounds of this type was described by Hayaishi and Kornberg (1954) in Scrratia plymiithica, and more recently Dawson ( 1956a) has studied a similar enzyme in rat liver preparations. A detailed investigation of the phosphodiesterase activity of fresh liomogenates of nervous tissues, using glycerylphosphorylcholine as a substrate, has been made by Webster and associates (1957). In rat brain the enzyme was found to be active between pH 6.5 and pH 10.5 with optimal activity at pH 9.5. In this respect the enzyme in brain differs from that in liver, for which Dawson (1956a) reported a p H optimum of 7.5. The p H activity curve of the brain enzyme, however, is closely similar to that found for Serrntia plyinuthica by Hayaishi and Kornberg ( 19'54) . Furthermore, the considerable degree of activity at physiological pH's shown by the phosphodiesterase differentiates it from the phosphomonoesterases of nervous tissue. At both p H 7.4 and 8.9 tlie diesterase liberated approximately G moles of free clioline for each mole of inorganic phosphate This indicates that the enzyme splits off choline from the substrate and that tlic appearance of inorganic phosphate results from the action of ~~liospliomonoesteraseon the glycerophosphate thus proclnced. Dialysis did not appear to affect the enzyme, but acetone-driecl preparations sliowcd a 36c/c8loss of activity. The enzyme was also sensitive to variations in ionic strength of the incubation medium, since activities measured at pH 7.5 in 0.06 A4 phosphate buffer were only 60% of those found in 0.025M bicarbonate. Increase in the ionic strength of tlie bicarbonate medium, or tlie addition to it of sodium chloride, also reduced the activity of the enzyme preparations. Dialysis of rat brain homogenates in the presence of Versene resulted in 907; loss of enzyme activity, which could, however, be substantially restored by addition of suitable concentrations of n/lnL+ or Mg++; C a + ~ +and C o t + partially reactivated the enzyme but Zn+ + and C u t + had only slight effects. Dawson ( 1956a)

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found that Zn4 + was strongly inhibitory towards the liver enzyme. It was of interest to determine whether the hydrolysis of glycerylphosphorylcholine b y the phosphodiesterase of nervous tissue was affected by any of the recognized inhibitors of cholinesterase. I t was found, however, that eserine, diisopropylfluorophosphonate, and triorthocresylphos~,hate,in concentrations milch greater than those required to inhibit cholinesterase, were without effect. The enzyme was also insensitive to fluoride and cysteine. The Michaelis constant, determined with glycerylphosphorylcholine as substrate, was about 2.7mM. Dawson (1956a) reported that the phosphodiesterase of liver also hydrolyzed glycerylphosphorylethanolamine and that this competitively inhibited the hydrolysis of the choline ester. Assays of the levels of phosphodiesterase activity in various regions of the nervous system in man, rat, guinea pig, rabbit, dog, and hen were also carried out by Webster, and associates ( 1957). The highcst levels, equivalent to 58 pmoles substrate hydrolyzed per gram fresh tissue per hour, were found in rat spinal cord. Rather lower activities of 36.4 and 25.0 ymoles per gram per hour were present in rat cerebrum and cerebellum, respectively. The lowest levels of activity of about 2.0 pmoles per gram per hour were found in rabbit brain and hen sciatic nerve. No marked differences were discernible between regions of the human nervous system consisting predominantly of white matter or gray matter. These investigations have tlius indicated a capacity in normal nervous tissue for the degradation of lysophosphatides by the successive actions of phospholipase B and phosphodiesterase. As regards the metabolic functions of the lipid phosphate monoesters in nervous tissue I t was concluded by Ansell and Dalvson ( 1951) that most of the ~'hosphoryletlianolamincin brain WAS not derived from the breakdown of pliosphatidyletlianolamine, and similar conclusions were reached concerning both phosphorylethanolamine and phosphorylcholine in liver ( Norman and Dawson, 1953; Dawson, 195617 ) . IVittenberg and Kornberg ( 1953) demonstrated the ability of acetone-dried preparations of brain and other tissues to phosphorylate choline and ethanolamine, but not serine, from ATP. The work of Kennedy and Weiss (1956) on the biosynthetic mechanisms leading to the formation of phosphatidylethanolamine and phosphatidylcholine in liver established

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that phosphorylclioline and phosphorylethanolamine were intermediates in these processes; similar reactions in brain involving phosphorylcholine have been described by McMurray ct ul. ( 1 9 5 7 ~ ) . A major function in lipid biosynthesis is thus indicated for the lipid phosphate monoesters. These substances are known, however, to be actively hydrolyzed by tissues including those of the nervous system. Baccari ( 1938) found that phosphorylcholine was split by phosphoinonoesterase of horse brain at pH 10-11, but was unaffected by the acid pliosphatase of this tissue. Stricklancl ct al. (1956a, b ) studied the hydrolysis of the phosphate esters of clioline, ethanolamine, and serine using a number of regions of the nervous system of man and various animal species. All three esters were actively split at an optimum pH of 8.9 but no significant hydrolysis took place below p1-I 7.5. In this respect the lipid phosphate monoesters contrasted markedly with such substances as phenyl phosphate, a-naphthyl phosphate, and a- and p-glycerophosphates, all of which were hydrolyzed by both the acid and alkaline pliosphatases of nervous tissue. Gray matter showed greatcr activity than white matter towards pliosphorylethanolamine at 111-1 8.9; mean activities equivalent to 726 and 261 pg phosphorus released per gram fresh tissue per hour, respectively, were found. It should be noted, however, that apparent lack of acid phosphatase activity toward the lipid phosphate monoesters is not confined to tissues of the nervous system but was shown also by human kidney, liver, intestine, and prostate ( Stricklancl ct al., 1956b). It appears therefore that these esters behave as relatively highly selective substrates for the alkaline phosphomonoes terases of a wide range of tissues including the nervous system.

IV.

The Action of Lysolecithin on Brain

The question whether there are present in nervous tissues enzymic mechanisms which could cause the production and subsequent destruction of lysophosphatides is of considerable interest in view of recent observations indicating that lysolecitlim can itself bring about marked destructive changes on the cells and other structures present in brain. The early studies of \Veil (1930) and later of Morrison and Zaniecnik (1950) and Birkmaytxr and

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309

Neumayer (1957) on the effects of cobra venom, which contains a highly active phospholipase A, on segments of fresh spinal cord showed that marked destruction of myelin was brought about by this agent. Morrison and Zamecnik suggested that, in view of the known hemolytic properties of lysophosphatides, these substances, produced in the tissue preparations by the action of the venom phospholipase A, might thcmsclves contribute to the myelin breakdown which they had observed. The hemolytic properties of lysolecithin have been extensively studied (Collier, 1952), and its effects on liver mitochondria have also been investigated (Nygaard et al., 1954). Grosse and Taubock ( 1942) briefly reported that lysolccithin could cause optical clearing of brain tissue suspended in saline; this phenomenon has more recently been studied in greater detail (Webster, 1957). Homogenates of whole rat brain in 0.025 M sodium bicarbonate (pH 8.3) were incubated at 38°C in the presence of various concentrations of pure lysolecithin and the clearing effect followed quantitatively by measurement of the change in optical transmission. There was an initial rapid increase in transmission during the first 10 minutes of reaction followed by a more gradual rise to a maximum value over the next 2 hours. The final degree of clarification reached rose with increasing concentrations of lysolecithin in the system to a limiting value, equivalent to about 80cjo of the optical transmission of water, in the presence of 0.007-0.008 M lysolecithin. Centrifugation (21,000 g for 60 minutes) of brain homogenates which had been maximally cleared in this manner resulted in only a slight further increase in optical transmission, and it was thus evident that the greater part of the components of the neurons, glial cells, and myelin sheaths of the brain tissue had been solubilized by the action of the added lysolecithin (see Fig. 1 ) . This clearing action of lysolccithin is presumably related to its known ability to form complexes with the lipid components of the tissues (Saunders, 1957) and also possibly with protein, much of which is present in nervous tissue as proteolipid (Folch et nl., 1958). In relation to this latter point Gent (1959) has recently shown in electrophoretic studies on soluble brain proteins that following treatment of brain homogenates with lysolecithin there is a considerable increase in the so-called albumin peak, accompanied by separation of this principal peak into several com-

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ponents of not very different mobilities; it was concluded that the peak showing these changes following exposure of the brain to lysolecithin is probably related to structural proteins. Apart from the direct destructive action of lysolecitliin on nervous tissue it seemed possible also that cytolysis might result in the release of intracellular enzymes in soluble form, and it was of interest, therefore, to determine what effects lysolecitliin might have on enzymic activities normally present in brain. Particular interest attaches to those enzymes which, if released into solution, might themselves be capable of having additional destructive action

0.005M

0.003M

10 30

60

I20 Tirne(min.)

/'

/ I / f I f

I80

FIG.1. Clcaring effcct of various concentrations of lysolecithin on l : ? O w/v rat brain honilogenates in 0.025 Af N a H C 0 3 ( p H 8.3) at 38" C (Webster, 19.557). S; optical transmission of supernatant fluids after centrifuging at 21,000 g h r 60 minutes; 1OOyo: optical transmission of distilled water.

on cells or cell membranes, or which might cause disturbance of normal processes of excitation and transmission in unaffected ncrve fibers. It is of interest in this last connection that Tobias (1956) reported that treatment of nerve fibers with phospholipase A or lysolecithin causes inexcitability and depolarization. Marples et nl. (1959) studied first the levels of activity of several enzymes in rat brain homogenates both before and after exposure to maximal clearing concentrations of lysolecithin. In the cleared preparations as compared with the uncleared, both true and pseuclocholinesterase, alkaline phosphatase, and phospliodiesterase showed very slight increases in activity amounting to 6, 12, 10, and 6$, respectively. Aliesterase (tributyrinase), acid phosphatase, and

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monoamine oxidase, on the other hand, all showed a reduction of about 40% in activity in the cleared homogenates. Glutamic-oxalacetic transaminase and the proteinasc (cathepsin) active at pH 3.53.8 (Ansell and Richter, 1951) were both virtually unaffected by treatment with lysolecithin. From the point of view of thc possible pathological significance of lytic agents in relation to degeneration of nervous tissue, a study was also made of the action of lysolecithin on brain slices, as representing a more intact tissue preparation than homogenates, to determine whether this procedure also resulted in the liberation of intracellular enzymes into solution. It was found that a concentration of 1.2 x lo-:{ A4 lysolecithin in a glucose-containing medium in which the slices were suspended caused the liberation into the surrounding fluid of about 20% of both thc true and pseudocholinesterase of the slices during incubation for 30 minutes at 38°C. Greater concentrations, u p to 18 10W3 A4 lysolecithin, resulted in a somewhat variable but progressive increase in the liberation of these enLymcs reaching 525% and 64% for true and pseudocholinesterase, respectively. In the absence of added lysolecithin in the incubation medium only about 3% and 6%, respectively, of the total activity ot these two enzymes in the slices were released into solution. The results were unaffected by the presence or absence of oxygen in the system during the incubation period. An even more extensive outflow of glutamic-oxalacetic transaminase from brain sliccs exposed to the action of lysolecithin has been found (McArdle ef aZ., 1960). In the absence of lysolecithin about 10% of the total amount of this enzyme in the slices was released into the incubation medium in 10 minutes at 38"C, this figure rose slowly with increasing periods of incubation to 18% in 2 hours. Addition of- various concentrations of lysolecithin to the system markedly incrcascd the outflow of the enzyme; 42%, 65%, and more than S0:h of the activity in the slices were released 6.0 x 10-3, and into solution by concentrations of 1.2 X 12.0 x lo-.' RI lysolecithin, respectively. Under these conditions maximal outflow of the ewyme was observed after 10 to 20 minutes incubation. The differences between glutamic-oxalacetic transaminase and cholinesterase as regards the extent to which they are liberated

x

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R. WEBSTER

from the slices both in the presence and absence of lysolecithin is most probably related to differences in their intracellular location. The major proportion of the former enzyme is present in the centrifuged supernatant fraction of sucrose homogenates of rat brain (McArdle et al., 1960), while Aldridge and Johnson (1959) showed that only 9% of the cholinesterase is in the supernatant fraction, 3540% in the microsomal fraction, and 40% in the mitochondria; Toschi (1959) and Hanzon and Tosclii (1959) also found cholinesterase to be associated with the microsomes. Several groups of workers [see McArdle et al. (1960) for references] have reported that lysolecitliin depresses the oxidative metabolism of liver mitochondria, while Braganca and Quastcl in 1953 found that cobra venom, previously heated to destroy enzymes other than phospholipase A, reduced the oxygen consumption of brain preparations and inhibited a number of oxidative enzymes in nervous tissue. Petrushka and associates (1959) have further shown recently that the oxygen consumption of rat brain slices is initially stimulated and later profoundly depressed by cobra venom phospholipase A. McArdle et al. (1960) found that A!) had no apparent effect on the oxygen lysolecithin ( 6 x consumption of the rat brain slices during the first 20 minutes incubation in a glucose-containing medium but subsequently caused a progressive fall in respiration. It was evident, therefore, that the maximal outflow of intracellular enzymes from brain slices under the influence of lysolecithin occurred before any effect on oxygen consumption was demonstrable. The marked cytolytic and solubilizing effects of lysolecithin on nervous tissue which these various investigations have revealed would indeed seem to support the suggestion of Morrison and Zamecnik ( 1950) that tissue damage including demyelination might occur if lysolecithin were to gain access to or to be produced locally in the nervous system in abnormal amounts. It would seem possible also that the direct disruptive effects of such lytic agents might well be accompanied by the release of intracellular enzymes in an active state which might further extend the structural and functional damage.

LIPID hlETABOLIShI

V.

313

Summary and Conclusions

Some aspects of the metabolism of cholesterol, cerebrosides, and phospholipids in tissues of the nervous system have been reviewed. Radioactivity from various labeled precursors is incorporated into each of these three classes of lipid in the nervous system of young animals both in vivo and in vitro. Recent long term studies on the fate of radioactivity incorporated into the lipids in young animals during the stage of myelination have further shown a considerable degree of persistence of labeling in the cholesterol, cerebroside, and phospholipid fractions of nervous tissue for long periods into adult life; this persistence of activity was more marked in tissue preparations rich in white matter than in gray matter. These findings have been interpreted as indicating that a proportion of the cholesterol, cerebrosides, and phospholipids in nervous tissue is relatively inert metabolically. In studies on the enzymic breakdown of phospholipids by nervous tissue slight but significant phospholipase B activity, hydrolyzing lysolecithin to free fatty acid and glycerylphosphorylcholine, has been demonstrated. The nervous system of man and of a number of animal species also contains an active phosphodiesterase which splits glycerylphosphorylcholine into choline and glycerophosphate. The lipid phosphate monoesters, phosphorylcholine, phosphorylethanolamine, and phosphorylserine are selectively hydrolyzed by alkaline phosphomonoesterases in nervous tissues and a number of other tissues. Lysolecithin has been shown to have a marked destructive action on the cells and other structures in the nervous system. Its ability to solubilize structural components and thus to produce optical clearing of brain homogenates has been investigated quantitatively. The activity of a number of enzymes in such cleared homogenates has also been studied; cholinesterase, alkaline phosphomonoesterase, phosphodiesterase, glutamic-oxalacetic transaminase, and a proteinase acting at pH 3.53.8 were found to be largely unaffected, but tributyrinase, acid phosphomonoesterase, and monoamine oxidase were partially inhibited. When brain slices were incubated in a medium containing lysolecithin, cholinesterase and glutamic-oxalacetic transaminase were released from the slices into the surrounding medium. Maximal outflow

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of these enzymes from the slices was observed after 10-20 minutes incubation and during this period the 0 2 consumption of the slices was unaffected although subsequently it was progressively inhibited. Thus, maximal release of these intracellular enzymes occurred before any interference with respiration was demonstrable, These observations on the cytolytic and solubilizing properties of lysolecithin lend support to the suggestion that it might play a part in the production of tissue damage, were it to gain access to or be produced locally in abnormal amounts in the nervous system. REFERENCES Abood, L. G., and Geiger, A. ( 1955). Am. J. Physiol. 182, 557. Aldridge, W. N., and Johnson, M. K. (1959). Biochem. J. 73, 270. Ansell, G. B., and Dawson, R. M. C. (1951). Bwchem. J. 60,241. Ansell, G. B., and Dohmen, H. (1957). J. Neurochem. 2, 1. Ansell, G. B., and Richter, D. (1954). Biochim. et Biophys. Acta 13, 87. Artom, C., Sarzana, G., Perrier, C., Santangelo, M., and Segre, E. (1937). Nature 139, 836. Baccari, V. ( 1948). Arch. sci. biol. (Napoli) 32, 391. Bakay, L., and Lindberg, 0. (1949). Acta. Physiol. S c a d . 17, 179. Baker, R. W. R., and Porcellati, G. (1959). Biochem. J. 73,561. Birkmayer, W., and Neumayer, E. (1957). Deut. Z. Nervenheilk. 177, 117. Bloch, K., Berg, B. N., and Rittenberg, D. (1943). J. BioZ. Chem. 149, 511. Braganca, B. M., and Quastel, J. H. (1953). Biochem. J. 53, 88. Brante, G. (1949). Acta Physiol. S c a d . 18, Suppl. 63. Burton, R. M., Sodd, M. A., and Brady, R. 0. (1958). J. Biol. Chem. 293, 1053. Changus, G. W., Chaikoff, I. L., and Ruben, S. (1938). J . Biol. Chem. 126, 493. Chargaff, E., Olsen, K. B., and Partington, P. F. (1940). J. Biol. C h m . 134, 505. Collier, H. B. (1952). J. Gen. Physiol. 36, 617. Contardi, A., and Ercoli, A. (1933). Biocha. Z. 261, 275. Davison, A. N., and Dobbing, J. (1958). Lancet ii, 1158. Davison, A. N., and Dobbing, J. (1959). Biochem. J. 73,701. Davison, A. N., and Dobbing, J. (1960a). Biochem. J. 74, 1P. Davison, A. N., and Dobbing, J. ( 1960b). Biochem. J. 76, 565. Davison, A. N., and Dobbing, J. (1960~).B i o c h a . J., 75, 571. Davison, A. N., and Wajda, M. (1959). Noture 183, 1606. Davison, A. N., Dobbing, J., Morgan, R. S., and Payling Wright, G. (1958). J . N ~ T o c3, ~89.. Davison, A. N., Dabbing, J., Morgan, R. S., and Payling Wright, G. (1959a). Lancet i, 658.

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Davison, A. N., Morgan, R. S., Wajda, M., and Payling Wright, G. (1959b). J . Neurochem. 4, 360. Dawson, R. M. C. (1953a). Biochem. J., 66, XII. Dawson, R. M. C. (1953b). Biochem. J. 55,507. Dawson, R. M. C. (1954a). Biochem. J. 67, 237. Dawson, R. M. C. (19.5413). Biochim. et Biophys. Acta 14, 374. Dawson, R. M. C. (1956a). Biochem. J. 62, 689. Dawson, R. M. C. (1956b). Biochem. J. 62,693. Dawson, R. M. C. ( 1 9 5 6 ~ ) .Biochem. J . 64, 192. Dawson, R. M. C., and Richter, D. (1950). Proc. Roy Soc. B137,252. Fairbairn, D. (1948). J. Biol. Chern. 173,705. Finean, J. B. (1957). In “Metabolism of the Nervous System” (D. Richter, ed.),p. 52. Pergamon, New York. Finean, J. B., and Robertson, J. D. (1958). Brit. Med. Bull. 14, 267. Finean, J. B., Hawthorne, J. N., and Patterson, J. D. E. (1957). J. Neurochem. 1, 256. Fdch, J. (1955). I n “Biocbemistry of the Developing Nervous System” ( H . Waelsch, ed.), p. 121. Academic Press, New York. Folch, J., and Lees, M. (1951). J. B i d . Chem. 191, 807. Folch, J., Lees, M., and Carr, S. (1958). Exptl. Cell Research Suppl. 6, 58. Francoli, M. (1934). Femntforschung 14, 241. Fries, B. A., Schachner, H., and Chaikoff, I. L. (1942). J. Biol. Chem. 144, 59. Geiger, A., Yamasaki, S., and Lyons, R. (1956). Am. J . Physiol. 184, 239. Gent, W. L. G. (1959). Biochem. J. 73, 6P. Gidez, L. I., and Karnovsky, M. L. ( 1954). J. Biol. Chem. 206, 229. Goebel, A., and Seckfort, H. (1948). Biochem. Z.319,203. Gronchi, V. ( 1936). Sperimentule 90, 223. Grosse, A., and Taubock, H. ( 1942). 2. Rheumforsch. 6, 429. Hanahan, D. J. (1952). J. Biol. C h m . 195, 199. Hannon, V., and Toschi, G. (1959). Exptl. Cell. Research 16, 256. Hayaishi, O., and Kornberg, A. (1954). J. Biol. Chem. 206, 647. Johnson, A. C., McNabb, A. R., and Rossiter, R. J. (1949a). Biochem. J . 44,494. Johnson, A. C., McNabb, A. R., and Rossiter, R. J. (194913). Can. J. Research E27, 63. Kennedy, E. P., and Weiss, S. B. (1956). J. Biol. Chem. 222, 193. King, E. J. (1931). Biochem. J., 25, 799. Kline, D., Magee, W. L., Pritchard, E. T., and Rossiter, R. J. (1958). J . N m r o c h m . 3,52. LeBaron, F. N. (1959). Ann. Rev. Biochem. 28,579. McAlpine, D., Oompston, N. D., and Lumsden, C. E. (1955). “Multiple Sclerosis.” Livingstone, London. McArdle, B., Thompson, R. H. S., and Webster, G. R. (1960). J . Neurochem. 5, 135. Macfarlane, M. G., and Knight, B. C. J. G. (1941). Bwchem. J. 36, 884.

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McIlwain, H. (1955). “Biochemistry and the Central Nervous System.” Churchill, London. McMillan, P. J., Douglas, G. W., and Mortensen, R. A. (1957). Proc. SOC. Exptl. Biol. Med. 96, 738. McMurray, W. C., Berry, J. F., and Rossiter, R. J. (1957a). Biochem. J. 66, 629. McMurray, W. C., Strickland, K. P., Berry, J. F., and Rossiter, R. J. (1957b). Biochem. 1.66, 621. McMurray, W. C., Strickland, K. P., Berry, J. F., and Rossiter, R. J. ( 1 9 5 7 ~ ) . Biochem. J. 66,634. Majno, G., and Karnovsky, M. L. ( 1958). J. Exptl. M e d . 107, 475. Marples, E. A., and Thompson, R. H. S . (1960). Biochem. J. 74, 123. hlarples, E. A., Thompson, R. H. S., and Webster, G. R. (1959). J. Neurochem. 4, 62. Morrison, L. R., and Zamecnik, P. C. (1950). A.M.A. Arch. Neurol. Psychiat. 63, 367. Moser, H. W., andKarnovsky, M. L. (1959). J. Biol. Chem. 234, 1990. Nicholas, H. J., and Thomas, B. E. (1959). J. Neumchem. 4, 42. Noguchi, S. (1944). J. Biochem. (Tokyo) 36, 113. Norman, J. M., and Dawson, R. M. C. (1953). Biochem. J. 54,396. Nygaard, A. P., Dianzani, hl. U., and Bahr, G. F. (1954). Ex$. Cell. Research 6, 453 Petersen, V. P., and Schou, hl. (1955). A d a Physiol. S c a d . 33, 309. Petrushka, E., Quastel, J. H., and Scholefield, P. G. (1959). Can. J. Biochem. and Physiol. 37, 975. Platt, B. S. (1955). Brit. Med. J. I , 179. Porcellati, G. (1958). J. Neurochem. 2, 128. Pritchard, E. T. (1958). Can. J. Biochmn. and Physiol. 36, 1211. Pritchad, E. T., and Rossiter, R. J. (1959). J. Neurochem. 3, 341. Radin, N. S., Martin, F. B., and Brown, J. R. (1957). J. Biol. C h e m . 224, 499. Robins, E., Eydt, K. M., and Smith, D. E. (1956). J. Biol. C h e m . 220,677. Rodnight, R. (1956). Biochem. J. 63,223. Rossi, A. (1935). Z. physiol. C h e m . 231, 115. Rossiter, R. J. ( 1957). In “Metabolism of the Nervous System” ( D . Richter, ed.), p. 355. Pergamon, New York. Rossiter, R. J., Mckod, I. M., and Strickland, K. P. (1957a). Can. J. Biochem. and Physiol. 35, 945. Rossiter, R. J., McMurray, W. C., and Strickland, K. P. (195713). Federation Pruc. 16, 853. Sachs, J., and Culbreth, G. G. (1951). Am. J. Physiol. 165, 251. Sadhu, D. P. (1953). Am. J. Physwl. 174, 238. Saunders, L. (1957). J. Pharm. and Pharmucol. 9, 834. Schachner, H., Fries, B. A., and Chaikoff, I. L. (1942). J. Biol. Chem. 146,s. Shapiro, B. (1953). Biochem. 3. 53,663.

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CONVULSIVE EFFECT OF HYDRAZIDES: RELATIONSHIP TO PY RI DOXlNElzz By Harry L. Williams3 and James A. Bain Department of Pharmacology, Division of Basic Health Sciences, Emory University, Atlanta, Georgia

I. 11. 111. IV. V. VI. VII.

Introduction ........................................... 319 General Pharmacological Responses to the Hydrazides . . . . . . . . 321 Neurophysiological Aspects of Hydrazide Action . . . . . . . . . . . . . 325 Biochemistry of Hydrazide Action . . . . . . . . . . . . . . . . . . . . . . . . . 329 Neuropathology and Clinical Aspects ....................... 339 Miscellaneous . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344

I. Introduction4

In the past ten years considerable interest has developed in the pharmacological and biochemical aspects of a group of compounds which we have called the convulsant hydrazides. These are, for the most part, unsubstituted or monosubstituted hydrazides of the general structure R-CO-NH-NH2 or their thio analogs. They are reducing agents and complex readily with carbonyl groups. These hydrazides will produce convulsions in animals and man 1 Supported in part by Grant Numbers B-660 and M-875 from the United States Public Health Service. 2 Survey of the literature for this review was completed July, 1960. We regret that some articles in the foreign literature were not available to us and being aware only of the titles we could not include them in our discussion. 3 Markle Scholar in the Medical Sciences. 4 Abbreviations used throughout this paper are : pyridoxal, PL; pyridoxine, PN; pyridoxamine, PM; pyridoxal phosphate, PLP; pyridoxine phosphate, PNP; pyridoxamine phosphate, PMP; isoniazid, INH; semicarbazide, SC; thiosemicarbazide, TSC; deoxypyridoxine, dPN; central nervous system, CNS; thiocarbohydrazide, TCH; y-aminobutyric acid, GABA; electroencephalogram, EEG; xanthurenic acid, XA.

319

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after a variable lag period of from 15 minutes to several hours after their administration. The study of the mode of action of these compounds, the best known of which is the antitubercular drug, isoniazid ( INH) , has led to investigations in epilepsy, pyridoxine metabolism, neuropliysiolog~~, and the biochemistry of amino acid transformations and actions in the central nervous system (CNS). The present review is concerned with the pharmacology of these coiivulsant hydrazides and more specifically with their probable relations to pyridoxine metabolism. The recent appearance of several excellent summaries of material closely related to the subject of the present review (Elliott and Jasper, 1959; Brady and Tower, 1960; Roberts, 1960; Roberts and Eidelberg, 1960; Tower, 1960) has made our task considerably easicr. Even though there will inevitably be some overlap between our presentation of the subject and those of others, we hope that our emphasis is sufficiently different to make it of interest. In 1943 Astwood reported that thiosemicarbazide ( T S C ) was toxic to rats, but he did not elaborate on the nature of the toxicity. Dieke (1949) commented on the convulsant activity of TSC and proposed its use as a rodenticide. When attempting to use semicarbazide (SC) as an iii v i m histaminase inhibitor, Lowell5 in 1949 came by chance upon its convulsant action. This observation prompted the addition of SC to the series of convulsants against which candidate anticonvulsant drugs were screened (Jenney and Lee, 1951), Another hydrazide, INH, was shown to be convulsant (Benson et aZ., 1952; Rubin ct nl., 1952; Schmidt et al., 1953). This action was used in the convulsaiit therapy of schizophrenia by Reilly and co-workers (1953), who had also used other hydrazides for this purpose (Stephens et aZ., 1951). This experimental finding of seizures produced by large doses of INH also showed up as a side effect in the therapy of tuberculosis (Pleasure, 1954). In the meantime the parent compound, hydrazine, had also been reported to be convulsant (Fine et nl., 1950). During this samc period interest in the tuberculostatic properties of TSC derivatives prompted studies on their mechanism of action (Dewey et nl., 1952). It was discovered that the inhibitory effects of this hydrazide on Tetrnhyniena geleii and its convulsant 2

D. J. Lowell, unpuhliahed observations ( 1949).

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OF HYDRAZIDES

321

effects in animals (Parks et al., 1952) could be reversed by pyridoxamine ( PM ). It was subsequently shown that pyridoxine (PN ) could reverse the convulsant c4ect of SC (Jenney et al., 1953) and that pyridoxine antagonists v oiild enhance the toxicity of SC.6 Convulsions produced by hydrazides were found to be accompanied by inhibition of vitamin B6-requiring enzymes in the brain (Killam, 1954). Hydrazide treatment was shown to produce xanthenmia, an indication of Pyridoxine deficiency, in tryptophan-loaded dogs ( M’illiams and Wiegand, 1955). The failure of reasonable doses of a variety of other metabolites (Dewey et al., 1952; Parks et al., 1952; Jenney and Lee, 1951) to antagonize SC and TSC effects accentuated the specificity of the pyridoxine reversal. Despite certain anomalous observations, such as the failure of pyridoxine-deficient diets to enhance the toxicity of TSC (Parks et al., 1952) and the failure of INH to produce xantlienuria in rats (Rosen, 1955), a relationship of vitamin BG to hydrazide-induced convulsions seemed clearly indicated. Parallel developments in the biochemical and clinical aspects of hydrazide action were also beginning about this time, but we will delay their description until later. It.

General Pharmacological Responses

to the Hydrazides

The most extensive basic data on the CNS effects of hydrazides as a group are those of Jenney and Pfeiffer (1958). These investigators surveyed 38 hydrazides of varying structure and found in the mouse LD;,<s ranging from 9.6 mmoles/kg for lactic acid hydrazide to 0.04 mmoles/kg for thiocarbohydrazide ( T C H ) , the most potent compound. Intraperitoneal and intravenous doses were about the same, and in the mouse the convulsant dose 50 was found to be only slightly less than the LDEo because the animals generally died from respiratory arrest in the tonic phase of the convulsion. There is a pronounced lag period of about 45 minutes between the administration of a convulsant dose 50 and the actual onset of convulsions. During this period animals become progressively more excitable and more sensitive to sensory stimuli. Audiogenic stimuli are especially effective in triggering seizures in mice6 (Quadbeck and Sartori, 1957), but other types of inputs may be 6

E. H. Jenney, unpublished observations (1953).

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used. The seizures are typically epileptiform with some accentuation of clonic phases. They usually begin with a frantic running episode culminating in a short period of violent clonus which is followed by a tonic extensor phase. If the animal survives this phase there then follows a variable clonic phase and a long period of postictal depression. Occasionally with borderline doses mice will survive long enough to have multiple seizures, and larger animals, including man, may be thrown into a state resembling status epilepticus. This typical pattern of response has been demonstrated in a variety of species including mice, rats, rabbits, dogs, cats, monkeys, birds, frogs, and man7 (Dieke, 1949; Jenney and Lee, 1951; Stephens et al., 1951; Lowell et al., 1952; Parks et al., 1952; Scarinci and Costantini, 1952; Allmark et al., 1953; Schmidt et al., 1953; Schalleck and Walz, 1954; Vysniauskas and Brueckner, 1954; Porcino, 1954; Wood, 1955). The convulsant close for the hydrazides does not vary much from species to species. For example, the dose of SC in milligrams per kilogram is 40, 60, 40, 75 in man, monkey, cat, and guinea pig, respectively. The dog is significantly more sensitive requiring only 10 mg/kg, and the rodent family is significantly more resistant. The doses for the rabbit, rat, and mouse are 175, 150, and 116 mg/kg, respectively. As was mentioned earlier there is little difference in the convulsant dose when administered by various parenteral routes at least in the mouse. However, the oral dose is somewhat higher being of the order of 175 mg/kg as compared to 125 mg/kg by parenteral routes (Jenney and Pfeiffer, 1958). The acute convulsive effects of the hydrazides are partially reversed by depressant and anticonvulsant drugs, a spectrum of which was studied by Jenney and Pfeiffer (1958). They found that phenobarbital, phenacemide, trimethadione, bromides, atrolactamide, and 3-methyl-5,5-phenylethylhydantoinproduced significant decreases in the incidence of convulsions and deaths. Diphenylhydantoin reduced the incidence of tonic extensor convulsions, but allowed persistent clonic convulsions which were ultimately lethal. Pure depressants such as pentobarbital, ethanol, paraldehyde, etc., also abort convulsions and decrease mortality from hydrazide administration; this is characteristic of their anticonvulsant actions in general when used in near-anesthetic doses. As might be expected 7

H. L. Williams, and E. H. Jenney, unpublished observations (1953).

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on chemical grounds, carbonyl-containing compounds, including naturally occurring metabolites such as pyruvate and a-ketoglutarate, significantly reduce the lethal effects of SC and increase the time to the first seizure (Jenney and PfeifFer, 1958; Prescott et al., 1958). Acetone, perhaps because of its depressant properties, is a very effective antidote but paradoxically acetone semicarbazone given by itself is a more potent convulsant than SC per se. It is pertinent, we believe, that the effective doses of these carbonyl compounds are very high (30-40 times) compared to those of the standard anticonvulsants, indicating a different mechanism of action. The hydrazides act in conjunction with other convulsant compounds to decrease their threshold doses, and the reverse is also true. For example, the convulsant threshold dose for pentylenetetrazol may be lowered to one-quarter that ordinarily required by pretreatment with SC (Jenney and Pfeif€er, 1958). On the other hand strychnine thresholds were not affected by pretreatment with SC nor were the thresholds for ammonium acetate seizures. This latter finding may have some bearing on biochemical considerations to be discussed later. Similarly, the effect of pretreatment with the hydrazides can be demonstrated as a decrease in threshold to electroshock production of seizures (Killam et al., 1960) and also as a reduction of the threshold for photostimulation in man (Stephens et al., 1951), a phenomenon which can similarly be demonstrated immediately after the administration of pentylenetetrazol. The antidotal effect of pyridoxine congeners on hydrazideproduced seizures has been reported by several groups of investigators since the first report by Parks and associates (1952) of the antidotal effect of PM on TSC poisoning. PN, PM, and pyridoxal ( P L ) are all approximately equally effective in antidoting the effects of the acute administration of SC to mice. TSC, however, was most effectively antidoted by PL followed by PN whereas PM gave little protection6 (Jenney et al., 1953). The doses of the latter compound used were lower than those used in the studies of Parks et al. mentioned above. An explanation for the lower potency of PM as a hydrazide antidote is now available in the data of Takahashi and co-workers (Bain and Williams, 1960) who showed that PL and PN were much more effective in increasing brain levels of vitamin BB than was PM. Although vitamin B6 is an effective anti-

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dote for hydrazide-induced seizures, it is not a general anticonvulsant since it does not protect against strychnine, pentylenetetrazol, ammonium acetate, or electroshock-induced seizures ( Jenney et al., 1953). Nor is Vitamin BG an effective anticonvulsant in the treatment of idiopathic epilepsy ( Livingston et nl., 1955; Tower, 1960). The simple hydrazides are easier to antidote with pyridoxine congeners than are such compounds as INH ( P a n et al., 1952; Pfeiffer et al., 1956; Prescott et nl., 1957; Quadbeck and Sartori, 1957; Ross, 1958). Reilly and co-workers (1953), for example, mention that B6 antidotal effects in the case of INH are incomplete and recommend the use of phenobarbital in human patients. Indeed Bukin (1960) reports that PL while antidoting completely toxopyrimidine convulsions does not affect INH poisoning. ( Toxopyrimithe pyrimidine is 6-amino-5-hydroxymethyl-2-methylpyrimidine, dine portion of vitamin B1 which acts as a vitamin B6 antagonist.) He also reports that L-glutamic acid and y-aminobutyric acid (GABA) do not prevent toxopyrimidine or INH poisoning but that GABA depresses the convulsions to a considerable degree. Parks et al. (19S2) reported that a B6-deficient diet did not enhance the susceptibility of mice to TSC-induced convulsions. However, it has been shown that animals kept on a B6-deficient diet are much more susceptible to the lethal action of INH (Caliari et al., 1955; Rosen, 1959) and that INH inhibits the storage of BG by liver leading to an inhibition of glutamine synthesis which inhibition could be reversed by B6. This substantiates the earlier report of Reilly and co-workers (1953) that INH lethal effects are reversed by PN. It has also been demonstrated that BG deficiency produced by feeding a poor diet to the mother of suckling rats resulted in a high sensitivity to sound-induced convulsions in the offspring ( Pilgrim and Patton, 1949), a condition which resembles the increased sensitivity to audiogenic stimuli produced by the hydrazides. Furthennore, there is a definite enhancement of the convulsive effectiveness of hydrazides by simultaneous administration of PN antagonists such as deoxypyridoxine ( dPN).6 Rats fed a vitamin B6-deficient diet and injected daily with a maximum oral dose of INH showed a greater retardation of growth than rats receiving the same diet plus the known antimetabolite dPN (Boone el nl.,

1955). Insofar as the pharmacodynamic effects of hydrazides are con-

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cerned these have been best studied in the case of INH. INH has been reported in man to produce constipation, difficulty of micturition, twitching of the extremities, exaggerated reflexes, headache, vertigo, drowsiness, dryness of the mouth, eosinophilia, and mild anemia (Robitzek and Selikoff, 1952). Despite these reports, careful studies in experimental animals with doses equivalent to therapeutic doses in man ( u p to 32 mg/kg) have failed to show any effect on intestinal motility, salivation, lacrimation, respiratory rate, metabolic rate, blood pressure, heart rate, electrocardiogram, peripheral resistance, hind leg volume, and postural hypotension; nor were any anticholinergic or antihistaminic or atropine-like effects seen. There was no evidence for an effect on the adrenal medulla or on the vagus. High concentrations of INH were shown to inhibit the motility of isolated intestinal strips but there was little effect on the intestine in situ (Benson et al., 1952; Mainardi and Semenza, 1952; Rubin and Burke, 1953). Similarly, TSC has no immediately demonstrable effects upon blood pressure, respiratory rate, or responses to histamine or acetylcholine in the dog (Jenney and Pfeiffer, 1958). INH has been reported to have a positive inotropic action on the isolated rabbit heart and also to increase the rate of coronary flow (Allmark et al., 1953). This group of investigators also reported that INH prolongs the sleeping time induced by hexobarbital, pentobarbital, and secobarbital in rats. However, Goldin and co-workers (1955) reported a prolongation of the anesthetic action of pentobarbital by INH, l-isonicotinyl-2-isopropyl hydrazine phosphate, isonicotinic acid amide, nicotinic acid hydrazide, 3-acetylpyridine, hydrazine hydrate, and glycine; this effect, then, does not appear to be specific for the convulsant structures.

111.

Neurophysiological Aspects of Hydrazide Action

The electroencephalogram ( E E G ) of animals treated with convulsant doses of hydrazides shows the typical high-voltage highfrequency pattern associated with grand ma1 epilepsy and with high doses of other CNS stimulants such as pentylenetetrazol (Stephens et al., 1951; Lowell et al., 1952; Reilly et al., 1953; Schalleck and Walz, 1954; Preston, 1955a). Local application of SC to the exposed cortex of the monkey was shown early to lead to spiking

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phenomena a t the point of application followed by spread and generalized seizure patterns in adjacent areas (Lowell et al., 1952). These investigators also noted that there was a lag period of 3 to 4 hours between the application of the hydrazide and the appearance of major seizures. They found that the appearance of spiking from topically applied hydrazide was accompanied by movements in the extremities corresponding to the point of application of the hydrazide to the motor cortex. Further, they determined that in the monkey 16 mg/kg of SC given intravenously produced, again after a 3- to 4-hour lag period, grand ma1 convulsions in the intact animal. These early studies have been followed up by much more detailed investigations by Preston (1955a,b),I n one study (Preston, 1955a), he contrasted the effects of pentylenetetrazol and TSC applied to a plug of cortex isolated, insofar as neuronal connections are concerned, from the rest of the brain. Again a lag period of about 30 minutes occurred between the topical application of TSC to the isolated cortical plug and the appearance of spike discharges. Such a lag period was not seen with pentylenetetrazol. Furthermore, intravenous injection of TSC resulted in seizures occurring in the intact cortex a long while before any seizure activity occurred in the isolated plug, in contrast to the action of intravenous pentylenetetrazol where seizures were seen simultaneously in the isolated cortical plug and the intact cortex. Preston (1955a) was led to postulate that the primary site of action of hydrazide was in some subcortical structure, whereas the action of pentylenetetrazol occurred simultaneously at both the cortical and subcortical levels. Following up this postulate, Preston ( 195513) showed by means of deep electrode recordings that the earliest spike activity after intravenous TSC occurred in the head of the caudate nucleus, appearing a short time later in the periaqueductal gray and finally spreading to the cerebral cortex, after appearance of synchronous spike discharges in the periaqueductal gray. These observations were later correlated with enzyme changes by Killam (1957) in studies to be discussed later. Killam and co-workers (Dasgupta et al., 1958; Killam et al., 1960) found that TSC increased the duration of seizures following cortical or hippocampal stimulation and lowered the threshold for cortical seizures. This action of the hydrazide was antagonized by topical application of GABA or by intravenous PL, and a similar antagonism by these

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substances to spontaneous scizures induced by TSC was demonstrated. An effect of the hydrazides has also been shown on cerebellar and cerebral activity in the cat by Dunlop and coworkers ( 1960). They found that, whereas pentylenetetrazol had little effect on cerebellar or neocortical direct-current potentials or on unit discharges, TSC produced irregularity in the neocortical wave activity and a regular progression of slow activity with increasingly negative dendrite to subcellular direct-current potentials tip to the point of seizure discharge, after which negativity decreased. Again PN gradually arrested the TSC induced seizures. Purkinje and cortical cells became inaccessible to cortical stimulation after TSC. TSC was also found to raise threshold for cerebellar cortical hyperpolarization and depolarization. Eidelburg and Buchwald (1960) demonstrated that TSC had an effect on spinal reflex activity consisting of an increase in the amplitude of monoand polysynaptic reflex responses. Bulbar reticular inhibition of these reflexes was no longer seen. PN (125 mg intravenously) reversed the effect of 30-50 mg/kg TSC given by the same route. These investigators conclude that TSC increases the level of excitability of spinal structures. Thus it would appear that the hydrazides, assuming that TSC is typical, increase the excitability of neurons at all levels within the CNS, and that this increase in excitability may be reversed by the administration of various forms of vitamin B6. Evidence that will be described below has led to the postulation that the excitatory cJffect of the hydrazides is brought about by a fall in the concentration of GABA, presumably in the vicinity of the neuronal membrane. Information bearing on this postulate has been extensively reviewed in a recent monograph (Roberts, 1960) and need not be recounted in detail here. GABA and to a lesser extent the three-carbon analog, p-alanine, were shown to have powerful depressant effects when applied directly to interneurons, motorneurons, and Renshaw cells of lightly nembutalized cats (Curtis et al., 1959; Curtis and Watkins, 1960). Using elegant microelectrode and micropipette techniques, these investigators obtained direct evidence of the effect of these agents on neuronal excitability. However, they also showed that dicarboxylic amino acids, such as aspartic, glutamic, and cysteic, excite all three types of the cells referred to above

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by producing depolarization (Curtis et al., 1960; Curtis and Watkins, 1960). The authors consider that t h s action is nonspecific and not related to synaptic processes because it is not blocked by P-erythroidine which blocks cholinergic excitation of Renshaw cells, and because D- and L-amino acids gave equivalent effects. A variety of possible enzyme inhibitors including a number of convulsant hydrazides were also applied iontophoretically but did not affect either synaptically or amino acid-evoked spikes. Killam and Killam (1'360) studied the effect of TSC on evoked potentials in the sensory motor cortex elicited by a variety of routes of stimulation. They found that all such potentials were increased irrespective of the direction or source of the stimulation. Again the augmentation of the responses was reversed by the administration of PL, this time with some regional differentiation in that pyramidal responses were the most easily, and cortical the least easily, reversed. At the time that seizures were abruptly halted by PL injection and the EEG nornialized only the evoked pyramidal response had returned to normal; the other evoked potentials were still enhanced for 15 to 20 minutes or longer before returning to control levels. The authors interpret this finding to mean that the seizure discharge, which they feel is due to inhibition of the production of GABA, is triggered when the soma is affected but that normal activity may return even when synapses are still conducting abnormally. This finding is compatible with the large body of data on sensitivity to seizures triggered by audiogenic or visual stimuli in animals and on seizures in man treated with doses of the hydrazides which by themselves would be subthreshold (cf. Reilly et al., 1953); it is also compatible with the prolongation of electrically induced seizures by such hydrazide treatment (Killam et al., 1960). It was also reported that when GABA is administered intraventricularly it has marked depressant effect on the TSC-induced high-voltage spike activity and also upon the appearance of spontaneous seizure activity (Killam et d.,1960). This degree of inhibition of TSC seizures is not brought about by topical application of GABA to the cortex. The author interprets this finding as support for the earlier conclusions that subcortical structures are most sensitive to hydrazides (Preston, 1955b; Killam, 1957). Local spikes or seizure discharges produced by the topical appli-

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cation of strychnine, pentylenetetrazol, dPN, or TSC to the cerebral cortex in cats could be changed in character by local application of GABA or p-alanine. The change was a rapid decrease in the negative component of the spike with an increase in the positive component. No change in the frequency of the spikes was noted, and these data were interpreted as not supporting an association between GABA levels and hydrazide seizures (Williams, 1958). This whole subject of the effect of co-amino acids on evoked potentials in the cortex has been intensively investigated by Purpura and co-workers (Purpura c>t d.,1959; Purpura, 1960). Purpura (1960) concludes his exhaustive review of this subject by indicating that “while the foregoing studies indicate the necessity of numbering certain amino acid drugs among the most potent synaptically active agents presently known they provide no evidence that the naturally occurring compounds, GABA, y-guanidinobutyric acid, etc. are directly involvcd in central synaptic transmission.” Similarly, Curtis and Watkins (1960) also cautioned against the facile interpretation of GABA action on synaptic processes as being the only possible explanation for hydrazide action as demonstrated by the neurophysiologists. It may be said with some certainty that the acute effects of the liydrazides are referrable to an action on the cell body or on interneuronal connections no matter what the mechanism by which these effects are brought about. With the doses usually employed and the time periods involved the axon itself is probably not affected since soaking peripheral nerve for extended periods in hydrazide solutions does not affect their conduction parameters.s

IV.

Biochemistry of Hydrazide Action

The fact that there was a considerable lag period between the administration of a convulsant dose of hydrazide and the appearance of the electroencephalographic and gross manifestations of the epileptiform convulsions suggested to us that this group of convulsants might provide a much needed tool which would make it possible to study the biochemical events in the brain that lead to 8

H. L. Williams, unpublished observations ( 1957).

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the seizure process. The developments in the pharmacology of hydrazide action outlined above took place, of course, against the background of information being gathered by investigators interested in the basic nutritional and biochemical aspects of vitamin BG. Most of the details of studics on these topics are beyond the scope of this review and the reader is referred to recent summaries on the general topic of pyridoxine biochemistry (Snell, 1955; Snell and Jenkins, 1959; Braunstein, 1060), the somewhat earlier monograph (Williams et nl., 1950), and the appropriate chapters of the Annual Reuiew of Biochemistry. It is sufficient to state here that one of the chief signs of vitamin BGdeficiency has long been recognized to be malfunction of the nervous system including the development of neuropathies, increased nervous system excitability, and u1tim;ately convulsions. Also paralleling the studies on hydrazides outlined in previous paragraphs was the development of our knowledge of amino acid transformations in the brain, particularly those involving the glutamic acid cycle and the importance of amino acid metabolism to CNS function. Early interest in this area may be seen in the papers cited in the reviews by Archibald (1945) and by Weil-Malherbe (1950). It was, in fact, the latter review which prompted Killam (1954) to initiate the amino acid studies which are described below. He undertook an investigation of the effect of hydrazide-induced convulsions on the amino acid metabolism of the brain. An impairment of the activity of glutamic acid decarboxylase, a PLP-requiring enzyme, in the brains of hydrazide-convulsed animals was demonstrated by a decrease in the concentration of the reaction product, GABA, and by direct measurement of the activity of the enzyme in excised brain tissue preparations (Killam, 1954, 1957; Killam and Bain, 1957). No changes were noted in the level of glutamic acid, nor in glutamic-oxalacetic transaminase. Likewise no changes in amino acid levels or in enzyme activity were produced by pentylenetetrazol convulsions. Convulsions produced by hydrazides and other agents such as pentylenetetrazol do have areas in common, as evidenced by the fact that hydrazide administered at subthreshold doses lowers the convulsant dose for pentylenetetrazol (Jenney and Pfeiffer, 1958). Supporting an opposite point of view is the fact that PN has no

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33 1

effect on pentylenetetrazol thresholds nor, for that matter, on idiopathic epilepsies (Tower, 1960). Because of the chemical reactivity between the aldehyde group of PLP and the hydrazides, studies on their in vitro activity as enzyme inhibitors cannot be unambiguously related to the intact animal since the conditions of pH, hydrazide concentration, etc., at the enzyme site in vioo are not exactly known. For example, Killam and Bain (1957) showed that glutamic acid decarboxylase and glutamic-oxalacetic transaminase were both inhibited in vitro by SC and TSC and to about the same dcgree with comparable conccntrations of inhibitor. On the other hand, apparently only the decarboxylase was inhibited i n tjioo. Bu deficiency produced by deprivation of the vitamin, however, is well known to produce a lowering of both glutamic-oxalacetic transaminase and glutamic decarboxylase in uiuo. This has been most recently demonstrated by Babcock and co-workers ( 1960). In a study of enzyme inhibition in various brain areas, Killam (1957) showed that the impairment of enzyme activity upon hydrazide treatment was greatest in subcortical structures, and he obtained evidence that the apornzyme in structures such as the caudate nucleus and corpora quadrigemina was saturated to a lesser degree with coenzyme than it was in cortical structures. This seemed to provide an explanation for the increased sensitivity of the subcortical structures to the action of the hydrazides. The distribution in the CNS of the one hydrazide, INH, for which data are available (Barlow et nl., 1957) is such that there is a somewhat higher concentration of the drug in cortical than in subcortical structures so that, like many other drug effects, specificity may not be a reflection of concentration of the drug in the affected structure. Coincident with the appearance of Weil-Malherbe’s review (1950) came the simultaneous publication from the laboratories of Awapara (Awapara et nl., 1950) and of Roberts (Roberts and Frankel, 1950) describing the discovery of GABA in the brain. This discovery has been followed over the course of the past ten years by numerous studies which have served to bring us to our present degree of knowledge regarding the biochemistry of this segment of amino acid metabolism. This work has been most recently summarized by Roberts and Eidelberg (1960) and by Tower (1!360). During this period the discovery of the Florey inhibitory factor I

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for the crayfish stretch receptor neuron, its tentative identification as GABA, and the demonstration that the latter substance had inhibitory properties in mammalian nervous systems ( Elliott and Jasper, 1959), heightened the interest of numerous investigators in these contiguous areas which culminated in tlie papers presented at a symposium on the inhibitory effects of GABA in the nervous system (Roberts, 1960). Still another aspect of the biochemistry of hydrazide effects began to be clarified with the studies of Biehl and Vilter (1954a) who, because of earlier work on the effect of PN in pregnancy (for review see Vilter et al., 1954), discovered that administration of INH caused an increase in the excretion of xanthurenic acid (XA) in the urine after administration of tryptophan. More important, they also observed a marked increase in the excretion of PN in the urine of isoniazid-treated patients. These workers postulated that INH formed a complex with PL which was subsequently excreted. These studies were later expanded to other species and a variety of hydrazides by Williams and Wiegand ( 1955, 1960). Williams and Abdulian (1956) demonstrated that the administration of SC to dogs resulted in the excretion of pyridoxal semicarbazone in the urine, thus confirming the postulation of Biehl and Vilter (1954a) that hydrazones of PL are formed and that PN, in the fomi of the hydrazone, is excreted in hydrazide-treated animals. Davison ( 1956) and Wiegand (1956a) studied the formation of PL and PLP hvdrazones. They concluded that because of the lower activation eriergy for the formation of tlie PLP hydrazone this was most probably the reaction which took place in tissues. It was postulated that the hydrazone was subsequently depliosphorylated before excretion, tlie dephosphorylation perhaps taking place in the kidney since Wiegand (1956b) had demonstrated the presence of a very active phosphatase in the kidney. Davison (1956) studied the kinetics of the inhibition of PLP-requiring enzymes by INH in vitro and concluded that this inhibition could be explained on the basis of the formation of a PLP hydrazone. Development of methods for the ion-exchange separation of vitamin BF congeners and their hydrazones in tissue extracts by Takahashi and co-workers (Bain and Williams, 1960) permitted them to demonstrate a marked fall in PLP accompanied by the appearance of PLP hydrazone and PL hydrazone in the brains of INH-treated animals. Wiegand (1956b)

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had earlier obtained preliminary evidence for the formation of PL and PLP hydrazones of various hydrazides in the brains of hydrazide convulsed animals. This information, together with that indicating a fall in glutamic acid decarboxylase in the area of the brain at which spiking first was produced by hydrazide treatment and the restoration of the activity of the enzyme by the addition of PLP (Killam, 1957; Killam and Bain, 1957), led to the postulate that treatment with the hydrazides through formation of PL and PLP hydrazones, particularly the latter, produced an acute pyridoxine deficiency in the tissues of the treated animals. It was further postulated that this deficiency was most critical to the function of the CNS and thus production of convulsions was the most outstanding manifestation of this deficiency. Discovery of the inhibitory properties of GABA on neuronal excitability discussed in Section 111 provided a rather facile explanation for the convulsant effect of the hydrazides and for the antidotal effect of PN on these convulsions. That this is perhaps a too facile explanation, at least insofar as GABA is concerned, has been pointed out by several authors. Baxter and Roberts ( 1960) have recently reported that, whereas they confirm the report of Killam and Bain (1957) that TSC treatment lowers GABA levels in the brain concomitant with the production of convulsions, nevertheless, simultaneous administration of hydroxylamine [which they previously reported elevated cerebral GABA levels (Baxter and Roberts, 1959)] prevented the fall or even produced a rise in GABA without preventing TSC-induced convulsions. Killam and Bain (1957) had previously reported that pentylenetetrazol-induced convulsions occurred without change in cerebral GABA levels, a finding which we interpreted at the time to indicate one of two things: ( a ) pentylenetetrazol and hydrazide convulsions are produced by two different mechanisms, or ( b ) GABA has no necessary relationship to the convulsant process. The findings of Baxter and co-workers (Baxter and Roberts, 1960; Baxter et al., 1960) support the second of these possibilities without, of course, having any necessary bearing on the first. No matter what particular metabolite or process is involved in the ultimate explanation of hydrazide-induced convulsions there seems little doubt that PN in some form plays a critical role. The most crucial evidence for this hypothesis is the antidotal effect of

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BG upon hydrazide-induced convulsions ( and neuropathies ) and the well-known effect of BG deficiency upon nervous excitability. Accepting for the moment the thesis that a decrease in the activity of some PLP-requiring enzyme is the key to the convulsant action of the hydrazides, two possibilities have been advanced to explain the decreased enzyme activity. The first possibility already referred to above is simply that formation of hydrazones of PL and PLP lowers the level of coenzyme to such a point that the apoenz,yme is no longer saturated. This explanation requires a ready dissociability of the coenzyme-apoenzyme complex, a condition which is satisfied by glutamic acid decarboxylase. A second somewhat more involved possibility has been advanced by McCormick and Snell (1959) on the basis of their finding that the PL hydrazones are effective inhibitors of PL kinase. Inhibition of this phosphorylation enzyme would also result in a lowering of PLP levels and a subsequent decrease in the activity of enzymes such as glutamic acid decarboxylase. Either mechanism gives the same result, and unless compartmentalization within the cell serves to limit diffusion of the hydrazones, the second mechanism can hardly be absent in the presence of the first. The McCormick and Snell (1959) mechanism may well account for the fall in PLP observed in the brains of hydrazide-treated animals ( Bain and Williams, 1960). However, these authors showed that the formation of pyridoxamine phosphate (PMP) and pyridoxine phosphate (PNP) was catalyzed by the same kinase, and it would be expected that PMP would likewise decrease under the influence of PL-INH. This does not occur in brain, at least, upon administration of the hydrazides (Bain and Williams, 1960). On the other hand, if PMP is firmly bound to some particulate structure as it may be (Rott, 1960) and is thus not in equilibrium with the cellular phase in which hydrazone formation and BF dephosphorylation occurs, then acute inhibition of phosphorylating processes might not lead to an appreciable fall in PMP. McCormick and Snell (1959) assume that there are sufficient levels of free PL in tissue to form PL hydrazones at a high enough concentration to inhibit the kinase. This is probably not so in brain where only traces of free PL were found (Bain and Williams, 1960). However, hydrazones form in vitro with greater ease with PLP than with PL (Wiegand, 1956a) and we assume that the primary event in hydrazide action is the formation of PLP hydrazone. It is interesting

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to speculate whether or not hydrazone formation can take place with PLP still in combination with the apoenzyme. Although the kinase itself occurs in the soluble fraction of the cell, PMP is found almost exclusively in the particulate fractions of brain, where about one-half the PLP is also found.9 PLP hydrazones are found in all particulate fractions and in the soluble fractions of brain from INH-treated animals. The sum of PLP and PLP-INH in the brain mitochondria of such animals, however, is less than the total PLP in the brain mitochondria of control animals. There is probably some sort of equilibrium between the PLP in the particulate fraction and that in the soluble fractions of the cell and it is quite possible that, as PLP hydrazones are formed in the soluble fractions, the PLP in the particulates moves into the soluble phase to satisfy the equilibrium conditions. Or it may be that the PLP is lost from the particulates after the formation of the hydrazone. Dephosphorylation of the PLP hydrazone may then occur giving rise to the appreciable amounts of PL hydrazones found in the soluble fraction. It is, of course, possible that the PLP is first dephosphorylated, followed by hydrazone formation. We are inclined to think that the hydrazides bring about a lowering of cellular levels of PLP by a combination of both mechanisms. It is especially so if it is assumed that the hydrazone of PLP (which is present in the brains of convulsed animals at about three times the level of PL hydrazone) also has an inhibitory action on the kinase. McCormick and Snell (1959) did not test this possibility, but it seems to us to be a reasonable assumption. Although the equilibrium PL + PLP lies far toward the right, as evidenced by the fact that there is very little free PL in normal brain, the PLP seems to be in constant flux. At least this must be so if our current concepts of dPN and toxopyrimidine as inhibitors, after phosphorylation, of the various PLP-requiring enzymes are correct since it has been shown that these inhibitors do not displace PLP from the apoenzyme, but must combine when no PLP is present ( Braunstein, 1960). Toxopyrimidine treatment has been shown to lower the level of glutamic decarboxylase (Rindi et al., 1959), but strangely, dPN which is presumed to have the same 9 W. Rott, H. L. Williams, H. Takahashi, and J. A. Bain, unpublished observations ( 1959).

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mode of action as toxopyrimidine did not have this effect on the liver or brain of acutely intoxicated animals ( Umbreit, 1955). Close examination of the data of Roberts and co-workers (1951) shows that they did, however, observe about a 20% decrease in glutamic acid decarboxylase activity in dPN-treated animals in addition to about a 20% decrease which was attributable to deprivation of BG per se. Furthermore, in comparing the current hydrazide investigations with the older dPN work it must be kept in mind that the hydrazide effects are really acute effects. These occur at parenteral doses that produce convulsions within about an hour and the animals are sacrificed at the peak of the convulsion. In contrast, the older dPN work was mostly done with low oral doses administered chronically over periods of days and weeks, and convulsive phenomena are rarely mentioned as ensuing from this regimen. If dPN or toxopyrimidine is given in the same manner as the hydrazides, i.e., in a parenteral dose which produces convulsions in about 90 minutes, a marked fall in the B, content of the brain occurs which is mostly attributable to a loss of PLP.lo This is in contrast to the older studies with chronically administered dPN where little change was seen in the tissue BC levels with the antimetabolite alone. Similar contrasting findings would probably be observed with easily dissociable holoenzymes such as glutamic decarboxylase when dPN is given acutely in high doses. There are, however, differences between the effects of hydrazides and the B6 antimetabolites even when both are given in essentially the same manner. For example, administration of hydrazides to tryptophan-loaded animals gives rise to a marked excretion of XA and Be. Deoxypyridoxine and toxopyrimidine do not lead to the excretion of XA nor apparently of R6 upon acute administrationlo (Williams and Wiegand, 1960). Furthermore, there is still inconsistency at the cellular level between biochemical and physiological effects of the “BC antagonists. Rosen and co-workers (1960) studied three hydrazides and five BC antimetabolites and found that all three hydrazides produced convulsions and a fall in glutamic acid decarboxylase in brain of rats. Two of the antagonists, toxopyrimidine and 3-dPN, produced convulsions without any effect on the enzyme. 10

H. Takahashi, H. L. Williams, and J. A. Bain, unpublished obaervations

( 1959).

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4-Deoxypyridoxine and 5-dPN did not produce convulsions or any change in enzyme at the doscs used. However, the fifth antagonist, co-methylpyridoxine, produced a marked fall in enzyme levels but did not produce convulsions. Vitamin B,; levels were not measured at the same time, but the antagonists were not affecting the apoenzyme directly in any instance because addition of PLP to the assay system restored total enzyme activity to its normal maximum. Dubinick and co-workers (1’360) have invoked the McCormickSnell hypothesis (1959) to elplain their findings that PL enhanced the toxicity of certain hydra7icles and hydrazines. These workers also found that the hydrazones formed from (3-phenylethylhydrazine plus PL and p-phenylisopropylhydrazine plus PL were more toxic than the parent hydrazines themselves. A similar finding had previously been reported by Jenney and Pfeiffer (1958) where they found that acetone semicarbazone was more toxic than SC itself. At that time the interpretation was that the hydrazone penetrated the CNS more rapidly than did the free hydrazide. If the explanation for the enhanced toxicity of the hydrazones over the free hydrazide or hydrazine is in fact their effectiveness in inhibiting the PL kinase, it is difficult to understand why PN, the alcohol form, should be an antidote for hydrazine or hydrazide-induced convulsions. One explanation may be that the PN has a higher affinity for the kinase than have either PL or the PL hydrazones, and thus its phosphorylation is not inhibited. After being phosphorylated as the alcohol PNP might then be transformed to PLP. This is, it must be admitted, a rather roundabout explanation. Similarly, if it be conceded that a fall in the level of PLP and consequent impairment of the activity of the apoenzymes is the crucial event, no matter which mechanism is the actual means by which the fall is brought about, we still must consider the fact that in hydrazide-treated animals appreciable amounts of PLP hydrazonc accumulate (Bain and Williams, 1960). Gonnard and co-workers have reported that PLP isonicotinic acid hydrazone is, in fact, more active than PLP per se as a coenzyme for kynureninase ( Gonnard and Baigne, 1960), for dihydroxyphenylalanine decarboxylase, or for glutamic-aspartic transaminase ( Gonnard and Nguyen-Chi, 1959a; Gonnard and Nguyen-Philippon, 1959). They also explored other hydrazones of PLP and found them to be active as coenzymes for dihydroxyphenylalanine decarboxylase ( Gonnard and Nguyen-Chi, 1959b). These unexpected findings are

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HARRY L. WILLIAMS AND JAMES A. BAIN

difficult to reconcile with those of Davison (1956) and Killam and Bain (1957) who found decarboxylases to be inhibited by INH; they need confirmation particularly since it is asserted that the hydrazones are not split during the process of their acting as coenzymes. 'This would, of course, rule out the formation of Schiff bases with the amino acids during the process of transamination as has been postulated (Snell, 1958; Braunstein, 1960). If true, however, since the total amount of Be in the brain of hydrazide-convulsed animals is anly reduced at most about 30% and since the fall in PLP is partially accounted for as PLP hydrazone, it would appear that, if the hydrazones are coenzymatic, there should still be adequate coenzyme activity to satisfy the need for the apoenzyme function. Still another consideration is the suggestion, put forth some time ago by Lichstein (1955), that a hydrazide, INH, could act per se as an inhibitor of B6-requiring enzymes. His data is open to another interpretation, namely, that the INH combined with PLP to form the hydrazone; but, if this were so, it would require the assumption that the hydrazone is inactive as a coenzyme and this also would not be in line with the data of Gonnard cited above. It now appears that the picture is not as simple as was first believed, even with respect to the assumptions regarding inhibition of PLP-requiring enzymes. The most crucial point at the moment appears to be the need for confirmation of the work of Gonnard using mammalian enzyme systems. There are a number of miscellaneous facts which bear upon the biochemical aspects of the problem. Other enzymes, for which vitamin BGis probably not involved as a coenzyme, have been reported to be inhibited by the hydrazides. Catalase ( Andrejew et al., 1959), glycolytic processes (Cafiero, 1953), and acetylation processes ( Garattini and Paoletti, 1954; Johnson, 1955) are examples. Again the relationship of these inhibitions to the effect of hydrazides on the CNS is far from clear and at the moment, a t least, does not seem to be as immediate as those effects involving B6 enzymes. Enzymes other than those concerned with the formation of GABA or with the glutamic acid complex in brain also require vitamin B, for their activity. For example, the process of transsulfuration requires BG (for review see Anonymous, 1958a). The activity of the cystathionine enzymes does not appear to be very high in brain. Nevertheless, the substance does occur as do the precursors,

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homocysteine and serine (Tower, 1960). Still another enzyme which requires BG is 5-hydroxytryptophan decarboxylase, the action of which is required for the formation of 5-hydroxytryptamine ( serotonin) which has recently enjoyed considerable notoriety as a possible neurohumor. This enzyme is similar to glutamic and other amino acid decarboxylases in requiring BG (for review see Anonymous, 195&b).The work of Cori and co-workers (Baranowski et al., 1957; Cori and Illingworth, 1957; Illingworth et al., 1958) disclosed that crystalline muscle phosphorylase also contains PLP. Whether or not the brain phosphorylase contains this same component is not known although it seems likely. However, the bond between the PLP and phosphorylase is evidently a very strong one (Fischer and Krebs, 1959) and it does not seem probable that it would be affected by acute administration of B6 antagonists or hydrazides.

V.

Neuropathology and Clinical Aspects

We have not found any reports of studies of the direct effects of the hydrazides on the morphology of the CNS. Vitamin BG deficiency in the diet of monkeys results in prominent abnormalities in the large nerve cells of the cerebral cortex which show swelling, eccentricity of nuclei, and loss of Nissl particles, a cellular change which bears a strong resemblance to that seen in human pellagra (Victor and Adams, 1956). Similarly, the administration of a pyridoxine antagonist, methoxypyridoxine, at 50 mg/kg produced within 2 hours marked shrinking, basophilia, and loss of intracellular architecture in hippocampal pyramidal neurons. This acute effect of a pyridoxine antagonist was specific for certain neurons, the so-called “end-blade” neurons. Various other experimental conditions such as hypoxemia, cardiovascular collapse, or synaptically active convulsants such as strychnine and pentylenetetrazol did not produce these effects (Purpura and Gonzalez-Monteagudo, 1960). Studies such as these employing the hydrazides are needed in order to complete our information concerning the relationship between the hydrazides and PN in this area of CNS neuropathology. INH, because of the prevalence with which neuropathies occur in the treatment of tuberculosis where about 40% of patients receiving 20 mg/kg/day develop peripheral neuritis (for review see Anonymous, 1954), has

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been extensively studied with respect to the neuropathology of peripheral nerve. Experimental studies in rats show that large oral doses of INH produce a severe focal form of degeneration with disintegration of the axis cylinders and proliferation of the Scliwann cell layers. Later a complete transformation of the myelin to the fat cells occurs. Similar demyelination processes apparently occur in man occasionally even involving the optic nerve leading to optic atrophy and loss of sight (Keeping and Searle, 1955; Sntton and Beattie, 1955; Dixon et d., 1956; Kass ct al., 1957). Thesc effects on periphcral nerve may be inhibited by the simultaneous administration of vitamin RG in various forms, but are not inhibited by glutamic acid or other members of the vitamin R group (cf. Zbinden and Studer, 1955a, b, 1956). These experimental studies in rats thus provide a morphological basis for the clinical observations in nian (Gammon et al., 1953; Jones and Jones, 1953; Lubing, 1953: Biehl and Vilter, 1954b). Such neuropathies are only observed upon chronic administration of the hydrazide and may, therefore, not be directly related to the acute convulsive effects with which we are primarily concerned herein. One point of resemblance is that these neuropathies are prevented, and, if treated early enough, reversed by the administration of vitamin B,, ( Biehl and Vilter, 195413; Oestreicher et al., 1954; Ungar et d,, 1954; Carlson ct al., 1956; Dixon et al., 1956). Indeed, it is now generally accepted practice to include PN in the therapeutic regimen whenever high doses of INH are employed (cf., David and Leahy, 1959; Mandel, 1959a). A pliarmaceutical preparation containing a combination of INH, PN, and p-aminosalicylic acid has been devised for such purposes ( blandel, 195917) . The biochemistry of these peripheral neuropathies has not yet been studied in sufficiently intimate detail to give any clues as to mechanism of action. Clinical investigators had early come to realize the importance of PN for the explanation of certain nervous disorders (Snyderman ct al., 1950). This realization was brought to a sharp focus when a series of convulsant episodes in infants was traced to the use of a liquid formula unusually low in PN (Coursin, 1954; Maloney and Parmalee, 1954). The studies of Hunt and co-workers (1954) on the occurrence of abnormal pyridoxine dependencies leading to convulsions in infants also emphasized the importance of the vitamin for normal CNS function in man. Penetrating reviews of the in-

CONVULSIVE EFFECT OF HYDRAZIDES

34 1

formation available from studies in man are those of Vilter (1956) and Coursin (1956). There are apparently three classes of infant convulsions related to vitamin BG. The first of these is simple deficiency. The second are those exemplified by Hunt’s cases in which abnormal requirement for B6 may have been induced by the administration of massive amounts of Be to the mother during the first trimester. The third group are those in which there is an aberrant metabolism of the vitamin; examples of these may be found in the cases of Bessey and co-workers ( 1957) (for review see Anonymous, 1 9 5 8 ~ )The . second of these types exhibit abnormally high excretion rates for vitamin BG in the form of PM and PL, which may be the explanation for their high requirements (Coursin, 1960). Production of vitamin BG deficiency symptoms by the use of dPN in man was demonstrated in 1949 and 1953 by Vilter and coworkers (Vilter et al., 1954)-indicating that dietary deprivation, abnormal metabolism or excretion, and B6 antagonists have all been shown to produce effects in man as they have in animals. A fourth regimen for the production of BF deficiency symptoms in man is presumably the administration of hydrazides. The neuropathies and CNS stimulation produced in man have already been discussed. The hydrazides do not, so far as we are aware, produce the dermatitis and lesions of the mucocutaneous junctions which are typical of BG deprivation or dPN-induced deficiency. Why they should be more specific for neural structures is unexplained except perhaps on the basis that in man the hydrazide effect is a relatively acute effect from high doses whereas lesions in other tissues may require a much more prolonged deficiency to develop. Because they can be prevented or reversed by the administration of BG there seems little doubt that the hydrazide neural effects are related in some way to the function of the vitamin.

VI.

Miscellaneous

Both B6 deficiency and INH administration cause adrenocortical hypertrophy (Beyer, 1954; Eisenstein, 1959a), but no impairment in steroid secretion response to the adrenocorticotropic hormone was noted in the B6-deficient rat. The gluconeogenic response to cortisol

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was shown to be repressed in B6-deficient rats, however, and this repression was apparently related to a failure of transaminase levels to respond with their normal increase to corticosteroids ( Eisenstein, 195913). Rosen and co-workers (1959) have studied the relationship between glucocorticosteroids and transaminase activity in detail (Rosen et uZ., 1959), but there seems to be no immediate relationship between this effect and the CNS effects of the hydrazides. Woodbury and co-workers (cf. Vernadakis and Woodbury, 1960) have studied the relationship between cortisol and CNS excitability and have found that the increase in excitability produced by cortisol is accompanied by a decrease in brain GABA. The hydrazide effects, however, are probably direct and not mediated through an adrenocortical mechanism. It has been reported (Camba et al., 1953) that, whereas liver damage has no effect on INH toxicity, kidney damage reduces the toxicity and the diuretic action of aminophylline increases the toxicity. This may well be a reflection of the importance of BF loss in the urine and it may be that, when this loss is impeded, enough hydrolysis of PL hydrazide occurs partially to reverse the toxic effects of INH. It has also been reported that BF deficiency decreases the rate of metabolism and excretion of INH (Boone et al., 1956). The excretion effect, at least, might be anticipated on the basis of the fact that some of the excretion of INH occurs as the PL hydrazone, and in deficiency there would be less PL available. It is also possible that PLP-catalyzed reactions are involved in INH metabolism. PN has definite effects upon the movement of materials across cellular membranes ( Christensen et aZ., 1954; Christensen, 1955; Riggs and Walker, 1958; Akedo et al., 1960; Jacobs et al., 1960), amino acids having been the most thoroughly studied. In addition to the transfer of organic molecules such as amino acids, ion transport (cf. Aikawa, 1960) into and out of various tissues are apparently also involved. The exact relationship of these phenomena to the functional activity of the brain is not clear, but since the brain depends largely upon membrane phenomena for its normal functioning, these possibilities should be kept in mind. There is no direct information concerning the action of hydrazides on this function of PN and without knowing the affinity of the “transport system” for PLP it is not possible to assess the degree to which it might be

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affected by hydrazides. This is obviously an important possibility which is deserving of more intensive investigation. The ability of INH to form complexes with metals (Fallab and Erlenmeyer, 1952; Albert, 1953; Foye and Duvall, 1958) has been suggested as being involved in its biological activity, but only by inference. Divalent metallic ions do catalyze the autoxidation of INH (Winder and Denneny, 1959) and complexes such as these may be involved in its biological transformation.

VII.

Summary

A group of unsubstituted or monosubstituted hydrazides will produce seizures in any animal thus far tested including man. These seizure states may be partially or completely prevented or reversed by administration of one of the naturally occurring forms of vitamin B6. A considerable body of evidence has accumulated linking these seizure states with the production of an acute B6 deficiency by the hydrazides, but the exact biochemical mechanisms involved are still in question. It has been postulated but not proved that a deficiency of GABA is involved in the hydrazide and other B6 deficiency states. I t may be that some metabolite other than GABA is actually directly involved in the seizure state. It is only necessary to review with Braunstein (1960) the manifold array of reactions in which pyridoxine has been implicated to realize that the evidence here presented is not sufficient to specifically link the CNS effects of the hydrazides to any one reaction. While not conclusive, the evidence is very strong that BG is involved in the convulsive effect of the hydrazides-a conclusion which reflects not only on the mechanism of action of the hydrazides, but also upon the intimate association between BG and CNS excitability. We believe that it may be fairly stated that this is one of the few convulsant phenomena (cf. Tower, 1960) which provides the possibility of a biochemical explanation that seems to be more or less connected to the cause of the disorder itself and not to its energy sources. As such it deserves an even fuller exploration than it has so far received.

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The Physiology of the Insect Nervous System By D. M. Vowles Institute of Experimental Psychology, University of Oxford, Oxford, England

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11. Sensory Physiology A . Chcmoreception ......................

349 351 351 35 1 355 358 364 371

I. Introduction

During the last decade interest in and work on the nervous mechanisms of insects has greatly increased. This is partly because of the development of more refined electrophysiological techniques, which has permitted recording from the small cells of the insect nervous system. But the use of insects as experimental subjects has always had many advantages. Not only are they numerous, inexpensive, and hardy, but their sense organs are extremely accessible, and their central nervous system is so conveniently arranged as to have the coordinating mechanisms for different types of behavior distributed in well-separated regions. The basic organization and evolution of the nervous system have recently been reviewed by Roeder (1959) and Vowles ( 1961). Figure 1 shows the anatomy of a typical insect central nervous system. The reviewer has therefore decided not to attempt any encyclopedic contribution, but rather to select for presentation those examples of recent work which seem to be most important. Some work, such as that on circadian rhythms, has been deliberately omitted since it has already been well reviewed by Harker (1958, b ) . Similarly, Hoylc (1957) has given a good account of 1960~1, 349

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neuromuscular activity. Such exclusions should not be thought to reflect on the importance of such work (indeed the problem of biological clocks is more likely to be solved in these animals than in vertebrates), but rather on limitations of space and aversion to re-reviewing more competent reviewers.

FIG.1. Nervous system of the head of the grasshopper Dissosteira Carolina. A, anterior view; B, lateral view. Ao., aorta; 1Br., protocerebrum; 2Br., deutocerebrum; 3Br., tritocerebrum; C.A., corpus allaturn; Coe.Con., circumesophageal connective; Cr., crop; Fr.Gng., frontal ganglion; LmA”., labral nerve; LO., lateral ocellus; M.U., median ocellus; Oc.Gng., occipital ganglion; 0. Pdcl., ocellar pedicel; Op.L., optic lobe; Phy., pharynx; SLD., salivary duct; Soe.Gng., subesophageal ganglion with nerves supplying the mouth parts; Tnt., tentorium. (Redrawn from R. E. Snodgrass, “Principles of Insect Physiology.” McGraw-Hill, New York, 1935.)

Two particular fields will be discussed in this article. The first will deal with sensory and perceptual mechanisms, and the second with the influence of the brain upon behavior and on segmentally coordinated activities. It is in these two fields that the most significant recent work has been done.

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II. Sensory Physiology

A. CHEMORECEPTION The work on mechanisms of taste in insects started by Dethier has continued. One advantage of the sensory hairs in insects is that behavioral and electrophysiological activities can be recorded from isolated units. Hodgson (1958) recorded from the contact chemoreceptors in the blow fly units which possess three sensory cells, two of which are sensitive to chemical stimulation and which run the whole length of the sensory hair, and a third which is connected to the base of the hair and which responds to mechanical displacement. Wolbarsht and Dcthier (1958) and Wolbarsht (1959) also investigated the singly innervated chemosensory hairs on the ovipositor, mouth parts, and antennae, and found that they were not sensitive to mechanical stimulation. One of the surprising observations which they made was the presence of the triply innervated hairs on the leading edge of the wing. Although the mechanoreceptor might be useful for detecting air movements, the function of chemical sensitivity in this position is obscure. Schneider (1957) was able to record from the antenna1 nerves of moths electric potentials which are possibly equivalent to generator potentials. It was significant that these receptors were specifically sensitive to the active component of the odor used in courtship by the moths.

B. PROPRIOCEPTION The most notable advance made in the last few years has, however, been in the analysis of proprioceptive mechanisms. The functions of sensory hairs, which are displaced by movements of joints, and of the campaniform sensillae, which are sensitive to strains in the exoskeleton, were reasonably well known from the early work of Pringle (1938). The functions of sensory hairs for recording the displacement of joints in the exoskeleton have been further studied by Pringle and Wilson (1952), who subjected such hair receptors to phasic sinusoidal displacements. They found the frequency of discharge was approximately proportional to the logarithm of the displacement,

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but reached its maximum slightly before the peak displacement was attained. It was suggested that this is an arrangement to overcome the disadvantage of subsequent sensory adaption occurring at maximum displacement. The hair receptors used for recording displacement of the head relative to the thorax of locusts have been described by Goodman (1959) and Haskell (1959). It was found that destruction of the hair plates--\vhich are normally stimulated by movement of the head-prevents the locust from showing the dorsal light reaction, because the body can no longer be turned and held in line with the head, after the head has been turned to bring its dorsal surface toward the light. Electrophysiological recordings showed that displacement of a single hair produced a high initial discharge of 200300 impulses per second which rapidly waned to a steady lower level. The initial frequency appeared to depend upon the speed and magnitude of the deflection, but bore no relation to the direction of deflection, whereas the final frequency was independent of the magnitude of the deflection also. It appears. therefore, that the receptors are extremely sensitive to change in their position but not to maintained distortion. The hair plates are arranged in such a way as to give information both of the equilibrium position and the direction of rotation of the head. Slifer and Finlayson (1956), and Finlayson and Lowenstein (1958) have described an insect stretch receptor consisting of sensory neurons lying in strands of connective tissue stretched across abdominal joints. This functions as a typical, slowly adapting receptor-the frequency of discharge being proportional to the stretch. In the most highly evolved forms the sense cells are closely associated with muscle fiber, which must take some of the tension from them. The receptor then bears close resemblance to the vertebrate muscle spindle. Lowenstein and Finlayson ( 1960) further extended their study of the abdominal stretch receptor by applying phasic sinusoidal deflections through an ingenious microstimulator. They found that with single step stimuli a change in stretch produced an increase in the frequency of discharge which later showed sensory adaption. The maximum frequency which they recorded was about 63 impulses per second. With continuous phasic stimulation of a frequency of about 2.5 cycles per second, the frequency of discharge

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of the nerve reached much higher levels (100 impulses per second) and the peak discharge occurred at the point of maximum velocity not at the maximum amplitude of displacement, and was proportional to the velocity of displacement. This shows that the stretch receptor can function both as a displacement and as a velocity receptor. This may be correlated with the control of respiratory movements by the abdomen. The most thorough investigation of the function of another type of proprioceptor (Johnston’s organ) has been carried out by Heran ( 1959). It had previously been shown that the honey bee (Heran, 1956) has an appreciation of wind speed while flying under natural conditions. The sense organs involved in this have been thoroughly investigated in bees, which were fixed by their thorax and caused to fly “on the spot” in a wind tunnel. Under these conditions the bee takes up a normal flying posture. Close observation of the antennae revealed that as the wind speed increased the antennae were held further forward so as to make a narrower angle with the anteriorposterior axis of the body. In intact bees changes in the amplitude of wing beat were observed to follow changes in wind speed. When the antennae had been amputated the correlation between wind speed and wing beat was lost. Amputation of other appendages did not influence this correlation. Enclosing the head in a transparent capsule with the antennae protruding through it did not disturb the relationship, showing that in bees the hair receptors on the head and cornea are not important, as they are, for example, in locusts. When the joints of various regions of the antennae were stiffened by the use of wax, etc., it was found that interference with only those joints between the flagellum and the scape (i.e., the pedicel, which contains Johnston’s organ ) disturbed the reflex movements of the antennae in response to changes of wind speed. When the joint was stiffened only on one antenna, the contralateral antenna was not influenced, showing that the responses were confined to the stimulated side. By fastening iron filings to the antennae and subjecting the bee to a magnetic field, Heran was able to show that deflections of the flagellum on the scape were reacted to by reflex movements of the antennae, as if the bee were in an air stream. These reflex responses were greatest when the magnetic field was fluctuating, particularly when the frequency of fluctuation

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was of the order of the normal vibration frequency of the antennae, which is in the neighborhood of 200 to 300 oscillations per second. This normal vibration seems to be at the resonance frequency of the antennae, considered as a mechanical system of levers, and is also in the neighborhood of the frequency of wing beat during fast flying. Electrophysiological recording from the antennal nerve showed large nerve impulses in response to deformation of the antennae, but these were mostly efferent and were correlated with the motor reflexes of the antennae. After section of the antennal nerve these impulses disappeared and smaller afferent, sensory activity was revealed in response to displacement of the flagellum. This activity was shown to originate with the pedicel. The potentials recorded did not take the form of typical nerve impulses but of electrical waves either synchronized with the frequency of vibration of the flagellum, or sometimes at double that frequency. It is not clear whether this electrical activity is the summated effect of impulses in many fine fibers, or represents something similar to the cochlear potentials in vertebrates. The response shows considerable adaptation to single step displacements. This is perhaps correlated with the demonstrated efficiency of the sense organ during vibration, which, by preventing adaptation, might allow absolute measurements over a long period. The magnitude of the potentials increases with the amplitude of vibration of the flagellum and with the wind speed. There is therefore a close relationship between the behavior and the electrophysiological data. The function of Johnston’s organ has also been studied in mosquitoes by Rassler (1957). In mosquitoes, too, the organ functions as a regulator for speed of flying and amplitude of wing beat (which functions are also controlled by means of the eye). It was shown that Johnston’s organ also functions as a statocyst and a wind speed detector. The insect could only maintain its equilibrium in space when the flagella on the two antennae were subject to the same strength of displacing forces. Burkhardt (1960) has recorded action potentials from the antennal nerve of the blow fly during mechanical stimulation. The potentials observed originated from Johnston’s organ. The amplitude and the latency of the potentials varied with the displacement, which suggested that he was recording the summated effect of sev-

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era1 synchronized individual impulses. There were two types of receptors with different responses; one of these was sensitive to torsional movement and the other to linear displacements-diff erent receptors being sensitive to different directions of movement. Both types of receptors, however, showed rapid adaption. This shows the sensitivity of Johnston’s organ to changes in antenna1 position.

C. VISUALMECHANISMS The mechanism by which light elicits nervous activity in the retina is being actively investigated in insects as well as in other animals. Goldsmith (1958) has extracted visual pigments from the honey bee. These consist of retinenel bound with protein to form a photosensitive pigment. The absorption peak lies in the region of 440 mp in the compound eye which agrees well with behavioral observations on sensitivity. The ocelli and compound eyes both contain other pigments, however, which have different peak sensitivities. The ultrastructure of thc insect eye has been studied in some? elegant preparations by FernBndez-MorLn ( 1958) using the electron microscope. He was able to show that the rhabdomeres have large numbers of minute parallel tubules orientated at right angles to the long axis of the rhabdome. In different rhabdomeres of the same ommatidium the tubules are orientated in different planes. In the Hymenoptera it seems likely that they are arranged radially from the central region of the rhabdome, whereas in Diptera the arrangement is rather irregular. It is possible, therefore, that this arrangement may be correlated with the mechanism for the perception of polarized light. It should be noted, as Kalmus (1958, 1959) has pointed out, that many of the behavioral experiments purporting to show the sensitivity of insects to polarized light have not always properly controlled the changes in the brightness of reflections from surrounding surfaces when a polarized light supply is rotated. Kalmus has shown that some insects which show optomotor reactions to such moving reflections will not do so if they are eliminated. Naka and Kuwabara ( 1959) have recorded the electroretinogram (ERG) from the compound eyes of Lucilia using a microelectrode with a tip diameter of approximately 3 v. This type of recording probably picks up intracellular potentials and is the only

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case in which the locus of an electrode in a primary visual sense cell has been unambiguously determined for any animal. I t was clear from their results that the ERG originates in the retinal cells and that it consists of two components which are, respectively, negative and positive in sign relative to the indifferent electrode placed within the head. With the low intensities of illumination only a positive wave was detected, but with higher intensities an additional negative wave with different and longer time relationships made its appearance. It seems clear, therefore, that at least two different types of potentials originate within the retinal cells themselves. The potentials are differently resistant to fatigue or desiccation. In the degenerating preparation the rcsponse to dim illumination became a negative wave. It may perhaps be assumed that the negative wave is normally inhibited, the inhibition being removed by degeneration. Studies on the electroretinogram have also been used to investigate the spectral sensitivity of the insect eye. Walther (1958), working with the cockroach (Periplaneta americana) and using monochromatic light for stimulation and adaptation, found that there seemed to be different spectral sensitivities in the upper and lower parts of the eye. The upper part of the eye has a sensitive peak toward the violet wavelengtlis in addition to the main peak at about 507 mp, while the ventral part of the eye shows only the latter peak, The peak toward violet wavelengths shown by the upper part of the eye, however, depends upon the state of adaptation of the eye. The upper part of the eye also shows differences in the form of the ERG when lights of different wavelengths are used. It is suggested that this rnight depend on a mechanism underlying color vision in the upper part of the eye. Dark adaptation has also been studied in the ocellus of the cockroach by Ruck (1958a) who found that the speed of dark adaptation is very rapid, being mainly complete within 2-3 minutes. Kirschfeld (1959) has shown that the relationship between the intensity of the testing light flash and the background illumin at'ion is important. The state of adaptation of the eye markedly influences the form of the ERG in Diptera. If the adapting light is maintained at a constant intensity, both the amplitude and the slope of the on-spike and off-spike increase with the intensity of the test flash. For a constant intensity of test flash and different adaptation bright-

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nesses, the amplitude of the on-spike and off-spike increases as the relative difference in intensities increases, but the slopes of the on-spike and off-spike are not altered. The results show clearly that the ratio AR/R does not follow the Weber-Fechner law, the Weber ratio being approximately two. In this context perhaps the important theoretical studies of light adaptation by Reichardt and Varju (1958, 1959) should also bc mentioned (see also Reichardt and Delbruck, 1956). The classic work of Autrum on the ERG has been recently reviewed (Autrum, 1958). It is suggested that there are two classes of eye: (1) Fast eyes which are found in rapidly flying insects and are characterized by rapid dark adaptation, diphasic ERG‘s, with on- and off-spikes, low absolute sensitivities, and high flicker fusion frequencies. ( 2 ) Slow eyes possessed by nocturnal walking insects and characterized by slow dark adaptation, monophasic ERG‘s, high sensitivity, and low flicker fusion frequency. However, Ruck (1958b) has studied the ERG’s of the compound eyes and ocelli in a range of insects, and has found agreement with Autrum’s more general hypothesis only in that the ability to resolve flickering stimuli is greater in swiftly flying diurnal species. There does not seem to be any correlation, however, between the flicker fusion frequency, form of the ERG, absolute sensitivity, or the rate of dark adaptation. Indeed the form of the ERG seemed to depend partly upon the state of adaptation of the eye and the strength of the stimulus. It seems clear, therefore, that Autrum’s hypothesis, although of elegant and satisfying simplicity, was unfortunately based on too small a sample of species and too small a range of experimental conditions.* Autrum and Hoffman ( 1960) have recently replied to some of Rucks criticisms. When subjecting the blow fly Calliphora to pressure or oxygen shortage they found that the “on” and the “off components can be separated from the steady negative wave in the electroretinogram. They confirmed the postulated relationship between adaption rate, sensitivity, and flicker fusion frequency. They suggest that the type of ERG recorded by Ruck may have been abnormal due to pressure on the eye during recording, or to general deterioration of the preparation during that time, although this seems unlikely to the reviewer. Until further evidence is available the matter will remain unsettled.* * See Adclendum on page 373.

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D. MOVEMENTPERCEPTION Although there have been many studies on the ERG there has been little work on the neurophysiological activity of the optic ganglia. Perhaps the sole exception is the study of Burtt and Catton (1954, 1956, 1959) who recorded nerve impulses from various parts of the nervous system of the locust in response to small moving stimuli. It was found that there were marked responses to movements of only a few minutes of arc, which are considerably less than the ommatidial angle. It seems probable, therefore, that the concept of the ommatidium as a functional unit is oversimple, and that insects may respond to changes of stimulus within the field of view of a single ommatidium, although it is possible that the slight movements producing the responses may occur on the boundaries of the ommatidial fields. This subject requires much further study. An important analysis of the perception of movement in insects has been made by Hassenstein and Reichardt (Hassenstein, 1950, 1951, 1957, 1958a, b, 1959; Hassenstein and Reichardt 1953, 1956; Reichardt, 1957). Their work is a result of the mutual cross-fertilization of ideas derived from cybernetics and behavior, the concepts from the former field being specified with precision and experimentally tested-a procedure much applauded but seldom before performed. The model which these authors propose is of a general application well beyond their restricted field of study. The experimental method used was to suspend a beetle (Chlorophanus)by its thorax, and to give it a “spangenglobus” to hold in its feet. The insect, when attempting to turn in one direction, then moved the spangenglobus in the opposite sense. The spangenglobus (Fig. 2 ) consisted of a series of 6 small, light, curved straws (or other material) which were fastened together so that at each junction three straws were joined to each other at an angle of 60 degrees. The spangenglobus, therefore, consisted of a series of Y‘s, which were interconnected upon the surface of a sphere. Thus, as the beetle walked along any straw pathway it repeatedly encountered the junctions of the Y‘s and was forced to turn left or right. The strength of the beetle’s tendency to turn could then be measured in terms of the numbers of choices made to turn left or right in a given time. It was found

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FIG. 2. Y-maze globe method. The beetle’s back is glued to a piece of cardboard which is held by a clip; the clip is fixed to a stand, so the beetle is freely suspended in the air. The beetle is given the “Y-maze-globe” which he carries of his own free will. The Y-maze-globe is made of six straw pieces which join in four points to form Y-like junctions. When the beetle starts walking he remains fixed, but the Y-maze-globe performs the negative of the movements the beetle would perform if he were walking freely. After a few steps the beetle reaches a Y-junction, or better, a Y-junction reaches him,and the beetle has to choose right or left. After passing the junction the animal is in the same position as before. After the next few steps it has to choose again, and so on. For the beetle the Y-maze-globe is an infinite Galton probability apparatus. In a given optical stimulus situation the ratio of choices of the right and left pathway has been proved to be a sensitive quantitative measure of optically induced ( optomotor ) turning tendencies. Beetles which were suspended in the center of a very slowly rotating striped cylinder showed clear reactions to angular movements of 0.02”/sec; this is a quarter of the angular velocity of the minute hand of the clock. (Drawing by E. Freiberg, after a photograph. )

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that at low intcmitic.s of turning there w a s a closc, agrecmelit between the turning tendency and the paramcter u s e d , 1)ut that at 1iighc:r turning tendencies a correction factor \\';is reciuirctl. The beetle, holding tlie spangenglobus, was suspcndcd inside various cylinders where precisely controlled moving visual patterns could be presented to it. A preliminary analysis first slio\ved that mo\wnent w u s perceived as a vclctor independently of its position in space. The basic functional unit in tliis perccption \vas f o r d to be adjacent pairs or triplcts of ommatidia. If a succession of stimulus clianges in time fell within a single ommatidium, no turning rcwltcd. Nor did turning occur if the pair of stimulated ommatidia \verc scparated by more than a single unstimulated ommatidium. It appears, tliercfore. that movemcnt is pcrceived by interaction hetween adjoining pairs or triplcts of ommatidia. If the succession of brightness changes in thc members of the pair were of the same kind, i.c. both going from light to dark or vice versa, the insect turned in the same sense as thc nioving cylinder. If, however, the temporal sequence of stimuli in tlie members of the pair imrolved opposite kinds of brightness cliangc, i t . one going from light to dark follotved by the other going from dark to light, then the insect turned in the opposite direction to the turning cylinder which produced the change. If a change froni light to dark is cxl>rcssed as and a change from dark to light as and A and B arc the neighboring ommatidia, then a succession of stimula A+ to B+, or A- to B- produces turning in the AB direction, while if the sequences are A+ to B--, or A- to B the direction of turning is in the BA direction. The strength of thc turning tendency is dependent quantitatively upon various factors:

"+",

"-"

+

1. The rclativt intcmsity cliange in cach single ommatidium. 9. The temporal intcrval b c t \ \ w n the stimulation of the m e n bers of functioning pairs of ommatidia. 3. The type of intensity change in the functioning pairs of ommatidia, (dark-lxiglit, or bright-dark). 4. The total number of ommatidia stimulated. In order to explain tlie mcdianism of movement perception that \vo,lld account for the behavior described, Hassenstein and Reichardt proposed the model which is illustrated in Figurc 3. This represents tlie information flow diagram for the components of t\vo

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neighboring ommatidia. It must be stressed that the “centers” are functional rather than anatomical entities. The various components of the model interact as follows: The elements ‘4 and B are sensitive to the brightness change produced by the moving object and, like a photocell, reproduce D

3

Y

W

A + -

+

c -

-

+

FIG.3. The theoretical mechanism underlying movement perception in the beetle ( discussion in text).

faithfully the pattern of change in intensity of illumination. The D elements are essentially differentiating components which react to the change of intensity, but not to the absolute value of the stimulus. The output from the differentiators is then fed, either straight on to the subsequent components for that ommatidial unit or across to the components of the adjacent unit. Interaction of the differen-

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tiator output with the H component produces a pattern of excitation which decays within a very short time constant. The longitudinal transmission produces in component E a pattern of excitation which decays with a long time constant. Thus H and E components may be regarded as providing time bases whose state is determined by the interval since the arrival of the original stimulus. The outputs from the H components then interact with the outputs of the E components in the neighboring unit. This interaction is said to be essentially multiplicative, partly because the stimulus successions A+ B+ and A- B- gives turning in the same direction, while stimulus successions A- B+ and A+ B- produce turning in the opposite direction, and partly on the basis of quantitative measurements of the turning tendency. In the S units the excitation tending to produce turning in a given direction is summated arithmetically, and presumably the excitation from all the stimulated units is further summated at a lower level. In a theoretical analysis of this model Reichardt (1957) was able to makc various quantitative predictions about the intensity of turning of the beetle in response to different parameters of stimulation, such as the angular velocity of the stripes, the relative intensities of successive stimuli, etc. These predictions were based on the assumption that the patterns of activity in the H and E components were governed by linear kinetic equations of the first order. The predictions were experimentally tested and good agreement was found with the predicted values if the time constants of the H and E elements are, respectively, 46 milliseconds and 35 seconds. It was suggested that the mechanism carried out an autocorrelation process as a function of time, and this was tested in one experiment by using a cylinder whose walls had a continuous variation in intensity following a sinusoidal distribution, and in another by using cylinders whose walls had patterns of stripes of fixed intensities (ranging from white to black) arranged in a statistically random sequence. The results of these experiments again strongly confirmed the predictions of the hypothetical model. It is obviously impossible here to do full justice to this elegant study, and readers would do well to consult the original papers. The importance of movement perception in the behavior of insects has been clear since the work of Hertz before World War 11, when it was shown that form perception was partly based upon

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the rate of stimulation of ommatidia when the insect moves relative to the object [for a good English review see Carthy (1958)]. A similar situation seems to exist for form perception in locusts (Wallace, 1958) and mantids (Rilling et al., 1959). The problem whether form perception in insects involves more than merely the total stimulation of the eye as the insect moves relative to the contours of the figure would well stand experimental re-investigation, particularly perhaps if it could be combined with hypotheses of the type proposed by Hassenstcin. Wallace (1959) has also shown that in locusts distance estimation is done in terms of movement. The insect sways from side to side in a peering motion prior to jumping and the relative movement of the image over the retina seems to determine the strength of its subsequent leap. An investigation of the prey catching behavior of the mantis has been made by Mittelstaedt also using control theory. Since Mittelstaedt (1957) has surveyed this recently, it will not be recapitulated here. This type of approach when it leads to quantitative predictions, is, however, very promising. The type of optomotor response studied by Hassenstein seems to act to keep the insect in a constant relationship to its visual environment. When the insect moves voluntarily it has to overcome this tendency. In the type of reaction described below the insect maintains a constant relationship to one source of stimulation, but moves relative to others. One of the more intriguing problems of visual orientation in insects has been the ability of some species to maintain a constant heading, which is at an angle to the direction of incident light. This is known as the “light-compass reaction,” and is shown, for example, by homing bees and ants who utilize the sun as source of light. The maintenance of such an orientation is not easy to understand, since it is asymmetrical with respect to the source of stimulation. Orientation to the plane of polarized light seems to present a similar problem. However, Jander (1957) has made an important contribution to the understanding of such mechanisms in the wood ant (Formica rufa). He showed that basically the wood ant behaves in a simple phototactic manner-being positively phototactic when leaving the nest, and negatively when returning. However, during their excursions they show typical compass reactions, and may also orientate by visual landmarks such as trees. If the ants

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were disturbed during their compass reactions they showed a reversion toward their basic simple phototactic reactions. Jander therefore suggests that the compass reactions are learned oricntations superimposed upon the more primitive taxis. A similar state of affairs for wasps and bumble bees has been demonstrated by Jacobs ( 1959). These insects, like ants, show spontaneous taxis and preferences for particular orientations relative to the plane of polarization of light. If the bees are presented with both polarized and directional sources of light their spontaneous orientation is a compromise between the two basic preferences. It is possible that such a compromise orientation might under natural conditions cause an apparently compasslike reaction. Since the position of the stin in the sky changes with the time of day, and hence also the pattern of polarization of the blue sky, insects that are away from home for long periods must be able to compensate for such solar revolutions. Birukov and Oberdorfer (1959) found that in the pond skater (Veliu czirrens) the spontaneous taxis orientations changed with the time of day-even the orientation shown toward gravity. It appears that the insect has some sort of internal clock which automatically resets its orientation mechanisms to match the time of day.

Ill. The Insect Central Nervous System

Previous work on the brain of insects has largely been confined to the effect of rather gross lesions upon behavior. This work has been well reviewed by Roeder (1953). The general hypothesis that emerged from his survey was that specific behavior mechanisms were coordinated at the segmental level in the ventral nerve cord. Sometimes the ventral ganglia possess spontaneously active centers associated with the coordinating systems, and their activity is regulated by descending inhibitory and facilitatory influences from the supra- and subesophageal lobes of the brain. Recent electrophysiological work tends to support this view. Weiant (1958) found that in the cockroach the spontaneous discharge down the leg nerves from the thoracic ganglia (probably associated with tonus ) increased markedly after decapitation. The spontaneous discharges in the last abdominal ganglion, however,

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were found to be inhibited from the subesophageal lobe (Roeder et al., 1960). Van der Kloot and Williams (1954) using the ablations method showed that in the silk worm larva the protocerebral lobes were necessary for the control of the complex coccoon weaving movements. A new technique developed by Huber (1955a, b, 1959, 1960a, b; Oberholzer and Huber, 1957) for use on crickets has imparted considerable impetus to the study of the brain. Huber initially used localized mechanical stimulation of the brain by inserting fine steel needles, but he later developed a technique for localized electrical stimulation of the brain using implanted electrodes. The electrodes which may be either uni- or bipolar are of steel wire about 30 p in diameter and insulated to the tip. Various electrical waves were used but the most effective were rectangular pulses of about one millisecond duration and low frequency (about 20 per second). The main discoveries of Huber were concerned with three types of behavior-simple movements and locomotion, stridulation, and respiration. These will be discussed below. Simple movements of the head, body, or appendages can be caused by localized stimulation in many parts of the brain, but more complex movements are elicited at low thresholds and with less variability when the electrodes are placed in the middle region of the protocerebrum, i.e. in the neighborhood of the mushroom bodies (corpora pedunculata ) , the central body, and the central commissure. The latency of the responses and their duration depend on the strength of the stimulus, and there is often an after effect, sometimes of the opposite kind to the elicited response. Thus, if the head has been turned to the right during stimulation, it may be turned to the left after the end of the stimulation. The direction of turning, both of the head and of the animal’s locomotor movements, depends upon the placement of the electrode; the direction may be different for different placings on the same side of the brain, partly depending on whether the electrode is in the calyx or the “stiel” system. With constant continuous stimulation the intensity of the movement often increases. When locomotion occurs, it may be concerned with different sorts of behavior. Walking may normally occur as part of the search for food or during fleeing. When locomotion is elicited by stimula-

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tion, different types of locomotion are sometimes seen, and these seem to be correlated with the two types of behavior. For example, when the insect is searching for food, or stimulated to do so by electrical impulses, it may make searching movements with its antennae, palpate the substrate with its mouth parts, and, if one places a piece of banana skin in the animal’s path, it will stop to feed. In the case of fleeing, however, the movements are more rapid, they are not influenced by the banana skin, and the searching movements of the head appendages do not occur. I t is clear therefore that the artificially elicited behavior can interact with the environment. The movements are not normally synchronized with the frequency of the stimulating impulses except in the case of the antennal movements when the electrode is near the antennal centers. In this case tetanus seems to occur at about 55-60 impulses per second. When the stimulus is placed laterally to the antennal centers, only the ipsilateral antenna responds, but during stimulation toward the mid-line the antennal movements are synchronized on both sides. This can be prevented by sectioning the commissure between the antennal lobes, when both antennae become independent of each other, and are frequently moved and cleaned. The antennae on both sides can, however, be excited to movement by stimuli on either side of the protocerebrum. There are many points within the protocerebrum from which the same sort of movement may be elicited or inhibited. After coagulation of these points, the behavior of the insect is changed only temporarily, if at all. The situation therefore seems similar to that in the vertebrate brain where multiple pathways also occur. The second type of behavior which Huber investigated was respiratory. In crickets-as in other insects-the respiratory movements of the abdomen are coordinated by the local segmental ganglia in conjunction with “spontaneously” active breathing centers. In crickets the breathing center lies in the subesophageal ganglion (unlike the situation in locusts, where the breathing center is in the thoracic ganglion) and apparently determines the “timing” of the discharge of the different segmental centers. The breathing center also controls the frequency and amplitude of the respiratory movements, and after removal of the ganglion the respiratory frequency sinks and remains permanently depressed. It appears to be

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sensitive to the concentration of carbon dioxide. Removal of the protocerebrum has only a slight effect on the respiratory rhythm. With localized stimulation in the protocerebrum the frequency, and to a lesser extent, the amplitude of the respiratory movements can be raised or lowered. Two regions of the brain (see Fig. 4) seem to be particularly involved: (1) The pars intercerebralis-the area adjacent to the central body between the corpora pedunculata. Stimuli in this region can produce respiratory changes, which even at high intensities of stimulation are accompanied by no other associated behavior changes. It is characteristic of the respiratory responses to such stimulation that they are of long latency and have a very long aftereffect. It seems possible that the control of respiration from this region involves some neurohumoral process. ( 2 ) The mushroom bodies and the adjacent lateral neuropil. It is characteristic of these regions that the respiratory changes elicited by stimulation are of a shorter latency and with a shorter aftereffect, and that with stronger stimulation at least they are accompanied by behavioral effects such as walking. With weak stimuli respiratory changes often occur before the other behavioral changes have built up. This is also a phenomenon normally occurring in behavior. After destruction of a stimulation point which elicits respiratory changes, the respiratory frequency is not definitely or lastingly disturbed, although it usually sinks temporarily when the central body is destroyed. This implies that the protocerebral centers must have pathways to the lower respiratory center independent of those going via the central body. It is clear that the respiratory mechanisms are fairly complex and that rapid activity of an animal leading to change in gas concentration due to expenditure of energy may act at the subesophageal and segmental level, whereas respiratory changes associated directly with other behavior require the action of supraorientated centers. The study of respiration in the locust by Miller (1960a, b, c ) largely supports these findings with the exception that the pacemaker center is in the thoracic ganglion. Its activity, too, is influenced by supra- and subesophageal centers, and all of the centers are sensitive to carbon dioxide concentration. The third type of behavior studied by Huber involves the co-

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D. M. VOWLES

ordination of stridulation in crickets. The song of this insect is essentially a high-frequency carrier wave, which is amplitude modulated. The characteristic of the song, which may be either conimon song, threat, or comtship, is determined by the temporal sequence of the changes in amplitude, as in other insects. The thoracic ganglion contains the mechanism for coordination of the rhythmic stridulatory movements and “inadequate stimulation” of the ganglion or mechanoreceptors will elicit a “ground form” of song, which is not typical of normal song either in its duration, its phrasing, or the shape of the pulse envelopes characterizing the song. Typical singing movements can be elicited, however, by stimulation of the “stiel” system and adjacent tracts of the mushroom bodies, and these produce apparently normal song. Different types of song can be produced by stimulation in different regions. Singing movements can also be inhibited or depressed by stimulation. Ablation of the mushroom bodies on one side still permits the production of typical songs. Stimulation of the central body produces singing, but of an atypical type. Three regions of the CNS seem to be involved, therefore, in the control of singing-the mushroom bodies, the central body, and the second thoracic ganglion. I t is suggested that the thoracic ganglion can maintain the basic rhythmic activity of FIG. 4. Optical sections for the brain of the cricket showing the points which, when stimulated, produced change in respiration rate; ( a ) frontal and ( b ) sagittal view. , Increased respiratory rate; 0, decreased respiratory rate; a, increased motor activity and respiratory rate; A, decreased motor activity and respiratory rate; PC, protocerebrum; DC, deutocerebrum; TC, tritocerebrum; nop, optic nerve; nu, antcnnal nerve; nom, frontal nerve of ocellus; nl, labral nerve; nc, nerve to corpora cardiacal 1; sk, circumoesophageal connective; PK, mushroom body; Z K , central body; B, protocerebral bridge; mk, antennomotor center; pi, pars intercerebralis. 1-3. Optic tract; 4, visual commissure; 5, tracts between protocerebral bridge and central body; 6, tracts of frontal nerve of ocelli; 7, sensory and, 8, motor branches of antenna1 nerves; 9, antennal commissure; 10, olfactoris-globularis tract ( fibers between deutocerebrum, mushroom body, and central body); 11, ascending and descending tracts between brain and ventral nerve cord; 12, fibers of labral nerves and Jrontal connections (connections to frontal autonomic nervous system); B, paths of corpora cardiacal nerves; 14, fiber bundles between frontal and caudal region of protocerebrum; 15, site of origin of descending paths; cu, caudal; cr, cranial. [From Huber (1960a).l

PHYSIOLOGY OF INSECT NERVOUS SYSTEM

369

stridulation and that the mushroom bodies select the type of song to be produced and control its duration, amplitude, etc., while the central body overlies the excitation from the mushroom bodies and determines the phrasing of the song and its syllables and rhythms. Using a similar stimulation technique in the honey bee, Vowles (in preparation) has found that stimulation of the “stiel” system of

370

D. M. VOWLES

the corpora pedunculata releases many types of behavior such as are normally regarded as instinctive-aggression, cleaning, combbuilding, feeding, regurgitation, etc. Activation of one such type of behavior suppresses other types and itself becomes more vigorous with continued stimulation. It seems clear that in the corpora pedunculata we have the insect analog of the diencephalon. In vertebrates the activity of the thalamus is closely linked with that of the hypothalamus, and this in its turn is involved with the chemistry of the body. It is interesting to consider what little is known of similar relationships in insects. The control of hunger in insects seems very much more simple than in vertebrates. Dethier and Bodenstein (1958) found that in the blow fly (Phormia regina) taste thresholds for sugar rise after feeding. The rate of feeding is largely determined by the concentration of the medium. However the rise in threshold following satiation was not due to any change in the sensitivity of the sugar receptors, or of blood concentration (Evans and Diether, 1957; Hudson, 1958). It was found that severing the nerve to the midgut did prevent the rise in threshold, and flies with the nerve so sectioned would drink continuously. I t is clear that stimuli in the mid-gut produce inhibitory action upon the drinking center in the head ganglia. As long as these stimuli persist, the concentration of blood sugar, etc., seems irrelevant to the flies’ drinking behavior. Chemicals do however play a part in other types of behavior. Haskell (1958, 1960) found that in some Orthoptera the responsiveness of the females to the males declined after mating. This decline was due to information passed from the genital region to the head ganglia. But severing the nervous pathways available did not disturb the effect, and Haskell therefore suggests a chemical influence. Ozbas and Hodgson (1958) had found that the activity of cockroaches could be depressed by injection of extracts of the corpora cardiaca. This depressant effect could also be observed in vitro by exposing a spontaneously discharging ventral nerve cord to the solution-this causing inhibition of the discharges. In a subsequent study Hodgson and Geldiay (1959) found that the activity of neurosecretion by cells of the corpora cardiaca could be released by forced hyperactivity and electrical stimulation of the brain. Milburn and associates ( 1960), following up the previously

PHYSIOLOGY OF INSECT NERVOUS SYSTEM

371

quoted work of Roeder et al. (1960), were able to show that the spontaneous activity of the abdominal ganglion concerned with the genitalia, which is increased by decapitation, is also increased by injection of corpora cardiaca extract. This may possibly act on the normally inhibitory center of the subesophageal ganglion. But part of its effect is puzzling since the extract seems to act when applied to the conducting pathways of the nerve cord, and specific internuncial neurons seem to be sensitive to it. It will be clear from the above survey that our knowledge of nervous mechanisms in insects is rapidly increasing. Many analogs with vertebrate systems are being discovered, but it appears that the details of the mechanisms are often much simpler in these invertebrates, although their behavior appears as complex. This simplicity of organization leads to the hope that we may one day have an adequate understanding of their nervous mechanisms. REFERENCES

Autrum, H. (1958). Exptl. Cell Research Suppl. 5, 426. Autrum, H., and Hoffman, W. (1960). J. Inst. Physiol. 4, 122. Bassler, U. ( 1957). Naturwissenschaften 44, 336. Bassler, U. (1958). Z. uergleich. Plzysiol. 41, 300. Birukov, G., and Oberdorfer, A. (1959). Z. Tierpsychol. 16, 693. Burtt, E. T., and Catton, W. T. (1954). J. Physiol. (London) 125, 566. Burtt, E. T., and Catton, W. T. (1956). J. Physiol. (London) 133, 68. Burtt, E. T., and Catton, W. T. (1959). J. Physiol. (London) 146, 492. Burkhardt, D. (1960). J. Inst. Physiol. 4, 138. Carthy, J. D. (1958). “An Introduction to the Behaviour of Invertebrates.” Allen and Unwin, London. Dethier, V., and Bodenstein, D. (1958). Z. Tie~psychol.15, 129. Dethier, V.,and Wolbarsht, M. ( 1956). Experientia 12, 335. Evans, D. R., and Dethier, V. G. (1957). J. Inst. Physiol. 1, 3. FernLndez-MorLn, H. ( 1958). Exptl. CeZZ Research Suppl. 5, 586. Finlayson, L. H., and Lowenstein, 0. (1958). PTOC. Roy. SOC. B148, 433. Goldsmith, T.H. (1958). Ann. N.Y. Acad. Sci. 74, 223. Goodman, L. (1959). Nature 183, 1106. Harker, J. (1958). BWZ. Reus. Cambridge Phil. SOC. 33, 1. Harker, J. (196Oa). J. Exptl. Biol. 37, 154. Harker, J. (1960b). J. Exptl. Biol. 37, 114. Haskell, P. T. (1958). Animal Behau. 6, 27. Haskell, P. T. (1959). Nature 183, 1106. Haskell, P. T. (1960). Animal Behau. 8, 76. Hassenstein, B. ( 1950). Naturwissenschaften 37, 45.

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Hassenstein, B. (1951). 2. vergleich. Physiol. 33, 301. Hassenstein, B. (1957). J. Inst. Physiol. 1, 124. Hassenstein, B. ( 1958a). 2. Naturforsch. 13b, 1. Hassenstein, B. (1958b). Z. uergleich. Physwl. 40, 556. Hassenstein, B. (1959). Animal Behau. 7, 109. Hassenstein, B., and Reichardt, W. (1953). 2. Naturforsch. 8b, 518. Hassenstein, B.,and Reichardt, W. (1956). Z. Nuturforsch. llb, 513. Heran, H. (1956). Z. cergleich. Pliysiol. 38, 168. Heran, H. (1959). Z. cergleich. Physiol. 42, 103. Hodgson, E. ( 1958). J. Inst. P/iy.~ioZ.1, 240. Hodgson, E.,and Geldiay, N. (1959). B i d . Bull. 117, 275. Hoyle, G. (1957). “Comparative Physiology of the Nervous Control of Muscular Contraction.” Cambridge Univ. Press, London and New York. Huber, F. (1955a). Z. Tierpsychol. 12, 12. Huber, F. ( 195513). Naturwissenschaften 42, 566. Huber, F. (19.59). Vcrhandl. deuf. zool. Ges. Munster., Zool. Anz. Suppl. 23, 248. Huber, F. (1960a). Z. uergleich. Physiol. 43, 3-59. Huber, F. (1960b). Z. uerglciclz. Physiol. 44, 60. Hudson, A. (1958). J. Inst. Physiol. 1, 293. Jacobs, U.F. (1959). Z. cergleich. Physiol. 41, 597. Jander, R. (1957). 2. cergleich. Physiol. 40, 162. Kalmus, H. (1958). Nature 182, 1526. Kalmus, H. (1959). Nature 184, 228. Kirschfeld, K. (1959). Z. Naturforsch. 14, 212. Lowenstein, O.,and Finlayson, L. H. (1960). J. Comp. Biochem. Physiol. 1, 56. Milburn, N., Weiant, E., and Roeder, K. D. (1960). Biol. Bull. 118, 111. Miller, P. L. (1960a). J . Exptl. Bwl. 37, 224. Miller, P. L. (1960b). J . Exptl. B i d . 37, 237. Miller, P. L. (196Oc). J . Exph?. Biol. 37, 264. Mittelstaedt, H. ( 1957). In “Recent Advances in Invertebrate Physiology” ( B. Scheer and G. Walsh, eds.), p. 51. Univ. Oregon Publ. Naka, K., and Kuwabara, M. (1959). J . Inst. Physwl. 3, 41. Oberholzer, R. J. H., and Huber, F. (1957). Helu. Physiol. et Pharmacol. Acta 15, 185. Ozbas, A., and Hodgson, E. (1958). Proc. Natl. Acad. Sci. U.S. 44, 825. Pringle, J. W. S. (1938). J. Exptl. B w l . 15, 114. Pringle, J. W. S., and Wilson, V. (1952). J. Ex$. BWl. 29, 220. Reichardt, W. ( 1957). Z. Naturjorsch. 12b, 448. Reichardt, W., and Delbnick, M. (1956). In “Cellular Mechanisms in Differentiation and G r o ~ t h ’ ’( D . Rudnick, e d . ) , p. 3. Princeton Univ. Press, Princeton, New Jersey. Reichardt, W., and Vaju, D. (1958). 2. pliysik. Chemie (Frankfurt) [N.S.] 15, 297. Reichardt, W.,and Varju, D. (1959). 2. Nuturforsch. 14, 210. Rilling, S., Mittelstaedt, H., and Roeder, K. D. (1959). Behauiour 45, 164.

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Roeder, K. D. (1953). “Insect Physiology.” Wiley, New York. Roeder, K. D. (1959). Smithsoniun Inst. Publs. Misc. Collections 137, 287. Roeder, K. D., Tozian, L., and Weiant, E. A. (1960). J. Inst. Ph!ysiol. 4, 45. Ruck, P. (1958a). J. Inst. Physiol. 2, 189. Ruck, P. (1958b). J. Inst. Physiol. 2, 261. Schneider, D. (1957). 2. uergleich. Physiol. 40, 8. Slifer, E. H., and Finlayson, L. H. (1956). Quart. J. Microscop. Sci. 97, 617. Van der Kloot, W. G., and Williams, C. (1954). Behauiour 6, 233. Vowles, D. M. ( 1961). In “Some Problcms in Animal Behaviour” (W. H. Thorpe and 0. L. Zangwill, ed\.) p. 5. Cambridge Univ. Press, London arid New York. Wallace, G. K. (1958). J. Exptl. Riol. 35, 765. Wallace, G. K. (1959). J. Exptl. Bid. 36, 512. Walther, J. B. (1958). J. Inst. Physiol. 2, 142. Weiant, E. A. (1958). Proc. 10th Intern. Congr. Entomol. 2, 81. Wolbarsht, M. L. (1959). J. Gen. Physiol. 42, 413. Wolbarsht, M. L., and Dethier, V. 6. (1958). J. Gen. Physwl. 42, 393.

ADDENIXJM

Since this review was written, the following papers have appeared which bear directly on the ERG, its propcrties, origins, and influence as a generator potential. Autrum, H., and Burckhardt, D. (1961). Nature 190, 639. Burckhardt, D., and Wendler, L. ( 1960). 2. uergleich. PhysioZ. 43, 687. Ruck, P. (1961a). J. Gen. Physiol. 44, 605. Ruck, P. (1961b). J. Gen. Ph!ysiol. 44, 629. Ruck, P. ( 1 9 6 1 ~ ) .J. Gen. Physiol. 44, 641. Ruck, P. (1961d). Biol. BUZZ. 120, 375.

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AUTHOR INDEX Numbers in italic indicate the pages on which the references are listed, Andy, 0. J., 179, 180,185 Angeleri, F., 195, 196, 246 Ansell, G. B., 294, 299, 307, 311, 314 Anthony, W. M., 340, 345 Apter, N. S., 320, 324, 325, 328, 347 Archibald, R. M., 330, 344 Arduini, A., 70, 71, 80, 91, 93, 94, 97, 118, 126, 131, 132, 136, 221, 222, 242, 250 Aring, C. D., 228, 242 Armstrong, J., 162, 183 Armstrong, M. D., 254, 260, 261, 2162, 263, 269, 270, 272, 288, 290 Artom, C., 299, 314 Asanuma, H., 225, 242 Aschcr, P., 118, 131, 224, 242 Astwood, E. B., 320, 344 Atwell, C. R., 253, 291 Auer, J., 203, 242 Autrum, H., 357, 371 Aves, E. K., 271, 272, 291 Awapara, J., 331, 344 Axclrod, J., 286, 291

A Abdulian, D. H., 332, 348 Abood, L. G., 304,314 Adam, D. J. D., 341, 344 Adams, R. D., 339, 348 Ades, H. W., 201,246 Adey, W. R., 188, 213, 218, 230, 242, 249, 327, 345 Adrian, E. D., 71, 72, 80, 81, 97, 101, 105, 110, 115, 118, 123, 131, 187, 196, 242 Agarwal, 1’. S., 282, 283, 288 Aikawa, J. K., 342, 344 Aird, R. B., 39, 61 Ajmone-Marsan, C., 95, 125, 133, 145, 150, 162,183 Akedo, €I., 342, 344 Alanis, J., 153, 183 Albe-Fessard, D., 67, 73, 80, 87, 97, 102, 126, 131, 132, 141, 183, 235, 242 Albcrt, A., 343, 344 Aldridge, W. N., 312, 314 Allen, J. N.,30, 31, 61 Allen, W. F., 201, 236, 239, 242 Allmark, M. G., 322, 325, 344 Alpers, B. J., 52, 61 Altschule, M. D., 271, 288 Alvarez, W. C., 253, 288 Alvord, E. C., 159, 183 Amassian, V. E., 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 86, 87, 88, 89, 95, 97, 98, 99, 101, 102, 103, 104, 111, 112, 113, 114, 115, 116, 117, 123, 124, 126, 128, 129, 130, 131, 134, 135, 192, 200, 201, 210, 211, 222, 235, 242, 247, 248 Andrejew, A., 338, 344 Andrew, B. L., 198, 242

B Babcock, M. J., 331, 344 Baccari, V., 308, 314 Blsslcr, U., 354, 371 Bagshaw, hf. H., 202, 206, 242 Bahr, G. F., 309, 316 Baigne, N., 337, 345 Bailey, P., 39, 64 Bain, J. A., 39, 64, 323, 330, 331, 332, 333, 334, 337, 338, 344, 346 Bairati, A., 15, 61 Bakay, L., 34, 39, 45, 47, 61, 62, 299, 314 Baker, R. W. R., 305, 314 Balakrishnan, S., 260, 277, 288

375

376

AUTHOR INDEX

Bale, W. F., 30, 45, 64 Ballantine, H. T., 47, 62 Ballard, A., 30, 31, 65 Banerjee, S., 282, 283, 288 Baranowski, T., 339, 344 Barbeau, A., 282, 288 Bard, P., 70, 134, 189, 190, 196, 230, 244, 246, 250 Barlow, C. F., 331, 344 Bamard, J., 188, 250 Barnard, J. W., 189, 209, 250 Baron, D. N., 261, 265, 266, 267, 268, 288 Barrcra, S. E., 139, 171, 184 Barron, D. H., 159, 183 Baruk, H., 253, 288 Bassett, D. L., 222, 246 Bangess, L. C., 258, 288 Baumgarten, R.v., 68, 70, 77, 120, 131 Baumgartner, G., 120, 121, 122, 123, 125, 126, 128, 132, 133,134 Baxter, C. F., 333, 344 Beattie, P. H., 340, 348 Becker, M. C., 133 Beerstecher, E., Jr., 330, 348 Bender, M . B., 192, 242, 24.3, 244, 245 Benjamin, R. M., 189, 202, 237, 243, 249 Bennett, H. S., 40, 50, 62 Bennett, M. V . L., 84, 131, 161, 183 Benson, W. M., 320, 325, 344 Berg, B. N.,295, 314 Berg, C. P., 258, 263, 288, 290 Bering, E . A., 45, 62 Berlin, L., 72, 76, 77, 95, 112, 113, 130, 131 Berlin, R., 262, 288 Bennan, A. L., 69, 71, 73, 74, 78, 79, 83, 89, 98, 100, 101, 104, 111, 112, 113, 114, 117, 134, 141, 150,184 Bemhard, C. G., 162, 183, 204, 210, 211, 212, 213, 243 Bemsohn, J., 282, 283, 286, 290 Berry, J. F., 299, 300, 308, 316 Bertaccini, G., 281, 288 Bertrand, G., 214, 243

Bcssey, 0. A., 341, 344 Beyer, A., 341, 344 Bianchi, L., 252, 289 Bicknell, F., 268, 289 Bichl, J. P., 332, 340, 341, 344, 348 Birkmaycr, W., 308, 314 Bimkov, G., 364, 371 Bishop, G. H., 89, 102, 103, 122, 128, 131, 132, 147, 148, 183 Black, D. A. K., 276, 289 Black, S. P. W., 229, 230, 247 Bloch, K., 295, 314 Blum, J. S., 130, 131 Bodenstein, D., 371 Boelter, M . D., 45, 63 Bornstein, W. S., 202, 243 Bogdanski, D. F., 253, 255, 289, 292 Bohm, E., 204, 210, 211, 212, 213, 243 Bok, S. T., 1'61, 183 Boldrey, E., 202, 247 Bond, D. D., 178,185 Bonnet, V., 201, 243 Boone, I . U., 324, 342, 344 Borden, J. H., 271, 289 Borenstein, P., 126, 132, 235, 243 Boshes, B., 211, 246 Boszormenyi, Z., 253, 289 Bowden, J., 215, 246 Boyce, R., 208, 243 Boyd, I . A., 197, 198, 243 Brady, R. O., 58, 62, 297, 314, 320, 344 Braganca, B. M., 312, 314 Branch, C. L., 78, 79, 81, 82, 83. 87, 94, 95, 107, 118, 119, 132, 134, 141, 146, 176, 183, 184 Brante, G., 294, 314 Braunstein, A. E., 330, 335, 338, 343, 344 Brazier, M. A. B., 75, 132, 327, 345 Bremer, F., 67, 89, 102, 103, 132, 162, 178, 183, 201, 202, 228, 239, 243 Brenner, C., 166, 183 Brinitzer, W., 271, 289 Brinlcy, F. J., Jr., 107, 132

AUTHOR INDEX

Brodal, A., 203, 206, 207, 208, 216, 229, 243, 245, 249 Brock, L. G., 79, 80, 87, 132 Brodie, B. B., 284, 291 Brodmann, K., 199, 243 Broman, T., 39, 62 Bromiley, R. B., 188, 200, 248 Brookhart, J. M . , 91, 93, 97, 119, 123, 126, 127, 128, 132, 136, 221, 222, 243, 250 Brooks, V. B., 105, 130, 132 Brown, A. W . , 35, 62 Brown, D. H., 339, 344, 346 Brown, D. V. L., 37, 62 Brown, J., 253, 291 Bro\vn, T. R., 297, 298, 316 Brown, R. R., 263,291 Brueckner, H. H., 322, 348 Brunrr, J., 235, 243 Brush, M., 331, 344 Bucliwald, N . A., 327, 345 Bukin, Y . V., 324, 344 Bullock, T . H., 159, 161, 183, 185 Bures, J., 178, 183 Burcsova, O., 178, 183 Burge, F . W., 340, 345 Burke, J . C., 320, 325, 347 Burkhardt, D., 354, 371 Burns, B. D., 101, 105, 124, 132, 166, 183 Burstein, L. S., 295, 317 Burton, R. M . , 297, 314 Burtt, E . T., 358, 371 Buscaino, G. A., 272, 282, 284, 289 Buscaino, V . M., 253, 285, 280 Buser, P., 67, 71, 73, 80, 87, 97, 102, 117, 118, 126, 131, 132, 133, 141, 183, 201, 224, 235, 242, 243 Butler, R. A., 188, 1913, 239, 243, 250

C Cafiero, M., 338, 344 Cajal, S. R., 160, 183 Caliari, W., 324, 344 Callan, D. A,, 329, 347 Calma, I., 71, 93, 94, 118, 132 Camba, R., 342, 344

377

Campbell, W.W., 45, 63 Cannon, W . B., 166, 178, 183, 185 Carlson, H . B., 340, 345 Carmichael, E., 322, 325, 344 Carr, S., 294, 309, 315 Carreras, M., 116, 132, 194, 243 Carrctero, R., 340, 348 Carthy, J. D., 363, 371 Casby, J . V.,201, 248 Catton, W . T., 358, 371 Chaikoff, I . L., 295, 299, 314, 315, 316, 317 Chaloupka, M. M., 268, 289 Chambers, W . W . , 207, 208, 209, 243 Chang, H.-T., 90, 101, 102, 105, 108, 126, 127, 132, 160, 183, 189, 190, 209, 222, 230, 231, 232, 243, 250 Chang, J . J., 48, 62, 63, 65, 164, 185 Changus, G. W., 299, 314 Chnrgaff, E., 299, 314 Chavez, M., 166, 183 Chiarugi, E., 208, 243 Chou, S. N., 165, 184 Cliow, K . L., 130, 132, 236, 237, 247 Christensen, H . N . , 342, 345 Christoff, M., 320, 322, 323, 325, 348 Chusid, J. G., 179, 184, 189, 243 Civen, M . , 258, 290 Clare, M . H., 89, 102, 103, 122, 128, 131, 132 Clark, C. T., 263, 289 Clark, S. L., 188, 249 Clark, W . E. Le Gros, 199, 243 Clasen, R. A,, 37, 62 Claycomb, C. K., 31 7 Clemente, C. D., 39, 44, 47, 62, 6 5 Contes, S., 269, 289, 292 Cocn, L. J., 342, 346 Cohen, H., 340, 346 Cohen, M . J., 69, 117, 132, 202, 244 Colin, W . E., 45, 63 Cole, J., 233, 237, 244 Collier, H . B., 309, 314 Combs, C. hl., 216, 228, 244 Compston, N . D., 294, 315 Cone, W . V . , 37, 64

378

AUTHOR INDEX

Conochic, J., 261, 289 Contardi, A,, 305, 314 Cooke, P. M., 227,228,245 Coombs, J. S., 79, 80, 87, 132 Cori, C. F.,339, 344, 345, 346 Cornelius, L. R., 180, 185 Costa, E., 255, 289, 342, ,344 Costantini, L., 322, 347 Conlter, N. A,, Jr., 45, 62 Coursin, D. B., 340, 341, 345 Courtois, G., 107, 181 Covian, M. R., 198, 199, 247 Coyne, B. A,, 342, 345 Cragg, 13. G., 161, 183 Crain, S. M., 82, 84, 131, 132, 161, 183 Crandall, G., 188, 250 Crawford, M. A., 2F1, 262, 267, 290 Creutzfcldt, O., 120, 123, 125, 126, 128, 132, 134 Culbreth, G. G., 299, 316 Cullen, C., 87, 88, 91, 95, 96, 97, 98, 99, 101, 111, 112, 113, 114, 128, 134, 220, 221, 246 Curtis, D. R., 51, 62, 327, 328, 329, 345 Curtis, H. J., 103, 104, 132, 230, 232, 244 Curzon, G., 272, 282, 289

D Dalgliesh, C. E., 255, 259, 2'60, 272, 275, 289,290 Dandy, W. E., 206, 246 Dasgupta, S. R., 323, 326, 328, 345, 346 David, G. B., 35, 62 David, J. D., 340, 345 Davies, P. W., 69, 71, 73, 74, 78, 79, 83, 89, 98, 100, 101, 104, 111, 112, 113, 114, 117, 119, 120, 133, 134, 141, 150, 184 Davison, A. N., 258, 270, 282, 289, 291, 295, 296, 297, 298, 301, 302, 332, 338, 314, 315, 345 Davson, H., 30, 31, 33, 34, 39, 46, 62 Dawson, A. M., 253, 289

Dawson, R. hl. C., 294, 295, 299, 305, 306, 307, 314, 315, 316 De Cnstro, F., 50, 62 Dbjerine, J., 215, 244 Delay, J., 253, 289 Delbrnck, M., 357, 372 28, 41, 44, 62, 91, Dempsey, E. W., 128, 1.32, 134, 218, 219, 221, 244, 246 267,289 Dcnko, C. W., Denncny, J. M., 343, 348 Dennis, D., 325, 345 266, 267, 268, Dent, C. E., 261, "63, 288 Der, P., 253, 289 De Robertis, E. D. P., 6, 18, 19, 28, 31, 33. 37, 38, 41, 44, 46, 50, 51, 52, 62, 63 Dcthicr, V. G., 351, 370, 371, 373 DcVito, J. I,., 75, 79, 89, 111, 131, 214, 215, 231, 232, 244, 248 Dcwcy, V. C., -320, 321, 322, 323, 324, 345, 346 Diamond, I. T., 239, 243 Dianzani, M. U., 309, 316 Dibner, I. M., 272, 289 Dick, M., 284, 292 Dieke, H., 320, 322, 345 Dillistone, F., 269, 292 Dimascio, A., 253, 291 Dimick, D., 320, 322, 325, 348 Dirkin, M. N. J., 68, 136 Dixon, G. J., 340, 345 Dixon, J. M. S., 275, 289 Doane, B., 69, 93, 130, 135 Dobbing, J,, 39, 62, 295, 29G, 297, 301, 302, 314 Dobson, R. L., 223, 224, 249 Dodt, E., 198, 242 Dohan, F. C., 272,291 Dohmen, H., 294, 299, 314 Douglas, G. \f7., 296, 316 Dow, R. S., 229, 230, 244 Dowben, R. M., 73, 132 Drake, C. G., 166, 183, 229, 230, 247 Dressler, S. H., 340, 346

379

AUTHOR INDEX

Dubinick, B., 337, 345 Duggan, D. E., 264, 289 Dunlop, C. W., 327, 345 Dunn, hl. S., 260, 289 Dusscr de Bnrenne, J. G., 176, 183, 228, 233, 244 Dutton, R. W., 255, 289 D ~ v a l lR. , N., 343, 345

E Eakin, R. E., 330, 348 Ecclcs, J. C., 49, 51, 62, 79, 80, 82, 87, 105, 132,133, 163,183 Ecclcs, R. hl., 51, 62 Echlin, F. A., 166, 183 Edstrom, R., 40, 46, 62 Ehrlich, P., 39, 62 Eidelberg, E., 156, 181, 183, 186, 3-70, 327, 331, 3.33, 344, 345, 347 Eisenmnn, G., 92, 135 Eisenstein, A. B., 341, 342, 345 Eldrcd, E., 72, 133, 226, 248 Elliott, K. A. C., 29, 30, 31, 33, 37, 62, 63, 64, 168, 180, 184, 185, 320, 332, 345 Ellison, J., 267, 290 Elvehjem, C. A., 2G8, 289 Engcr, P. S., 105, 132 Enomoto, T. F., 95, 133, 145, 150, 162, 183 Ercoli, A,, 305, 314 Erickson, T. C., 179, 184, 188, 213, 244 Erlenmeyer, H., 343, 345 Erspamer, V., 255, 289 Erulkar, S. D., 119, 120, 133 Escolar, G. J., 216, 244 Esplin, D. W., 155, 168, 186 Etzwiler, D. D., 269, 290 Evans, D. R., 371 Evans, J. P., 237, 244 Evarts, E. V., 254, 289 Evered, D. F., 266, 289 Ewins, A. J., 260,289 Eydt, K. M., 294, 316 Eyzaguirre, C., 147, 153, 154, 184

F Fabing, H. D., 253, 289 Fairbairn, D., 305, 315 Fairmnn, D., 188, 191, 250 Fallab, S., 343, 345 IJarrphar, hl. C., 6, 14, 19, 28, 63 Fatt, P., 82, 133, 148, 163, 18.3 Fay, J., 188, 250 Feldstcin, A., 272, 279, 280, 282, 283, 289 Fellman, J. H., 270, 289 Fernindez-hlorin, H., 29, 63, 355, 371 Fine, E. A,, 320, 345 Finean, J. B., 29, 63, 294, 315 Fink, M., 192, 243, 244 Finlayson, L. H., 352, 371, 372, 373 Fischer, E. H., 339, 345 Fisher-Williams, M., 16G, 184 Fishman, V., 271, 289 Fleischauer, K., 15, 63 Flesner, L. B., 46, 65 lilorenz, A,, 139, 184 Foerster, O., 188, 244 Folch, J., 294, 309, 315 Folin, O., 252, 289 Forbcs, A., 68, 74, 135 Forbes, H. S., 69, 133 Formica, J. V., 58, 62 Forrest, A. D., 272, 289 Forster, F. M., 167, 1S4 Foye, W. O., 343, 345 Francoli, hl., 304, 315 Frank, K., 49, 63, 72, 81, 82, 83, 84, 86, 88, 108,133 Frankel, S . , 331, 336, 347 Frnnkl, W., 260, 289 Frceman, H., 279, 280, 282, 283, 289 French, J. D., 156, 181, 183, 186, 215, 216, 217, 244 Frey, A., 255, 290 Freygnng, 15'. H., Jr., 49, 63, 79, 83, 86, 133 Friedemann, U., 39, 63 Friedman, B. I., 332, 341, 348 Friedman, B. K., 263, 292

380

AUTHOR INDEX

Fries, B. A., 299, 315, 316 Fuerst, R., 331, 344 Fujimori, B., 72, 133 Fulton, J. F., 129, 135, 202, 228, 237, 242, 244, 247 Fuortes, M. G. F., 72, 81, 82, 83, 84, 88, 108, 133, 159,183 Furlow, L. T., 192, 242

G Galambos, R., 126, 133 Galston, A. W., 261, 262, 290 Gammon, G. C., 340,345 Carattini, S., 324, 338, 344, 345 Cardner, E., 198, 244 Carol, H. W., 188, 244 Gasser, H. S., 77, 133 Gastaut, H., 166, 184 Cauthicr, C., 222, 244 Geiger, A., 304, 314, 315 Celdiay, K.,370, 372 Gellhorn, E., 213, 244 Cent, W. L. G., 309, 315 Gerard, R. W.,158, 178, 184, 294, 317 Ceren, B., 32, 63 German, W. J., 129, 135 Gernex-Rieux, C., 338, 344 Cerschenfeld, H. hl., 6, 18, 19, 31, 33, 37, 38, 41, 44, 46, 52, 62, 63 Ceachwind, N., 50, 64 Glarman, N. J., 280, 281, 290 Cibsen, J., 267, 290 Giclez, L. I., 295, 315 Cillett, E., 235, 242 Girado, hl., 71, 104, 105, 125, 127, 128, 135, 329, 347 Girao, C. B., 261, 262, 267, 290 Glees, P., 2, 63, 206, 233, 237, 244 Goehel, A,, 303, 315 Coldenberg, H., 271, 289 Coldin, A., 325, 345 Coldring, S., 158, 174, 184, 185 Goldsmith, G. A., 267, 290 Golcl\mith, T. H., 355, 371 Gonnard, P., 337, 345, 346 Gonzalez-Monteagudo, O., 339, 347

Gooclman, L., 332, 371 Gordon, hl. W., 164, 185 Gortatowski, M. J., 254, 260, 262, 263, 288, 288 Cowers, W. R., 137, 184 Grafstcin, B., 104, 124, 132, 133, 166, 183 Granit, R., 72, 133, 199, 218, 244, 245 Gray, E. G., 13, 63, 162, 184 Green, J. D., 94, 133, 156, 162, 184,

185 Green, J. P., 280, 281, 290 Green, hl., 192, 243, 244 Greenberg, D. M., 45, 63 Greenberg, J. B., 261, 262, 290 Griffiths, R., 269, 292 Gronchi, V., 304, 315 Grosse, A., 309, 315 Grdsser, 0.-J.,121, 122, 133 Griitzner, A., 121, 122, 133 GnindEest, H., 77, 83, 84, 87, 104, 105, 106, 107, 109, 127, 131, 133, 135, 147, 148, 161, 162, 183, 184, 329, 347 Grundy, W.E., 267,289 Guazzi, G., 195, 196, 246 Gullotta, S.,271, 290 Gygax, P. .4.,202, 247

H Haege, L. F., 30, 64 Hagbarth, K. E., 206, 207, 210, 218, 245 Haimovici, H., 166, 183 Hampson, J. L., 225, 245 Hampton, J. C., 40, 62 Hanahan, D. J., 304, 315 Hanbery, J., 219, 245 Hansen, A. E., 341, 344 Hanzon, V., 312, 315 Hardisty, R. hl., 265, 290 Harker, J., 349, 371 Harris, H., 261, 265, 266, 267, 268, 269, 288, 290 Harrison, C. R., 198, 199, 225, 245, 247

381

AUTHOR INDEX

Hart, E. W., 261, 265, 266, 267, 268, 288 Hartmann, J. F., 6 , 14, 19, 28, 63 Haskell, P. T., 352, 370, 371 Hass, C. M., 37, 62 Hassenstein, B., 358, 371, 372 Hassert, C. L., 320, 347 Hastings, A. B., 30, 64 Hawkins, J. R., 253, 289 Hawthome, J. N., 294, 315 Hayaishi, O., 305, 306, 315 Haymaker, W., 52, 61 Head, H., 192, 245 Heinze, C., 201, 243 Henneman, E., 227, 228,245 Henry, G. W., 271, 275, 290 Henson, C. O., 126,133 Heran, H., 353, 372 Hering, H. E., 212, 245 HemBndez-Peh, R., 207, 216, 217, 218, 244,245, 248 Herrlin, L., 35, 63 Hersov, L. A., 265, 266, 267, 290 Herter, C. A., 252, 290 Hess, A., 36, 40, 63 Hess, S. M., 254, 290 Hild, W., 48, 52, 62, 63, 164, 183 Hillman, R. S. L., 342, 346 Hoagland, H., 272, 279, 280, 282, 283, 289 Hoche, A,, 215, 245 Hodgkin, A. L., 49, 63 Hodgson, E., 351, 370, 372 Hoffman, M., 261, 292 Hoffman, R., 320, 322, 347 Hoffman, W., 357, 371 Hofmann, A., 255,290 Holst, E. A., 47, 62 Holt, L. E., Jr., 340, 348 Homing, E. C . , 259, 260, 272, 275, 290 Horstmann, E., 29, 49, 50, 63 Houser, E., 272, 291 Howland, B., 90, 133 Hoyle, C., 349, 372 Hsu, J. M., 324, 346

Hubel, D. H., 69, 70, 72, 73, 74, 93, 122, 126, 129, 133 Huber, F., 365, 368, 372 Hudson, A., 370, 372 Hueter, T. F., 47, 62 Hughes, H. P., 320, 322, 347 Humphreys, S. R., 325, 345 Hunt, A. D., Jr., 340, 346 Hunt, C. C . , 87, 133 Hutchens, T. T., 317 Hyde, J., 213, 244 HydCn, H., 18, 52, 63

I Illingworth, B., 339, 344, 345, 346 Imbert, M., 71, 117, 133 Ingram, W. R., 195, 248 Inskip, W. M., 282, 283, 286, 290

J Jabbur, S. J., 108, 135 Jackson, J. H., 177, 184 Jacobs, F. A., 342, 346 Jacobs, U. F., 364, 372 Jaeger, J. C . , 51, 62 Jaffe, J., 192, 245 James, W. D., 323, 324, 347 Jameson, D., 272, 291 Jander, R., 363, 372 Jansen, J., 229, 245 Jansen, J., Jr., 226, 229, 245 Jansz, N. S., 339, 346 Jasper, H. H., 30, 37, 63, 68, 69, 70, 71, 77, 78, 79, 87, 88, 91, 93, 94, 95, 96, 97, 98, 99, 101, 111, 112, 113, 114, 125, 128, 130, 133, 134, 135, 138, 140, 141, 149, 150, 152, 180, 184, 185, 189, 191, 214, 218, 219, 220, 221, 237, 245, 246, 247, 320, 332, 345 Jenkins, W. T., 330, 347 Jcnkner, F. L., 68, 135 Jenney, E. H., 320, 321, 322, 323, 324, 325, 328, 330, 337, 346, 347 Jepson, J. A., 260, 261, 262, 265, 266, 267, 268, 288, 290

382

AUTHOR INDEX

JOh115011, A. C., 294, 303, 315 Johnson, hl. K., 312, 31,l Johnson, N., 2G7, 290 Johnson, \Y. J., 338, 346 Jolly, W. A,, 215, 248 Jones, E. R. H., 262, 290 Jones, C. P., 340, 346 Jones, W. A., 340, 346 Jung, R., 68, 70, 93, 120, 122, 126, 128, 131, 133, 134, 150, 176, 184 Jus, A., 279, 280, 290

K Kaada, B., 102, 132 Kaada, B. R., 218, 24.5 Kabakow, B., 282, 291 Knkimoto, Y., 282, 201 Kalmus, I I . , 355, 372 Kandel, E. R., 107, 132 Karnovsky, M. L., 29.5, 298, 300, 315, 316 Kass, I., 340, 346 Katsuki, Y., 120, 134 Kaufmann, G., 323, 334,347 Keeping, J. A,, 340, 346 Kelly, W., 259, 260, 290 Kennard, M. A., 167, 179, 184, 189, 215, 245, 2'19 Kennedy, E. P., 300, 307, 315 Kennedy, T. T., 111, 125, 134 Kerr, D. I. B., 206, 218, 2/15 Kerr, J. D. B., 188, 210, 242 Kety, S., 283, 290 Kidder, C . W., 320, 321, 322, 323, 324, 345, 346 Killam, E . K., 323, 326, 328, 345, 346 Killam, K. F., 320, 321, 323, 324, 325, 326, 327, 328, 3.30, 331, 333, 338, 345, 346, 347 Kimmig, J., 262, 290 King, E. E., 219, 245 King, E. J., 303, 31 5 King, G., 340, 345 King, L. S., 39, 63 King, W., 255, 258, 259, 260, 263, 276, 278, 292 Kirman, B. H., 271,290

Kirschfeld, K., 356, 372 Klatzko, J., 37, G:7 Klemme, R. hl., 216, 246 Kline, D., 295, 315 Knight, H. C . J. G . , 303, 315 Knighton, R. S., 188, 195, 196. 245 Knowlcs, W. B., 215, 246 Knox, W. E., 258, 290 Koch, A., 30, 31, 65 Kolliker, A,, 52, 63 Kolmodin, G. M., 163, 184 Konigsmm:trk, B., 156, 183 Kono, II., 188, 247 Kopeloff, L. hl., 139, 171, li':), 184, 189, 243 KopcloiT, N., 139, 140, 167, 171, 176, 179, 184, 186, 189, 243 Kopin, I. J., 282, 283, 290 Korey, S., 58, 63 Kornberg, A,, 305, 306, 307, 3 IS, 31 7 Kovnl, G. J., 58, 62 Krcbs, E. G., 339, 345 Kretchmer, N., 269, 290 Krieg, \Y. J. S., 216, 231, 2-15 Kristianscn, K., 167, 184 Kiiijccvic, K., G9, 133 Krogh, A,, 45, 64 Kruger, L., 102, 117, 134, 189, 1'30, 196, 2.37, 245, 246 Kumcr, S. W., 147, 153, 154, 184 Kudo, Y., 282, 291 Kunkel, A. M., 320, 345 Knno, M., 87, 133 Kuwabara, M., 355, 372 Kuypcrs, 1-1. C . J. hl., 203, 204, 205, 206, 207, 210, 216, 245

L Ladnn, A. J., 331, 344 Laidlaw, P. P., 260, 289 Lance, J. W., 209, 245 Landau, W. M., 49, 63, 108, 109, 134, 209, 245 Landgren, S., 69, 117, 132, 202, 244, 245 Lanc, W. A., 252,290 Langham, W. H., 324,344

383

AUTHOR INDEX

Langncr, R. R., 263, 290 Laskowska, D., 279, 280, 290 Laskowski, E. J., 37, 63 Lassek, A. hl., 211, 246 Latimer, C. N., 125, 134 Lauer, J. W., 282, 283, 286, 290 LaVallee, A., 322, 325, 344 Leahy, D. J., 340,345 Lerro, A. A. P., 178, 179, 184 Leavitt, S., 37, 62 LeBaron, F. N., 293, 315 Leduc, E. H., 44, 65 Lee, J., 47, 64 Lcc, L. D., 320, 321, 322, 346 Lccs, RI., 204, 309, 315 Leeson, G. A., 337, 345 LempcriPre, T., 253, 289 Lende, R. A., 188, 256 Lconarcli, A., 324, 344 Lcpage, L., 212, 249 Lc-ttvin, J. Y., 90, 133 Leverton, R. M., 267, 290 Lcvin, P. M., 215, 246 Lcvinc, R., 320, 322, 323, 325, 348 Lcvitt, M., 116, 132, 194, 243 Lewis, D., 206, 246 Leyton, G. B., 272, 276, 277, 200 Li, C.-L., 68, 69, 70, 71, 73, 77, 78, 79, 80, 81, 82, 86, 87, 88, 91, 92, 93, 91, 95, 96, 97, 98, 99, 101, 107, 111, 112, 113, 114, 117, 118, 126, 128, 134, 141, 142, 150, 164, 105, 184, 220, 221, 225, 246 Libet, B., 158, 178, 184 Lichstcin, H. C., 338, 346 Lilly, J. C., 189, 246 Lin, E. C . C., 258, 290 Linclberg, O., 299, 314 Lindsley, D. B., 215, 246 L issman, ',, H. W., 158, 184 Liu, C.-N., 207, 208, 209, 243 Livingston, R. B., 216, 217, 218, 242, 244 Livingston, S., 324, 346 Lloyd, D. P. C., 206, 210, 246 Loewenstein, W. R., 153, 184 Long, R. G., 224, 225, 246

Lorente dc N6, R., 92, 98, 108, 125, 126, 128, 134 Loughbridge, L., 261, 262, 267, 290 Lovenberg, W., 254, 262, 263, 282, 292 Lowell, D. J., 322, 325, 326, 346 Lowenstcin, O., 352, 371, 372 Lu, F . C., 332, 325, 344 Lubing, H. N., 340, 346 Lnft, J. H., 40, 62 Lumsden, C. E., 39, 40, 64, 294, 315 Lnse, S. A,, 6, 15, 29, 41, 52, 64 Lyons, R., 304, 315

M hlcAlpine, D., 294, 31 5 McArdle, B., 311, 312, 315 hlacchi, G., 193, 196, 246 hlcCormick, D. B., 334, 335, 337, 346 McCrory, \Y. W., 340,346 hlcculloch, W. S., 90, 133, 140, 167, 171, 176, 179, 183, 186, 189, 229, 230, 233, 244, 245, 246, 248 hlacfarlane, M. G., 303, 315 hlcIhvain, H., 30, 33, 64, 265, 266, 267, 291, 295, 316 hlcIntyre, A. K., 198, 246 hlcKibben, P. S., 37, 65, 188, 246 Macklin, C. C., 47, 64 Macklin, hl. T., 47, 64 hlclaren, J., 253, 289 McLennan, H., 68, 134 RIcLeod, I. M., 300, 316 McMillan, P. J., 296, 316 McMurray, W. C., 299, 300, 308, 316 hlcsabb, A. R., 294, 303, 315 htacy, J., Jr., 72, 74, 76, 77, 95, 112, 113, 130, 131 Magee, hl., 342, 344 hlagec, W. L., 295, 315 hlagoun, €1. W., 215, 218, 219, 229, 244,246, 247, 248, 249 hlahnke, J. H., 157, 159, 169, 184, 186 hlainardi, L., 325, 346 Majno, G., 300, 316 Malis, L. I., 102, 117, 134, 189, 190, 246

384

AUTHOR INDEX

Maloney, C . J., 340, 346 hlancia, M., 126, 132 Mandel, W., 340, 346 Mannery, J. F., 30, 39, 45, 64 Manning, R. L., 209, 245 Mark, R. F., 113, 134 hlarkaroglu, L., 324, 346 Marples, E. A., 305, 306, 307, 310, 316, 317 hiarshall, C., 139, 185 Marshall, W. H., 70, 80, 88, 97, 107, 111, 132, 134, 139, 178, 179, 184, 196, 246, 320, 324, 325, 328, 346, 347 Martin, A. R., 78, 79, 81, 82, 83, 87, 94, 95, 107, 118, 119, 132,134, 141, 146, 176, 183, 184 Martin, F., 37, 62 hlartin, F. B., 297, 298, 31 6 hlaruyama, N., 120, 133 hlarvel, c. S., 258, 288 Mast, G. W., 261, 292 Matthews, B. H . C., 153, 159, 183, 184 Mauss, E. A., 281, 292 Maxwell, D. S., 94, 133 Mayer, S. E., 39, 64 Maynard, E. A., 6, 28, 41, 64, 65 Mayrand, E., 320, 322, 323, 325, 348 Meltzer, €I. I., 295, 299, 31 7 Menniken, G., 255, 292 hlerritt, H. H., 166, 183 Mettler, F. A., 203, 215, 231, 246 Mcves, FI., 29, 49, 50, 63 Meyer, D. R., 189, 201,239, 246, 250 Mickle, W . A., 201, 246 Middlebrook, G., 340, 345, 346 Milburn, N., 370, 372 Milch, E. C., 231, 246 Miles, R. C., 189, 249 Milholland, J., 336, 347 Millen, J. W . , 41, 65 Miller, P. L., 367, 372 Milne, M. D., 261, 262, 267, 290 Minckler, D., 216, 246 hlincklcr, J., 216, 246 hliner, N., 200, 238, 249

Mittelstaedt, H., 363, 372, 373 Mollica, A,, 229, 249 Moncrieff, A., 269, 292 Morgan, R. S., 295, 296, 297, 298, 302, 314, 31.5 Morison, B. R., 68, 74, 135 Morison, R. S . , 91, 128, 132, 134, 218, 219, 221,222, 244, 246 Morita, H., 159, 184 Morlock, N. L., 155, 186 Morrcll, F., 139, 184 Morris, A. A., 180, 185 Morrison, L. R., 308, 312, 316 Mortensen, R. A., 296, 316 Moruzzi, G., 70, 71, 72, 80, 91, 93, 118, 123, 126, 131, 132, 136, 215, 221, 222, 228, 229, 230, 244, 246, 247, 249, 250 Moser, H. W., 295, 298, 316 Mott, F. W., 223, 247 Mountcastle, V. B., 69, 71, 73, 74, 77, 78, 79, 83, 89, 98, 100, 101, 104, 110, 111, 112, 113, 114, 115. 116, 119, 129, 134, 135, 141, 150, 155, 184, 185, 192, 193, 194, 195, 196, 197, 198, 199, 200, 215, 247, 248 Mucllcr, J. F., 332, 341, 348 Muggleton, P. W . , 340, 348 Munro, T. A., 269, 291 Murayama, M., 45, 63 Murtas, L., 344 Myers, R. E., 200, 238, 247, 249

N Nagy, T., 253, 289 Naka, K., 355, 372 Nakahama, H., 98, 104,134, 188, 191, 192, 223, 231, 232, 233, 234, 247, 282, 291 Nakamura, K., 223, 247 Naquet, R., 219, 24.5 Natori, S., 188, 231, 232, 247 Nautn, W. J. H., 127, 134, 202, 231, 232, 247 Neff, W. D., 239, 243 Nelson, P. G., 82, 83, 84, 133 Neumayer, E., 308, 314

AUTHOR INDEX

Nguyen-Chi, J. P., 337, 346 Nguyen-Philippon, C., 337, 346 Eichol, C. A., 336, 342, 347 Nicholas, H. J., 295, 296, 316 Nicolai, H., 258, 261, 291 Nielsen, A., 139, 185 Niessing, K., 29, 36, 64 Nims, L., 139, 176, 183, 185 Nisino, Y., 203, 247 Nissl, F., 35, 64 Noguchi, S., 305, 316 Norman, A. P., 269, 289 Norman, J. M., 307, 316 Nulsen, F. E., 229, 230, 247 Numberger, J. I., 164, 185 Nygaard, A. P., 309, 316

0 Oberdorfer, A., 364, 371 Oberholzer, R. J. H., 365, 372 Ochs, S., 72, 105, 107, 134 Oestreicher, R., 340, 346 Okamoto, K., 225, 242 Okamoto, T., 282, 291 Okey, R., 299, 317 O’Leary, J., 158, 174, 184, 185 Olscn, K. B., 299, 314 Olszewski, J., 47, 64, 124, 132, 166, 183, 216, 247 Orbach, J., 236, 237, 247 Orrego, F., 79, 80, 83, 86, 87, 88, 102, 135, 141, 147, 155,185 Ostenso, R., 188, 189, 250 Otsuka, J., 260, 291 Ott, H., 255, 290 Ozbas, A., 370, 372

P Paasonen, M. K., 280, 281, 290 Pacella, B. L., 179, 184 Page, I. H., 255, 291 Palade, G. E., 29, 50, 64 Palay, S. L., 19, 50, 64, 162, 185 P’an, S. Y., 324, 346 Pandolfi, S., 37, 62 Paoletti, R., 338, 345 Pappenheimer, J. R., 45, 64

385

Pappius, H. A., 31, 33, 64 Pare, C. M. B., 270, 271, 290, 291 Parkin, K. R., 340, 348 Parks, R. E., Jr,, 320, 321, 322, 323, 324, 345, 346 Parma, M., 222, 244, 247 Parmalee, A . H., 340, 346 Partington, P. F., 299, 314 Patek, P. R.. 41, 64 Patterson, J. D. E., 294, 315 Patton, H. D., 70, 71, 74, 78, 80, 81, 87, 98, 99, 101, 102, 103, 104, 107, 118, 123, 124, 128, 131, 134, 135, 136, 200, 201, 202, 206, 210, 211, 214, 215,222, 242, 247,248 Patton, R. A,, 324, 347 Payling Wright, G., 295, 296, 297, 298, 302, 314, 315 Pazur, J., 267, 290 Peacock, S. M., Jr., 104, 125, 135, 231, 247 Pease, D. C., 6, 28, 41, 64, 65 Pcele, T. L., 231, 233, 247 Penfield, W., 2, 37, 64, 138, 140, 149, 152, 180, 185, 188, 189, 191, 202, 213, 214,237, 247, 248 Perl, E. R., 89, 97, 98, 101, 104, 116, 125, 135, 194, 195, 201, 232, 248, 249 Perri, V., 335, 347 Perrier, C., 299, 314 Peters, J. P., 260, 291 Petersen, D. C., 324, 346 Petersh, I., 210, 211, 243 Petersen, V. P., 295, 316 Petrushka, E., 312, 316 Petrzilka, T., 255, 290 Pfaffmann, C., 202, 243 Pfciffer, C. C . , 320, 321, 322, 323, 324, 325, 328, 330, 337, 346, 348 Phillips, C. G., 69, 70, 73, 74, 78, 79, 80, 81, 82, 84, 86, 87, 88, 94, 107, 108, 123, 135, 142, 164, 165, 185, 225, 248 Phillis, J. W., 327, 345 Piazza, S., 322, 325, 326, 346 Pichot, P., 253, 289

386

AUTHOR INDEX

Pigon, A., 18, 52, 63 Pilgrim, F. J., 324, 347 Pinto Hamuy, T., 188, 189, 200, 213, 248, 250 Piraux, M. D., 37, 63 Pitt, B. hl., 258, 290 Pitts, W., 90, 133 Platt, B. S., 298, 316 Pleasure, H., 320, 347 Pletschcr, A., 278, 280, 284, 291, 292 Poggio, G. F., 73, 116, 13.5, 179, 180, 185, 193, 194, 248 P o k y , E. H., 79, 80, 83, 86, 87, 88, 102, 135, 141, 147, 155,185 Pomcrat, C. M., 15, 48, 64 Pope, A., 180, 185 Porcellati, G., 303, 314, 316 Porcino, F., 322, 347 Porter, P., 196, 209, 237, 245 Powell, T. P. S., 71, 111, 112, 113, 114, 115, 116, 119, 129, 134, 135, 192, 193, 194, 196, 197, 198, 199, 200, 243, 247, 248 Prescott, B., 323, 324, 347 l’rescott, F., 268, 289 I’rclston, J. B., 325, 326, 328, 347 l’ribram, K. H., 117, 130, 132, 134, 189, 190, 202, 206, 242, 246 Price, J. M., 203, 291 Price, S. A. P., 251, 291 Pringle, J. W.S., 351, 372 Pritchard, E. T., 295, 300, 301, 315, 316 Probst, M., 212, 248 Purpum, D. P., 67, 71, 90, 91, 94, 97, 104, 105, 106, 107, 109, 119, 125, 127, 128, 135, 149, 163, 185, 329, 339, 347

Q Quadbeck, G., 321, 324, 347 Quastel, J. H., 312, 314, 316

R Radin, N. S., 297, 298, 316 Rall, W., 149, 185 Ramhn y Cajal, S., 50, 64, 108, 135 Ranson, S. W., 195, 248

Rapport, M. M., 263, 293 Rasmnsscn, T., 188, 189, 191, 248 Rayport, M., 74, 77, 135 Redlich, E., 206, 248 Register, U. D., 267, 290 Reichnrdt, W., 357, 358, 362, 372 Reichel, M., 303, 317 Reilly, J., 324, 34G, 347 Reilly, R. H., 320, 325, 328, 347 Rkinoncl, A. G., 223, 224, 249 Renshaw, B., 68, 74, 135 Rcynolds, bl. S., 368, 289 Rhodin, J., 29, 64 Ricci, G. F., 69, 89, 93, 102, 105, 130, 135, 148, 156,185 Richter, D., 294, 293, 311, 311, 315, 317 Riegc.lhaupt, L., 272, 291 Riggs, T. Ii., 342, 345, 347 Rilling, S., 373, 373 Rindi, G., 335, 347 Rinkel, hl., 253, 291 Rio IIortega, P. dcl, 2, 18, 19, 30, 52, 64 Rittcnberg, D., 293, 314 Rolxrts, E., 320, 327, 331, 332, 333, 336, 344, 347 Robcrts, G. B., 340, 345 Roberts, N. R., 342, 347 Robcrts, T. D. M., 197, 198, 243 Robertson, J. D., 294, 31 5 Robins, E., 294, 316 Robinson, K. S., 269, 288 Robinson, J. R., 30, 64 Robitzek, E. H., 325, 347 Rodnight, R., 2’60,262, 263, 264, 265, 266, 267, 271, 272, 276, 277, 278, 280, 288, 290, 291, 304, 31 6 Rodriguez, L. A,, 39, 64 Roe, M. D., 320, 325, 344 Roeder, K. D., 349, 363, 364, 365, 370, 371, 372, 373 Roger, A,, 71, 117, 133 Rose, J. E., 73, 111, 113, 119, 120, 132, 133, 135, 155, 185, 193, 194, 195, 196, 199,201,215, 248

387

AUTHOR INDEX

Rose, W.C., 258, 288 Rosen, F., 321, 324, 336, 342, 347 Rosenblueth, A,, 178, 185 Roscnthal, S., 159, 185 Ross, G., 282, 291 Ross, K. R., 324, 347 Ross-Dnggan, J. K., 322, 325, 326, 346 Rossi, A,, 303, 316 Rossi, G., 228, 248 Rossi, G. F., 208, 216, 243, 248 Rossiter, R. J., 293, 294, 295, 299, 300, 301, 303, 308,315,316 Roth, L. J., 331, 344 Rott, \V. H., 334, 347 Rouged, A., 126, 131, 235, 242 Rowntrec, L. G., 38, 64 Rozdilsky, B., 47, 64 Ruben, S., 299, 314 Ruhin, B., 320, 325, 347 Ruch, T. C., 129, 135, 200, 202, 206, 247, 248 Ruck, P., 356, 357, 373 Rudin, D. O., 92, 135 Rupert, A,, 126, 133 Russcll, W.F., Jr., 340, 345 Ruthven, C. R. J., 258, 282, 291

S Sachs, E., 281, 291 Sachs, J., 299, 316 Sadhu, D. P., 298, 316 Saito, M., 188, 231, 232, 247 Sand, R., 215, 248 Sandler, M., 258, 270, 271, 282, 289, 290, 291 Sano, I., 271, 282, 291 Santangelo, M., 299, 314 Sarctt, H. D., 267, 290 Sartori, G. D., 321, 324,347 Sarzana, G., 299, 314 Sasaki, T., 260, 291 Sato, M., 153, 185 Saunders, L., 309, 316 Saxon, S. V., 228, 244 Scarinci, V., 322, 347 Schachner, H., 299, 315, 316 Schadk, J. P., 29, 50, 65

Schaeffer, K. P., 77, 131 Schalleck, W., 322, 325, 347 Schappell, A. W., 192, 242, 24.3 Schatelbrand, G., 39, 64 Schcrrer, H., 207, 218, 245, 248 Schindler, W. J., 94, 133 Schlag, J. E., 74, 80, 87, 98, 99, 101, 102, 103, 104, 128, 131,222, 242 Schmidt, L. H., 320, 322, 347 Schmidt, R. P., 95, 135, 142, 143, 145, 150, 151, 152, 163, 168, 170, 171, 172, 181, 185, 186 Schmitt, F. O., 50, 64 Schneider, D., 351, 373 Sclioen, L., 123, 125, 132 Scholefield, P. G., 316, 316 Schoolar, J. C., 331, 344 Schon, M., 295, 316 Schreiner, L. H., 215, 246 Schultz, R. L., 6, 28, 41, 64, 6.5 Schwarz, V., 298, 31 7 Scott, c.,337, 345 Scale, B., 331, 344 Searle, C. W., 340, 346 Seckfort, H., 303, 315 Segre, E., 299, 314 Segundo, J. P., 218, 242 Seidenstein, S., 188, 2-19 Selikoff, I. J,, 325, 347 Semenza, F., 325, 346 Sencer, W., 104, 135, 189, 2.50 Settlage, P., 188, 189, 250 Shanes, A. M., 165, 185 Shapiro, B., 305, 31 6 Shapiro, M., 192, 242, 243 Sharlit, H., 260, 291 Shaw, K. N. F., 254, 260, 261, 262, 263, 272, 288, 290, 292 Shcrlock, S., 253, 289, 291 Sherrington, C . S., 213, 223, 247, 248 Shciwood, W. K., 276, 291 Shive, W., 330, 348 Sholl, D. A., 124, 135 Shore, P. A., 284, 291 Simpson, S., 215, 248 Sindberg, R. M., 201, 249

c.

388

AUTHOR INDEX

Singer, H., 254, 260, 262, 263, 272, 288 Sird, R. B., 45, 63 Sjiiqvist, O., 230, 248 Sjoerclsma, A., 254, 255, 258, 259, 260, 262, 263, 276, 278, 282, 292 Skoglund, C. R., 165, 184, 198, 248 Slifer, E. H., 352, 373 Sloane-Stanley, G. H., 293, 303, 304, 317 Smith, D. E., 294, 316 Smith, 0. A., 214, 215, 231, 232, 244, 248 Smith, R. P., 321, 323, 324, 346 Smith, T. G., 163, 185, 329, 347 Snell, E. E., 330, 334, 335, 337, 338, 346, 347 Snider, R. S., 226, 227, 228, 229, 245, 248 Snodgrass, R. E., 350 Snyderman, S. E., 340, 348 Sodd, M. A., 297, 314 Sosa, D., 47, 62 Sostman, E., 331, 344 Spatz, H., 39, 65 Spaziani, E., 30, 31, 33, 46, 62 Spector, R. G., 258, 282, 291 Sperry, R. W., 200, 238, 239, 248, 249 Spcrry, W. M., 295, 299, 303, 317 Spicgel, E. A., 166, 183, 185 Sprince, H., 272, 291 Srere, P. A., 295, 31 7 Stacey, R. S., 265, 270, 271, 280, 290, 291 Stafford-Clark, D., 253, 291 Stamm, J. S., 238, 249 Starzl, T. E., 215, 219, 249 Stavraky, G. W., 166, 183, 185 Stefanchi, L., 272, 282, 284, 289 Stefko, P. L., 320, 325, 344 Steiner, J., 113, 134 Stephens, R. R., 320, 322, 323, 325, 348 Stephenson, M., 258, 291 Stetten, D., 299, 317 Sticherling, W., 262, 290 Stilwell, D. L., Jr., 198, 249

Stokes, J., Jr., 340, 346 Stoppani, A. 0. hl., 261, 291 Stoupel, N., 89, 102, 103, 132 Stoyanoff, V. A., 295, 317 Strait, L. A., 39, 61 Streicher, E., 294, 317 Strickland, K. P., 299, 300, 308, 316, 317 Strom, L., 69, 117, 132, 202, 244 Stromberg, V. C . , 257, 291 Stroud, H. H., 340, 346 Strumwasser, F., 159, 185 Stucler, A., 278, 280, 292, 340, 348 Stumpf, C., 94, 133 Suda, M., 342, 344 Suga, N., 120, 134 Sugawa, T., 342, 344 Sutton, P. N., 340, 348 Sveatichin, G., 153, 185 Swank, R. L., 208, 249 Sweeley, C . C . , 259, 260, 290 Sweet, W. H., 202, 249 Szabo, T., 203, 206, 207, 243 Szara, S., 253, 286, 291 Szekely, E. G., 166, 185 Szentiigothai-Schimert, J., 208, 249

T Tacquet, A,, 338, 344 Tait, A. C., 284, 292 Tasaki, I., 48, 63, 65, 79, 80, 83, 86, 87, 88, 102, 135, 141, 147, 155, 164, 185 Taubock, H., 309, 315 Tausig, T., 320, 324, 325, 328, 347 Taylor, C. B., 37, 62 Taylor, C. W., 215, 249 Taylor, J. L., 45, 65 Taylor, R. M., 295, 299, 31 7 Taylor, U'. C., 262, 290 Teasdall, R. D., 166, 185 Temperley, H. N. V., 161, 183 Teorrel, T., 153, 185 Terry, R. D., 38, 65 Terzuolo, C., 201, 213, 230, 243, 249 Terzuolo, C. A., 159, 185 Teuber, H. L., 189, 192, 242

389

AUTHOR INDEX

Thannhauser, S. J., 303, 31 7 Thomas, B. E., 295, 296, 316 Thomas, B. C. H., 320, 347 Thomas, L. B., 68, 95, 131, 135, 142, 143, 145, 150, 151, 152, 163, 170, 171, 172, 185, 186 Thomas, 1%.P., 132, 186 Thompson, R. F., 201, 237, 243, 249 Thompson, R. H. S., 293, 305, 306, 307, 308, 310, 311, 312, 315, 316, 317 Timiras, P. S., 30, 31, 65 Titus, E., 260, 282, 292 Tobias, J. M., 310, 317 Todrick, A., 284, 292 Tokizane, T., 72,133 Tolbert, M. E., 299, 31 7 Tomich, E. C., 340, 348 Torack, R., 38, 65 Torvik, A., 203, 206, 207, 243, 249 Toschi, C., 312, 315, 317 Towe, A. L., 69, 70, 71, 74, 75, 78, 79, 80, 87, 98, 99, 101, 102, 103, 104, 107, 108, 111, 112, 113, 114, 115, 116, 118, 128, 129, 131, 134, 135, 222, 242 Tower, D. B., 167, 168,185,320, 324, 331, 339, 343, 344, 348 Tower, S. S., 208, 249 Townsend, A. D., 271, 292 Tozian, L., 365, 371, 373 Tramezzani, J. H., 19, 44, 63 Travis, A. M., 188, 189, 213, 214, 249, 250 Treitman, S. S., 295, 317 Trevarthen, J., 263, 292 Troxler, F., 255, 290 Tschesche, R., 262, 290 Tschirgi, R. D., 39, 45, 65 Tunturi, A. R., 120, 135, 200, 249 Turner, W. J., 281, 292 Turney, D. F., 324, 342, 344 Twitchell, T. H., 223, 249 Tyler, F. H., 269, 270, 288 Tyrrell, L. W., 295, 303, 31 7 Tyrrell, W. G., 340, 345

U Udenfriend, S., 253, 254, 255, 2.58, 259, 260, 262, 263, 276, 278, 282, 289, 290, 292 Umbreit, W. W., 336, 348 Ungar, J., 340, 348 Urbach, H. C., 262, 290

V Valcourt, A. J., 284, 292 Vanasupa, R., 174, 185 van Breemen, V. L., 39, 44, 65 Van Bmggen, J. T., 295, 317 Van cler Kloot, W.G., 365, 373 van Harreveld, A,, 29, 49, 50, 6.5 Van Slyke, D. D., 260,291 Varju, D., 357, 372 Varma, S. N., 298, 317 Velasco, M., 207, 245 Venditti, J. M., 325, 345 Ventura, V., 335, 347 Vcrhaart, W. J. C., 215, 249 Vernadakis, A,, 342, 348 Verzeano, M., 215, 244 Victor, M., 339, 348 Vilter, R. W., 332, 340, 341, 344, 348 Vogel, W., 29, 36, 64 von Amerongen, F. K., 215, 244 von Baumgarten, R., 229, 249 von Euler, C., 89, 102, 105, 135, 148, 156, 185 Vowles, D. hl., 349, 373 Vysniauskas, C., 322, 348

W Waalkes, T. P., 264, 292 Wada, J. A,, 180,185 Waelsch, H., 295, 317 Wajda, M., 296, 297, 298, 302, 314, 315 Walberg, F., 206, 207, 208, 249 Wald, F., 6, 18, 19, 31, 33, 37, 38, 41, 44, 46, 52, 62, 63 Walker, A. E., 189, 211, 228, 246, 249 Walker, L. Yl., 342, 347

390

AUTHOR INDEX

Wall, P. D., 90, 133, 186, 223, 224, 249 Wallace, G. K., 363, 373 W'allcr, H. J., 72, 74, 76, 77, 95, 112, 113, 130,131 Walter, A. E., 179, 180, 185 Walther, J. B., 356, 373 Walz, D., 322, 325, 347 Walzl, E. M., 201, 250 Wang, C. H., 188,250 Ward, A. A., Jr., 95, 135, 138, 140, 141, 142, 143, 145, 150, 151, 152, 155, 157, 159, 163, 167, 168, 169, 170, 171, 172, 176, 179, 181, 184, 185, 186 Ward, J. W., 188, 249 Watanabe, T., 120, 134 Watkins, J. C., 327, 328, 329, 345 Welnter, G. R., 293, 294, 306, 307, 308, 309, 310, 311, 312, 315, 316, 317 Wced, L. H., 37, 46, 65 Weiant, E., 564, 365, 370, 371, 372, 3 73 Weil, A., 308, 31 7 Wed-Malherbe, H., 270, 292, 330, 331, 348 Weinstein, B., 282, 291 Weinstein, E. A., 230, 248 Weiss, S. B., 300, 307, 315 Weissbach, H., 255, 258, 259, 260, 263, 276, 278, 282, 289, 292 Welch, K., 188, 213, 248 Welker, W. I., 188, 189, 243, 249 Werle, E., 255, 292 Wertheimer, E., 212, 249 "Vest, E. S . , 295, 317 West, G. B., 254, 291 Wheelis, D. R., 188, 246 White, J. C., 156, 181, 186 Whitlock, D. G., 70, 80, 89, 91, 93, 97, 98, 101, 104, 116, 125, 131, 135, 136, 194, 195, 219, 221, 222, 232, 242, 248, 249, 250 Whittier, J., 271, 289 Wiegand, R. C., 321, 332, 334, 336, 348

Wiesel, T. li., 122, 129, 133 William?, C., 365, 373 Williams, H. L., 321, 323, 329, 332, 334, 336, 337, 344, 348 Williams, J. N., 268, 289 Williams, R. J., 330, 348 Wills, J. H., 320, 345 Wilson, V., 351, 372 Winder, F. G., 343, 348 Windle, W. F., 2, 6, 65 Wislocki, G. R., 28, 41, 44, 62, 65 Witkofl', L. J., 282, 288 Wittenberg, J., 307, 317 Wolbarsht, M., 351, 371, 373 Woldring, S., 68, 136 Wolfe, W. J., 168, 181, 185 Wood, M. M., 322, 348 Woodbury, D. M., 30, 31, 65, 155, 168, 186, 342, 348 Woodbury, J. W., 70, 74, 80, 81, 87, 98, 99, 101, 102, 103, 104, 128, 131,136, 222, 242 Woods, D. D., 260, 292 Wooldridge, W. E., 261, 292 Woolf, L. I., 269, 289, 292 Woollam, D. H. M., 41, 65 Woolsey, C. N., 70, 97, 117, 134, 136, 188, 189, 190, 191, 193, 194, 195, 196, 200, 201, 209, 211. 213, 225, 239, 242, 244, 245, 246. 248, 249, 250 Wyckoff, R. W. G., 28, 36, 65

Y Yamasaki, S., 304, 315 Yoshikawa, S., 342, 344 Young, J. Z., 28, 36, 65, 162, 183 Younger, F., 336, 347

Z Zadunaisky, J. A., 31, 33, 37, 38, 44, 46, 63 Zamecnik, P. C., 308, 312, 316 Zanchetti, A., 91, 93, 97, 119, 123, 127, 128, 132, 136, 208, 221, 222, 243, 244, 247, 250

AUTHOR INDEX

Zartman, H., 320, 322, 323, 325, 348 Zbinden, G., 278, 280, 292, 340, 348 Zeller, E. A,, 282, 283, 286, 290 Zimmermann, H. M., 38, 65

391

Zimny, S., 279, 280, 290 Zotterman, Y., 69, 117, 132, 202, 244 Zubek, J. P., 236, 237, 250 Zucker, M. B., 263, 292

SUBJECT INDEX A

Arnnnita mappn, 257 Aminoaciduria renal, 266-267

Astrocy-tes ( Astroglia: sce also Neuroglia ) contraction of, 48 morphological ch;trac.tcristic.; ,:)f, 6. 18, 59 permeability of, 49 relation to blood-brain barrier, 39-44 capillaries in CNS, 44-47 cerebrospinal flnid, 34-35 estrncellular spec:, 28-32 water-electrolytr mc+nbolisn~ of brain, 33-34 role in edema of CNS, 37-39 summary of functional significkunce, 60-61 Auditory area histology, 232 unit responses in, 119-120 Augmenting response, 221-22::3 Autointoxication, intestinal as cause of mental illncss, 25'7-253. 284-285 Axon collaterals, 160-161, 17F Asons, 126 dorsal column, 92 pyrxmidal, 91, 94, 123 collntc~rals of, 108

y-Aminobutyric acid (Galla), 155, 168-169, 324, 343 action on synaptic I)roccsses, 326329 biochemistry, 330-333 p-Aminosalicyclic acid; 340 Anesthesia barliiturate, 70-72 chloralose, 70-72 120, 179 and neuronal activity, 70-73, 114, Area postrema, 44 Arousal reaction, 215 Aspartic acid, 327-328

B Barbitmt-e spincllcs, 91 Barium ion, 304 Bascmciit mcsmhrane, 41, 60 Behavior conditioning studies, 236-240 insect, chemical control of, 370-37 1 insect-, control by locd 11rnin stiniulation, 365-371 Bctz cells, 70, 118, 209 spikes from, 78, 80-84, 87, 94-95, 123-124, 141-142, 146 spontaneous discharge from, 94

Ablation studies, 167, 189, 202, 206, 208, 213-215, 228, 236.240 Abstraction, 182 Acetate, 300 Acetone scmicarbazone, 323, 337 Acetylcholine, 51, 165 denervation hypersensitivity to, 166 metabolism, in epilepsy, 166-168 Acetylation, 338 Acctyltryptophan, 263 Action potentials' (see Spikcs ) Adenosinc triphosphate, 300 Adrenal cortex hypertrophy, 341 Afterpotentials axonal, 92 fi-Alanine, 327 Aliesterase, 310, 313 Alpha rhythm, 220 Alumina ercam, 139- 140, 155, 157, 166-167 Antcnnac, 351, 353-355, 366 Antcnnal nerves, 351, 354 Anticliolinesterases, 167

392

SUBJECT INDEX

Blood-brain barrier, 30, 36, 51, GO, 299 as active transport mechanism, 4647 relation to astrocytes, 34, 39-44, 60-61 Ivhere absent, 44, GO Brain waves (see Spontaneous activity and Electroencephalogram) Bufotenidin, 257 Bufotenine, 253, 257, 279 Bumble bee, 364

C C14, 295-300 3-C14-serine, 298 Cahoba, 257 Calcium ion, 304, 306 Calliphora, 351, 357 Campaniform sensillae, 351 Capillaries of CNS, filtration coefficient, 45-46 morphological classification, 4041 peimcability, 45-47 submicroscopic analysis, 40-44, 69

Carbon dioxide, analysis of expired, 72 Catalasc, 338 Catechol amines, in phenylketonuria, 270 Central body, 365, 368 Central commissure, 365 Cephalin, 303, 305 Cerebellum (including various ;matomical subdivisions), 226-230, 327 Cerebral pulsations, control of, 69-70 Cerebrosidase, 303 Cerehrosides, 313 metabolism in brain, 297-299 Cerebrospinal fluid, 34 Cerveau isolC, 73 Chemoreceptors, in blow fly, 351 Chlorpromazine, 282

393

Cholesterol metabolism in brain, 295-297, 313 hvo types in brain, 297 Choline, 300-301, 305, 307, 313 Cholinesterases, 310-313 Chromatin of astrocytes, 6 of oligodendrocytes, 18-19 Chronometers, electric, 74-75 Cobalt ion, 306 Coppcr ion, 306 Corpora cardiaca, 370 Corpora pedunculata, 365, 367-368, 370 Corpora quadrigcmina, 331 Corpus callosum, 103-105, 125, 179180, 230-232, 238-239 Cortex (scc also Cortical evoked response) areas of (see under Visual area, Auditory area, etc. ) cerebral “isolated slab” preparation, 124, 166-167, 326 layers of, 98-99, 232, 233 relation to cerebellum, 225-230 topographically organized, role in discrimination, 130 epileptic lactic acid in, 176 oxygcn tcnsion of, 176 pH of, 167 unit recordings from, 95, 137183 unit responses in (see Neurons, Cortical unit responses i n ) Cortical evoked response to antidromic stimulation of pyramidal tract, 107-109 in auditory cortex, 102, 126 effect of thiosemicarbazone on, 328 interactions of, 125-126 intercortical, 156, 233-235 to locd cortical stimulation, 105107, 181 in somatosensory areas, 71, 97103, 117,202,220, 235

394

SUBJECT INDEX

from specific thalamocortical volley, 97-103, 126 transcallosal, 103-105, 125 in visual cortex, 102-103, 125, 128 Cortisol, 341-342 Crickets, 366-368 Cystathione enzymes, 338 Cysteic acid, 327-328 Cytoplasm, of ostrocytes, 6

D Dark adaption, of insect eye, 356-357 Data, automatic handling of experimental, 74-76. Deafferentation, 223 Decarboxylases, 255, 259-260, 263, 270, 330, 331, 333, 335, 337, 339 Demyelination, 295, 312 and isoniazid, 340 Dendrites, 32, 81, 87, 101-103, 105, 108-109, 127 epileptic activity, 151-156, 163-164, 176-177 normal activity, 147-149 role in spreading depression, 178179 synchronization, 160-165 Deoxypyridoxine, 324, 329, 335-337, 34 1 3-Deoxypyridoxine, 336 4-Deoxypyridoxine, 337 5-Deoxypyridoxine, 337 Depolarization by amino acids, 327-329 by mechanical stimuli role in epileptogenesis, 152-156, 157, 164 Depolarizing agents, use in unit recordings, 72 Depth reversal, of cortical evoked responses, 90-91, 95-105, 127 Diencephalon, analog in insects (corpora pedunculata), 370 Diisopropylfluorophosphonate, 307 Diphosphoinositide, 299

Dipoles, 90, 101 Diptera, 355 Discrimination somatosensory, 236-238 visual, 238-239 Distance estimation, in insects, 363

E Edema, of brain, 37-39, 60 Effercnts (see Projection) Electroencephalogram, 171-181 in epilepsy, 149-152 and hydrazides, 325-326 Electron microscope, 3-28 pass., 36, 41, 50, 52, 162-163 advantages of, 2-4 on brain slices, 31-32 on insect eye, 355 Electroretinogram, in insects, 355-357 Electroshock treatment, 280-281 Encephale isolC, 70, 73, 202 Encephalopathy, hepatic, 253 Ephaptic interaction, 161-163 Epilepsy, 95, 137-183 experimental method of study, 139140 Jacksonian, 177-178 neurochemistry of, 167-169 relation of experimental to natural, 138-140 Epileptic focus, neuroanatomical features of, 140-141, 160-161 Epileptic seizure mechanism of origin, 169-177, 181 mechanism of propagation, 177-181 role of environmental stress, 181 Eserine, 307 Esters, of phosphoric acid in brain, 305-308, 313 Ethanolamine, 300-301, 305, 307 Evoked response (see Cortical evoked response) Extinction, 192 Extracellular space determination by physiological methocls, 29-31 lack of, 16-18, 35-36, 46, 49-50

SUBJECT INDEX

relation to “ground substance” of CNS, 35-36

F Facilitation, 114, 128, 212, 224-225, 228-230, 234-235, 239 “Feed-back,” 166, 210, 218, 240-241 Fibers ( s e e Projection) Fibrils, glial, 15, 59, 140 Field effects role in epileptogenic focus, 157-160 interaction of neurons, 161-165 spread of epileptic seizure, 178 Flagellum, 353-354 Flaxedil, 72 Flicker fusion frequency, 357 Fornzicu rufa, 363-364

G Galactose, 298 Gates, electronic, 75-76 Glucocerebrosides, in brain, 298 Glucocorticosteroids, 342 Gluconeogenesis, 341-342 Glucose, incorporation in brain lipids, 298, 300 Glutamic acid, 167, 327-328, 330, 340 L-Glutamic acid, 324 Glutamic acid cycle, 330 Glutamic-aspartic transaminase, 337 Glutamic-oxaloacetic transaminase, 311-313, 330, 331 Glycerol, 300-302 Glycerylphosphorylcholine, 301, 304, 306-307, 313 Glycerylphosphorylethanolamine, 301, 306-307 Glycine, 300 Glycolysis, 338 Golgi complex of astrocytes, 7-8 of oligodendrocytes, 19 Golgi-Cox stain, 141, 232 Ground substance ( intercelltilures grau), 36, 60

395

as blood-brain barrier, 40 y-Guanidinobutyric acid, 329 Giymnarchus niloticus, 158 Gyrus anterior sigmoid, 95 postcentral, 111, 117, 189

H Hair plates, 352 Hartnup disease, 255, 261, 265-269 Hippocampus, 68, 339 theta rhythm from, 94 His-Held ( pericapillary ) space, 41, 44, GO Homocysteine, 339 Honey bee, 369-370 Hunger, control in insects, 370 Hydrazides, 319-348 biochemistry of, 329-339 neuropathology, 339-340 neurophysiology of, 325-329 pharmacodynamic effects of, 324325 potentiation of other convulsant compounds by, 323 reversal of convulsant effects by depressants, 322-325 pyridoxine congeners, 323-325 Hydrazine, 320 Hydrazones, 332-338, 342 5-Hydroxyindolylacetic acid, 255, 258, 262, 263, 270, 281-284, 286 6-Hydroxyindolylacetic acid, 286 Hydroxylases, 269, 270 6-Hy droxy -&‘-dimethy ltryptamine, 286 6-Hydroxyskatole, 272 5-Hydroxytryptamine, 253 5-Hydroxytryptophan, 271, 286 Hymenoptera, 355 Hypothalamus, 203 Hypothesis Autrum’s, 357 Chang’s three-component of callosal response, 231-232 Cragg and Temperley’s, 161

396

SUBJECT INDEX

I Indican, 258, 260-261, 266, 267, 271 Inclicanuria, 252, 266-268, 275 Indigo, 262 Indirubin, 262 Indole, 258 Indoles abnormal tissue, 253, 285-286 mctabolism in plicnylkctonuria, 26927 1 normal blood, 263-265 normal urinary, 260-263 role in mental illness, 251-288 Indolylncetic acid, 255, 258, 259-260, 262, 263, 264, 266, 267, 269, 276, 2881-283, 286 Indolylacetylgliiciironide, 262, 265 Indolylacrylic acid, 262 Indolylformic acid, 262, 277 Indolylformylglucuronide, 277 Indolylglycolic acid, 262 Indolyllactic acid, 258, 260, 262, 263, 269, 277 Indolylpropionic acid, 260 Indolylpyruvic acid, 2ij8, 262 Indoxyl, 258, 261 Inhibition, 114-116, 119, 122, 123, 165-166, 176-177, 192, 196, 224225, 229-230 Inositol monophosphate, 304 Inositol phosphatidc, 304 Integration, sensorimotor, 223-225, 24 1 Intracellular cleft, 18, 29, 49-50 Ionic exchanges of brain capillaries, 45-46 effect on pyridoxine, 342-343 of glia, 33-35, 61 of intracellular clcft, 49-52 of neurons, 165 Isoniazid, 320, 324-325, 331, 342-343 and ncuropathy, 339-340

J “Jelly roll” theory of myelinogenesis, 52 Johnston’s organ, 353-355

K a-Ketoglutarate, 323 Kidney, 342 metabolism of amincs by, 263, 286 Kynurenic acid, 267 Kynuronine, 254, 267

L Lactic acid, 176 Latency, 111, 117-119, 122, 123, 125, 129, 365 postsynaptic, 79 presynaptic, 79 Lateral geniculate body, spikes from, 79, 83 Lateral neuropil, 367 Learning, conditioned, 217-218, 230, 23G-240 Lecithin, 299-305 Leminiscus, medial, 193 “Light-compass reaction,” 363 Lipid breakdown in brain, 303-308 synthcsis in brain, 294-303 Lipoproteins, role of biosynthcsis in myelinogenesis, 58 Locomotion, insect, control by local brain stimulation, 365-366 Locust, 3G3 Lysergic acid diethylamide, 253, 285 Lysolecithin, 305 action on brain, 308-313 Lysophosphatides, 308

M Magnesium ion, 304, 306 Malabsorption syndrome, 272, 276 Manganesc ion, 306 hlantid, 363 Membranc cell, 342 neuronal, as electrically inexcitable, 83, 147, 162-163 Membrane current, 83-84, 153-155 Membrane potential, 80, 152-156, 161 fluctuations of, role in cpileptogenesis, 165 glial, 164-165

397

SUBJECT INDEX

hfetabolism brain in epilepsy, 167-169, 181 indoles, 251-291 lipids in brain, 293-317 water-electrolyte of brain, 33-34 Meter, “Berkeley Universal,” 7 5 Methoxypyridoxine, 339 o-Methylpyridoxine, 337 Michaelis constant, 307 hficroelectrode, 51, 67-121 p s s . , 141172 pass., 327 analysis of pyramidal activity, 225 analysis of recruiting response, 220 determination of position of tip of, 74 glass, 73-74 Hubel’s tungsten, 73 metal, 73-74 position of tip of, 48-49, 61, 83-84, 141 size of tip of, 78-79, 98, 141, 355 Microglia, characteristics of, 28 Microsome, 58, 297, 312 “Mirror” focus, 180 Mitochondria, 32, 295, 300, 309, 312, 335 of astrocytes, 7 of microglia, 28 of oligodendrocytes, 18-19 hlodels, role in scientific procedure, 182 Monoamine oxidase, 255, 281, 284, 311, 313 Monro-Kellie doctrine. 45 Motoneuron, effect of Gaba on, 327328 Motor-sensory are‘), 191-241 puss. activation of, 224-225 Movement perception in insects, 358-364 theoretical mechanism for, 360362 Mushroom bodies, 365, 367-368, 370 Myelin destruction by lysolecithin, 309 disposal of, 58-59

hf yelination lipid metabolism in, 296 f role of biosynthesis of Iipoproteins in, 58 role of glin, 52-59, 61

N Nauta-Gygu method, 202 f, 231-233 AT-diethyltryptamine, 253 N-dimethyltryptamine, 253,279, 286 Nerve degeneration, 59 Neuroglia contraction of, 164-165 clectrical responses of, 164-165 membrane potentials of, 88 pathology in epileptic focus, 169 relation to neuronal activity, 47-52 Neurohypothesis, 44 Ncuronography, evoked potentid method, 196-202, 216-218, 230, 233-234 Neurons comparison of two or more simultaneously, 77, 94-95, 112, 116, 130 cortical effect of anesthesia, 72 field effects of, 157-160, 161-165 input-output function of, 113, 130, 241 sensitive to joint rotation, 198-199 topographical patterns of, 199 types of spontaneous discharge of, 93-95, 141-146 unit responses in auditory area, 119-120 to corticocortical volleys, 124125 to direct cortical stimulation, 123-124 in motor areas, 117-119 patterns of, 93-95, 112f, 123126 in somatosensory areas, 110117, 129, 197-202 in vibual area, 120-122, 129 “end-blade,” 339

398

SUBJECT INDEX

epileptic, 152-157 geniculate, 102 Golgi type 11, 92, 101, 103, 124, 128 pyramidal, 127 pyramidal tract projection, 70-71, 77, 97, 107-109, 118, 123, 125, 128 Neuropathy and isoniazid, 339-340 Nicotinamide, 254-255, 266-268 Nuclci cranial nerve, 203-206, 207, 215 thalamic, 193-196 Nucleus caudatc, 326, 331 cuneate, 79 of microglia, 28 of oligodendrocytes, 18-19 solitary, 206 spinal of fifth nerve, 193

0 Oligodendrocytes, 41 “acute” swelling of, 37 contraction of, 48 function of, 52-59, 61 perineuronal, 18 two types, 59-60 in white matter, 19 Oligophrenia, 269 Ommatidium, 355, 358-364 Optic atrophy, 340 Oxidase. 311 Oxygen tension, of epileptic cortex, 176

P P32, 299-302 Pars intracerebralis, 367 Pedicel, 353-354 Pentylenetetrazol, 326-327, 329, 330, 333, 339 Periplaneta Americana, 356 Perfusion, of brain, 304 Pellagra, 265-268, 339 Phenolic acids, in phenylketonuria, 269-271 Phenothiazines, 282

Phenylalanine, 269-271 Phenylalanine hydroxylase, 269 fi-Phenylethyl hydrazine, 337 Phenylketonuria, 269-271, 285 fLPhenylisopropylhydrazine, 337 Phosphatase, alkaline, 311 Phosphatidylcholine, 307 Phosphatidylcthanolamine, 299-300, 307 Phosphatidylserinc, 299-301 Phosphodicsterase, 304-308, 310, 313 Phospholipases, 304-305, 307, 309, 310, 312, 313 Phospholipids, 295 f hydrolysis of, 303-308 metabolism in brain, 299-303, 313 Phospliomonoesterase, 305-308, 313 Phosphorylase, 339 Phosphorylcholine, 300, 305, 307308, 313 Phosphorylethanolamine, 305, 307308, 313 Phosphorylserine, 305, 313 Phototaxis, in insects, 363-364 Pia mater, dimpling by microelectrode, 74 Pioglial membrane, as blood-brain barrier, 39 Piptadenis peregrinu, 257 Polarized light, reaction of insects to, 355, 363364 Pond skater, 364 Porphyria, 262 Potassium, 167 Potentials, action ( s e e Spikes ) membrane, 48, 88 post-synaptic, 87, 89, 91, 94, 97, 101-105, 108, 127 standing in epileptic cortex, 157160, 174-176 standing in normal cortex, 158-159 Projection, afferent from muscle, 198-199 to auditory area, 119-120 cortical to cranial nerve nuclei, 203-206

399

SUBJECT INDEX

cortical to diencephalon, 203 cortical to dorsal column nuclei, 206-208 corticocerebellar, 226-230 corticocortical, 179 corticospinal, 208-213 frontal corticoreticular, 215-218 ipsilateral intercortical, 232-235 of medial lemniscal afferents, 193196 of motor cortex, 117-119 specific thalamocortical, 97-103, 109-117 specific thalamocortical, mode of termination of axons, 126 of spinothalamic afferents, 193196 of splanchnic afferents, 200 transcallosal, 103-105, 125, 230232 unspecific thalamocortical, 218-223, 225, 241 to visual area, 120-122 Proprioception, mechanisms in insects, 351-355 Protocerebral lobe, 365-367 Psilocybine, 253, 255 Psychosis, body fluid indoles in, 271-284 in Hartnup’s disease, 266 “Pyramidal” cat, 70, 73, 221 Pyramidal tract, 80 Pyridine nucleotides, 268 Pyridoxal, 323, 326-328, 332-338, 341 Pyridoxal kinase, 337 Pyridoxal phosphate, 331-338 Pyridoxal semicarbazone, 332 Pyridoxamine, 321, 323, 341 Pyridoxine, 321, 323-324, 327, 330, 339-341, 343 biochemistry, 332-339 neuropathology, 339-340 seizures produced by diet low in, 340-341 Pyruvate, 323

R Receptive fields, of somatosensory neurons, 192-193, 196-200 Redox potential, in epileptic cortex, 167 Reflex, grasp, 213 Reflexes, spinal, effect of thiosemicarbazone, 327 Renshaw cells, 51, 327-328 Reserpine, 253, 284 effect on blood serotonin, 280-281 Respiration, insect, control by local brain stimulation, 366-367 Response, conditioned, patterns of cortical unit activity in, 130 corticocerebellar, 226-227 recruiting, 91, 126, 128, 218-223 recruiting, relation to unit recordings, 95-97 spreading burst, 124, 166-167 unit (see under Neurons, Spikes, and Cortex) Reticular formation, brain stem, 215-218, 224-225, 229, 241, 327 thalamic, 218-223 Retinene, 355 Rhabdome, 355 Rhabdomeres, 355 Ribose nucleic acid, of astrocytes, 44 of oligodendrocytes, 18-19 Ribosomes, of astrocytes, 7, 44 of oligodendrocytes, 18, 60

S S;lz, 297 Scape, 353-354 Schiff bases, 338 Schizophrenia, indoles in, 272, 276, 282-283

400

SUBJECT INDEX

Schwann cell, 50, 58, 340 Seizures, audiogcnic, 321-322, 328 hydrazide, 333 produced by low pyridoxine intake, 340-341 Semicarbazide, 302 f pass. Sensory hairs, of insects, 351-352 Sensory-motor areas I and 11, 191241 pass. Serine, 300, 305, 339 Serotonin, 253-258, 263, 264, 270, 271, 280-281, 283-284, 286, 339 levels in blood, 279-281 levels in urine, 277-279 Serratiu plymuthica, 306 Skatole, 259 Skatoxysulphate (see 6-Sulphatoxyskatole) Slow waves, 81-82 see also Spontancous activity and Electroencephalography comparison of cortical with spinal, 87-88 postsynaptic, 92 relation to unit activity, 89-109, 149-152, 160-165, 173-176 Sodium chloride, 30, 33, 60 Sodium ion, cquilibration of, 45 equilibration potential of, 49 Somatosensory areas, auditory functions, 200-201 evoked potentials in, 97-103 functional organization, 188-241 unit responses in, 110-117 “vertical columns” in, 115-117, 200 Spangenglobus, 358-360 Spectral sensitivity, of insect eye, 356 Sphingomyelin, 299-303, 305 Sphingomyelinnse, 303 Spike-and-wavc discharges, 151 Spike (E.E.G.), 149-152, 160-165, 181

Spikes (neuronal), 221 Spikes, A-B, 82-83 analysis of components of, 82-85, 145-14G antidromic, 81 from axons, 81-87 from Betz cells, 78, 80-84, 87, 9495, 118-119, 123-124, 141142, 146 from cortical neurons, 77 f, 141 f from “epileptic” neurons, 142-146, 149-152, 156, 163 cxtracellular, 78, 83-87 initially negative, 78, 89 initially positive, 80 of injury discharge, 88-89, 143 interspike internal, 75, 94, 11:3 intracellular, 78, 80-84, 97, 123124, 165-166 neuronal, classification of, 76-80 positive-negative, 79, 84, 88-89, 120 relation to slow waves, 89-95, 149152, 160-165, 173-176 from soma, 81-87, 120, 122, 141147, 150, 156 Spindles, 91, 93, 95, 221-222 Spontaneous activity, 77 see also Slow waves and Electroencephalography automatic analysis of, 76 microelectrode analysis of, 89-109 Spreading depression, 178-179 Stiel system, 368 Stretch receptors, crayfish, 332 insect, 352-353 Stridulation, insect, control by local brain stimulation, 367-371 Subesophageal lobe, of insect brain, 364 f pass. Subthalamus, 203 Succinylcholine, 72 6-Sulfatoxyskatole, 259, 271-276

401

SUBJECT INDEX

Superficial cortical response ( SCR),

105-107 Supraesophageal lobe, of inscct brain, 364 f pass. Synapses, role of field effects, 161-165, 176 Synaptic cleft, 51 Synaptic endings, 32, 127, 139, 147-

149, 162-163 Synaptic junction and glial processes,

50-51, 61 Synaptic noise, 79, 87 Synaptic transmission, monosynaptic cersus polysynaptic,

211-213, 215 relation to astrocytes, 50-52 relation to superficial cortical response, 105-107

T Technique, “closcd Iiead”, 69-70 wax chamber, 69-70 Thalamus, anterior nucleus, 219 centromedian nucleus, 126, 203,

219, 235, 241 dorsomedial nucleus, 203 posterior nuclei, 193-194, 201, 219 reticular nucleus, 203, 219 ventral nuclei, 83, 97, 194-196,

199, 201, 203, 222 Thioscmicarbazide, 320 f pass. Thoracic ganglia, 364, 366, 368 Toxopyrimidine, 324, 335-336 Tract, pyramidal, 103, 107, 123-124, 207-

213 see also Neurons, Pyramidal projection electrical responses of, 210-211, 214, 222-223 phylogenetic development of, 211-213 spinothalamic, 193

Transaminases, see Transamination Transamination ( transaminases ) , 258,

260, 262, 311-313, 330, 331, 337, 342 “Transfer”, transcallosal, 238-239 Transsulfuration, 338 Tributyrinase, 310, 313 Triorthocresylphosphate, 307 Tryptamine, 254-255, 259-260, 262,

263, 277-278, 281-282, 286 Tryptophan, 254, 266, 283-284, 336 metabolism, by bactcria, 258-263,

275-276 metabolism, in Hartnup’s disease,

267-269 normal metabolism, by tissues, 2.55-

258, 260-261, 286 Tryptophanase, 258 Tuliocurarine, 72

U Unit, “cut-off type, 114, 116 Unit rcsponse (see under Spikes, Neurons, and under specific anatomicaI area) Uridine diphosphogalactose, 297

v Velia currens, 364 Venom, cobra, 312 Ventral ganglia, 364 “Vertical columns” of neurons, 115-117, 129, 200 Visual area, activation of motor cortex by, 224225 histology of, 232 unit responses in, 120-122, 129 Visual mechanisms, in insects, 355-357 Voltage clamp, 83

402

SUBJECT INDEX

W Wasps, 364 \l’ater intoxication, 155 Waveform generator, Tcktronix, 75 W’eber-Fechner law, 357 Weber ratio, 357

X Xanthenuria, 321 Xanthurenic acid, 267, 332, 336 X-rays, action on blood-brain barrier, 47

Z Zinc ion, 306-307