Progress in Brain Research Volume 16 Horizons in Neuropsychopharmacology

PROGRESS I N B R A I N RESEARCH V O L U M E 16 HORIZONS I N NEUROPSYCHOPHARMACOLOGY PROGRESS IN BRAIN RESEARCH ADVIS...

Author: W.A. Himwich | J.P. Schade

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PROGRESS I N B R A I N RESEARCH V O L U M E 16 HORIZONS I N NEUROPSYCHOPHARMACOLOGY

PROGRESS IN BRAIN RESEARCH

ADVISORY BOARD W. Bargmann

H. T. Chang E. De Robertis

J . C . Eccles J. D. French

H. HydCii

J. Arieiis Kappers S . A. Sarkisov

Kiel

S haiigliai Buenos Aires Canberra Los Angeles

Goteborg Amsterdam

Moscow

.I. P. Schad@

Amsterdam

F. 0. Sclirnitt

Cambridge (Mass.)

T. Tokizane

Tokyo

H. Waelsch

New York

J. Z. Young

London

PROGRESS I N BRAIN RESEARCH V O L U M E 16

HORIZONS IN NEUROPSYCHOPHARMACOLOGY EDITED BY

W I L L I A M I N A A. H I M W I C H Galeshurg State Research Hospital, Galeshiirg, Ill. ( U.S.A.) AND

J. P. S C H A D E NetherlarirlA C m t m l InJtitiile for Brain RPxarch, Anisteurlarn (The Netherlands)

ELSEVIER PUBLISHING COMPANY AMSTERDAM

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1965

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NEW YORK

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335 J A N

V A N G A L E N S T R A A T , P.O.ROX

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AMSTERDAM

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ALL KIGHTS RESERVhD T H I S B O O K O R A N Y P A R I ' T I I E R E O F M A Y N O T BE R E P R O D U C t D I N A N Y F O R M .

I N C L U D I N G P H O I O S T A T I C O R M I C R O F I L M FORM, W I T H O U T W R l l FEN P b K M I S S I O N F R O M T H t P U B 1 I S H E R S

PRINTED I N THE NETHERLANDS

List of Contributors

APRISON, M. H., The Institute of Psychiatric Research and the Departments of Biochemistry and Psychiatry, Indiana University, Indianapolis, Ind. BERLET, H. H., Neurologische Klinik und Poliklinik, University of Goettingen, Goettingen (Germany). BOROFF,D. A., New England Institute for Medical Research, Ridgefield, Conn. BRUNE,G. G., Neurologische Universitatsklinik und Poliklinik, Hamburg (Germany) BULL,C., Butler Hospital, 333 Grotto, Providence, R.I. DI PERRI,R., Clinica Neurologica, Policlinic, Napoli (Italy). DRAVID,A. R., Institut de Chimie Biologique, FacultC de MCdecine de Strasbourg, Strasbourg (France). HAMBRECHT, T. F., Biomedical Engineering, Johns Hopkins University, Baltimore, Md . HIMWICH, H. E., Thudichum Psychiatric Research Laboratory, Galesburg State Research Hospital, Galesburg, Ill. HIMWICH,W. A., Thudichum Psychiatric Research Laboratory, Galesburg State Research Hospital, Galesburg, Ill. KNAPP,F. M., Department of Biology, Duuesne University, Pittsburgh, Pa. KOBAYASHI, T., Neuropsychiatric Research Institute, 9 I Bentencho, Shinjuku-ku, Tokyo. MORILLO, A., Laboratory of Electroencephalography and Neurophysiology, Universidad Javeriana, School of Medicine, Bogota, Colombia. MORPURGO, C., Research Laboratories, J. R. Geigy Ltd., Basle (Switzerland). POLLACK,S. L., Thudichum Psychiatric Research Laboratory, Galesburg State Research Hospital, Galesburg, Ill. PSCHEIDT, G. R., Thudichum Psychiatric Research Laboratory, Galesburg State Research Hospital, Galesburg, Ill. RINALDI, F., Neuropsychiatric Institute of the University of Naples, Naples (Italy). SAMSON, JR., F. E., Department of Comparative Biochemistry and Physiology, The University of Kansas, Lawrence, Kan. SCHMIDT, JR., H., Department of Psychiatry, Washington University, School of Medicine, St. Louis, Mo. SMYTHIES, J. R., Department of Psychological Medicine, University of Edinburgh, Edinburgh (Great Britain). STEINER, W. G., Department of Psychology, Yale University, New Haven, Conn. STONE,W. E., The Department of Physiology and the Epilepsy Research Center, University of Wisconsin Medical School, Madison, Wisc. TEWS,J. K., The Department of Physiology and the Epilepsy Research Center. University of Wisconsin Medical School, Madison, Wisc. VALCOURT, A. J., Veterans Administration Hospital, Brockton, Mass. WHITE,R. P., Department of Pharmacology, University of Tennessee Medical Units, Memphis, Tenn.

Otlrer voliritres in this series:

Volume I : Brain Meclranisttis Spect'fic and Unspecif'ic Mechanistns of Sensory Motor Integration Editcd by ci. Muruzzi, A. Fessard and H. H. Jasper '

Volume 2: Nerve, Brain and Memory Moc1eI.r Edited by Norbert Wieiicrt and J. P. Schadd

Volume 3 : The Rhinencephalon and Relaieii Slrrietirres Edited by W. Bargmann arid J. P. Schade

Volume 4: Growth and Mufuration of the Braiii Edited by D. P. Purpura and J. P. Schade

Volume 5 : Lectures on the Diencepliulon Edited by W. Bargmann and J. P. Schadd Vulume 6 : Topics in Basic Neurology Edited by W. Bargmann and J. P. SchadE

Voltrme 7: Slow Electrical Processes in the Brain by N. A. Aladjalova

Volume 8 : Biogenic Amities Editcd by Harold E. Iiimwich and Williamina A. Himwich

Volume 9: The Developing Brain Edited by Williainina A. Hiniwich and Harold E. Himwich

Volume 10: The Structure and Function of the Epiphysis Cerehri Edited by J. Ariens Kappers and J. 1'. Schadd

Volunie 11 : Organization of iiie Spinal Cord Edited by J . C. Eccles and J. P. Schadk

Volume 12: Physiology of Spinal N e w o m Edited by J. C. Eccles and 5. P. SchadB

Volume 1 3 : Mec1innistii.s of Neural Regeneration Edited by M. Singer and J . P. Schade

Volume 14: Degeneration Patterns in the Nervous System Edited by M. Singer and J. P. Schadk Volume 15: Biology of Neuroglia Edited by E. D. P. De Robertis and R. Carrea Volume 17: Cybernetics of the Nervous System Edited by Norbert Wiener? and J . P. SchadC Volume 18 : Sleep Mechanisms Edited by K. Akert, Ch. Bally and J. P. SchadC Volume 19 : Experimental Epilepsy by A. Kreindler

Volume 20: Pharmacology and Physiology of the Reiicular Formation Edited by A. V. Valdman Volume 21 : Correlative Neurosciences Edited by T . Tokizane and J. P. Schade Volume 22: Brain Reflexes Edited by E. A. Asratyan

Volume 23 : Sensory Mechanisms Edited by Y. Zotterman Volume 24: Carbon Monoxide Poisoning Edited bv H. Bow. and I. McA. Ledinnham

DR.HAROLDE. HIMWICH

Preface

The Thudichurn Psychiatric Research Laboratory was formally dedicated on October 17, 1953 almost two years after Dr. Harold E. Himwich became the director. Since then the laboratory has developed rapidly with research contributions ranging from clinical studies through precise chemical and electrophysiological measurements. The papers gathered together in this book are both the contributions of those who are calumni’ of the laboratory and of those who were members of the staff in the spring and summer of 1963. Frequent changes in scientific personnel is one of the characteristics of the laboratory. If papers were to be added from those working on the publication date of this volume at least 4 or 5 more papers would appear. The success of the laboratory has depended not only upon the genius of its director, the creativity and perseverance of the individual scientists but also upon the loyalty and devotion of the rest of the staff. It is impossible in a volume such as t h s to mention each and every technician who has contributed to the development and continuation of the laboratory. I am sure, however, that I am voicing the sentiments of all the authors when I say that certain supporting groups are so essential that they should be mentioned here. The animal house, which during most of this time was under the direction of Mr. Richard W. Bailey, was essential for much of the research reported here; the photography and medical art work required was carried out by Mr. Tenneson and his staff. The construction of equipment and maintenance of electrical equipment are due to the services of Mr. Floyd Saunders and Mr. Eugene Ginther. It goes without saying that the library and the prompt intelligent assistance of a competent librarian form an important part of any reseach program. Mrs. Yvonne Chambers has fulfilled this position admirably. We would be far from realistic if we did not attempt to discharge here a portion of our debt of gratitude to the ladies who have made up the secretarial staff during this l0-year period. Without their devoted and meticulous attention to detail, papers could not have been prepared for publication and the general administration of the laboratory could not have been conducted. I am sure that Dr. Harold Himwich, to whom this volume is dedicated would want to join us in the whole-hearted appreciation of the support that the laboratory has received from the hospital staff as a whole. In a class by themselves we should rank the understanding and the appreciation which the laboratory received from Dr. Lester H. Rudy when he was superintendent and from Dr. Thomas T. Tourlentes,

x

PREFACE

the current superintendent. Miss Florence 0. Johnson, as Business Manager and later as Assistant Superintendent, has also made valuable contributions to the administrative problems of the laboratory. The clinical research, represented in this volume largely by Dr. Bull’s paper, has depended i n the final analysis upon the whole-hearted cooperation of the nursing staff. Every portion of the clinical studies has required the active participation of this group and i n most cases of the activity therapy staff as well. The laboratory’s appreciation and gratitude also goes to all the other hospital and administrative staff both on the local and state level too numerous to mention here. For summarizing the scientific career of Harold Himwich we let the bibliography printed in the back of this volume speak for itself. J. P. SCHADB

Contents

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

V

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

TX

List of contributors Preface

Neurobiology and psychiatry J. R. Sniythies (Edinburgh, Great Britain)

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

Development of the experimental psychiatry program at the Thudichum Psychiatric Research Laboratory C. Bull (Galesburg, Ill.) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1

39

Research approaches to problems in mental illness: Brain neurohumor-enzyme systems and behavior M. H. Aprison (Indianapolis, Tnd.) . . . . . . . . . . . . . . . . . . . . . . . . . 48 Metabolism of biogenic amines and psychotropic drug effects in schizophrenic patients G. G. Brune (Hamburg, Germany) . . . . . . . . . . . . . . . . . . . . . . .

. .

81

Limited usefulness of EEG as a diagnostic aid in psychiatric cases receiving tranquilizing drug therapy W. G. Steiner and S. L. Pollack (Galesburg, Ill.) . . . . . . . . . . . . . . . . . . . 97 Behavioral changes of dogs following injection of neurotropic drugs into the arachnoid space overlying the cerebral cortex T. Kobayashi (Galesburg, Ill.) . . . . . . . . . . . . . . . . . . . . . . . . . . . 106 Antiparkinson drugs and neuroleptics C . Morpurgo (Bade, Switzerland) .

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

121

Free amino acids and related compounds in brain and other tissues: Effects of convulsant drugs J. K. Tews and W. E. Stone (Madison, Wisc.) . . . . . . . . . . . . . . . . . . . . 135 The excretion of 5-hydroxyindoleacetic acid in mental patients A. J. Valcourt (Brockton, Mass.) . . . . . . . . . . . . . Some motor and electrical signs of drug action R. P. Whitc (Memphis, Tenn.) . . . . . . .

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

164

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

169

Aspects of amino acid metabolism in phenylketonuria and other amino acidopathies H. H. Berlet (Galesburg, Ill.) . . . . . . . . . . . . . . . . . . . . . . . . Energy flow in brain F. E. Samson, Jr. (Lawrence, Kan.)

. . .

184

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

216

Direct action of atropine on the cerebral cortex of the rabbit F. Rinaldi (Naples, Italy) . . . . . . . . . . . . . . . .

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

229

Chicken brain amines: Normal levels and effect of reserpine and monoamine oxidase inhibitors G. R. Pscheidt and H. E. Hiniwich (Galesburg, Ill.) . . . . . . . . . . . . . . . . . . 245 Spinal input to the midbrain reticular formation : Pharmacological investigation A. Morillo, A. R. Dravid and R. Di Perri (Galesburg, 111.) . . . . . . . .

. . . . . . 250

CONTENTS

XI1

Bacterial neurotoxins D. A. Boroff (Philadelphia, Pa,)

. , . , , . . . . . . . . . . . . . . . . . . . . . 256

Variations in water ingestion : The response to barbiturates H. Schmidt, Jr. (St. Louis, Mo.) , , , , ,

. .. . ..

.

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

. 263

The cerebral circulation: Some hemodynamic aspects F. M. Knapp (Galesburg, Ill.) , .

. . . . . . . . . . . . . . . . . . . . . . . . . 285

Multi-channel telemetry systems F. Terry Hambrecht (Baltimore, Md.)

. . . . . . . . . . . , . . . . . . . . . . . . 297

Electrical activity of the dog’s brain: Telemetry and direct wire recording W. A. Himwich, F. M. Knapp and W. G . Steiner (Galesburg, Ill.) ..

. . . , . . . . 301 Bibliography of Harold E. Himwich. . . . . . . . . . . . . . . . . . . . . . . . . . 318 AuthorIndex , . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . , . . . 333 Subject Index . , . . . . , . . . . . . . . . . . . . . . . . . . . . , . . . . . . 343

1

Neurobiology and Psychiatry J. R. S M Y T H I E S Deparfment of Psychological Medicine, University of Edinburgh, Edinburgh (Great Britain)

INTRODUCTION

The last ten years has been a period of real advance in biological psychiatry. The causes of this have been two-fold. In the first place the haphazard and undirected researches of the previous decades have given place to research directed by specific hypotheses as to the causes of particular psychiatric illnesses. As the history of medicine from Pasteur to this day indicates progress attends most swiftly when research is directed by some hypothesis. The hypothesis itself may be based on some chance observation, such as when Fleming noticed that a mouldy patch on his agar plate was surrounded by a clear zone in which the bacteria had been killed. The observation of such trivial events becomes valuable when it becomes the basis of an hypothesis that can be tested e.g. that ‘certain moulds produce substances that can kill bacteria’. We now have some useful hypotheses in biological psychiatry. The second cause for this advance has been the creation of the proper organizations where research in biological psychiatry can be carried out. Biological psychiatry needs basic scientists in sufficient numbers - biochemists, neurophysiologists, neuropharmacologists, etc. - working on the problems of the causation and cure of psychiatric disease. Basic scientists working in basic science departments will tend to work on growing points in their own specialty, which, in most cases, will have nothing to do with psychiatry. It is, of course, always possible that some new advance in fundamental brain chemistry may lead directly to developments of interest to psychiatry. Indeed the very development of specific hypotheses in a clinical science depends on this basic work: otherwise there would be no bricks from which the specific hypotheses could be built. But in general the acquisition of these basic facts requires one type of organization and their exploitation and development in clinical science needs another type of organization. Organizations engaged in biological research in psychiatry operate in University Departments of Psychiatry, State Institutions and Bureaux, Federal and other Government supported research organizations of all kinds. Out of the vast range of phenomena which can be studied, some will represent growing points of the various sciences concerned, others will be relevant to the search for the biochemical events that determine mental illness. It is unlikely that these two will encompass by chance the same phenomena. Therefore psychiatric research institutes need to keep their own function continually in view. Their main function is not to conduct basic research but to discover the biological basis of mentalillness. References p . 38

2

J.

K. S M Y T I I I E S

Experience in the United States has clearly shown that the best type of organiLation to ensure this is the multidisclipinary team where neurochemists, pharmacologists, physiologists, psychiatrists, psychophysiologists, etc., can work together on intcrrelated problems. It is thus that the development of the specific hypotheses of the aetiology of psychiatric illricss can be furthered. Discoveries made in psychopharmacology lead immcdiately to questions that can be answered only by biochemists or neurophysiologists. Discoveries made by the latter gain significance when tested by the psychopharmacologists and so on. The building up of such a team requires a grasp of these disciplines and an ability to coordinate them. 11 was my privilege to work for one year in Dr. Harold E. Himwich's laboratory 2nd there to observe these principles in operation in one of the three leading psychiatric research laboratories in the United States. Dr. Himwichs iaboratory fulfills thcse criteria not only as to quality but also as to quantity. A laboratory should not be too sinall for obvious reasons but it should also not be too large, losing cohesion and direction of purpose in the process. Furthermore Dr. Himwich combines the rare qualities of leadership, drive, scientific acumen, grasp of organization and warm hunianily that are necessary for such an enterprise. My own research has been directed by two considerations. The first has been to develop the hypothesis of the cause of schizophrenia that I published in collaboration with Humphry Osmond and John Harley-Mason in 1952. The second has been to obtain a n undcrstanding of, and experience in, those basic sciences relevant to psychiatry. If one wishes to understand the nature of schizophrenia one must study psychiatry. In order lo apply iieurobiology it is necessary to work oneself in one or morc of its disciplines. I have worked in two - neuroanatomy and psychology as well as in biological psychiatry itself including neuroplrarmacology and I will deal with these - all of which I worked on at Galesburg - separately. ~

~

UIOLOGICAL PSYCHIATRY 111 1952 Humphry Osmond and 1 published a paper in which we drew attention to two facts. The fir51 was that there are certain marked similarities between the p5ychological effects of mescaline and the symptoms of an acute attack of schizophrenia. The second was that the chemical formula of mescaline is remarkably similar to that of adrenaline (Fig. I). We therefore suggested that the essential biochemical lesion in

0

CH2.CH,.NH,

CH,O

HO QCH2.C';.

NH,

HO

CH, 0

Fig. I . Formulae of' mescaline aiid adrenaline.

schizophrenia might be a disorder of the metabolism of adrenaline, in particular a disorder of methylation, whereby methylation o f the phenolic hydroxyl groups could occur and a substance like mescalinc ('M-subatance') would thus be produced.

NEUROBIOLOGY A N D PSYCHIATRY

3

The first criticism levelled at this theory was that the symptoms of mescaline intoxication are more like those of an acute toxic psychosis than schizophrenia. It was stated, for example, that mescaline did not induce affective blunting or catatonia and that it produced visual rather than auditory hallucinations. However, a careful study of the effects of mescaline plus a consideration of certain obvious factors enables us to make the following assessment of the situation. Mescaline does not only cause visual hallucinations but also disturbances of thinking, feeling and behaviour very similar to those seen in schizophrenia (Wolbach et al., 1962). Secondly the effects of mescaline vary very much according to the circumstances under which it is given and the personality of the subject. This enables US to see why the symptoms of schizophrenia would be different from those of an acute mescaline intoxication even if mescaline was the actual compound produced by the aberrant schizophrenic metabolism. Factors of time, chronicity, adaptation, environmental attitudes, secondary stresses and so on are clearly operative and are sufficient to counter this criticism. Having formulated this hypothesis the next step is to test it and develop it. Testing the hypothesis involves establishing the following facts : ( i ) that the abnormal metabolite or metabolic reaction occurs in schizophrenics and not in normals, or that it occurs to a greater extent in schizophrenics than it does in normals; ( i i ) the amount of the abnormal metabolite or the degree of activity of the abnormal reaction (or inactivity of a normal reaction) should vary concomitantly with the clinical state of the patient; (iii) the abnormal metabolite injected into normal people, or the abnormal reaction induced in normal people, will induce in them symptoms as similar to schizophrenia as the different environmental and other factors outlined above allow. The difficulty here of course is to decide what metabolite to look for. A great number of people have made chromatographic surveys of schizophrenic urine and reports have appeared from time to time of variously coloured abnormal spots that have not carried much conviction. Only very recently have reports appeared that an abnormal metabolite - dimethoxyphenylethylamine (Fig. 3,11) - is present in schizophrenic urine (Friedhoff and Van Winkle, 1962; Takesada et al., 1963). The former report that it is present in 79 % of schizophrenic urine and in 0 % of normal: the latter used a different and possibly more sensitive technique and reported that it is present in 92% of schizophrenic urine and in 46% of normal. This suggests that there may be quantitative differences and that a minor pathway in normal metabolism may have become a major one in schizophrenics. This question can only be resolved when quantitative methods for estimating dimethoxyphenylethylamine become available. It is of interest that this compound differs in only one detail from the original ‘M-substance’ postulated. The significance of these findings is, however, still not certain, as Friedhoff and Van Winkle point out, and requires further study, However, the possibilities for interesting developments are there. The next step then is to narrow down the field in which to search. We do this by extending our source of data by developing the specific hypotheses of the cause of the illness. The adrenaline hypothesis was soon joined by the serotonin one. This was published independently by Gaddum and by Woolley and Shaw in 1954 and was based on the anti-serotonin action of LSD. This hypothesis suggests that some disReferences p . 38

J. R. S M Y T H I E S

4

order of serotonin metabolism or action is concerned in psychosis. These two hypotheses have put serotonin and noradrenaline in the centre of the psychiatric picture both in the case of schizophrenia and, from other evidence, depression. For drugs that can induce depression (reserpine, methyl-DOPA) reduce the levels of these amines in the brain whereas drugs that alleviate depression probably raise the levels of the free form of these amines in the brain. With regard to the relationship of serotonin and schizophrenia a further important clue was provided by the fact that the hallucinogenic drugs (exclusive of sernyl and the atropine group which have in any case different clinical effects) are close chemical relatives of serotonin (as well as noradrenalinej as is shown in Fig. 2. An inspection

Serotonin i Tryptamine)

Bufotenin

PSiIOCyn

5-MethOXy - D M T

(DMJI

Fig. 2. Formulae of hallucinogenic relatives of serotonin and tryptamine.

of this figure reveals the fact that this chemical relationship is based on one common mechanism namely methylation: that is to say, the hallucinogenic drugs (mescaline, bufotenin, dimethyltryptamine (DMT), psilocyn and 5-methoxy-DMT) are O-methylated or N-methylated (or both) derivatives of the neurohumours. This fact has led to a further and more general hypothesis (of which the other two can be regarded as special instances) that schizophrenia is characterized by excess methylation. This latter hypothesis has received support from the observation by Pollin et al. (1961) and Brune and Himwich (1963) that feeding the methyl donors methionine and betaine to schizophrenics makes their symptoms worse. A further hypothesis by McIsaac (1961) is based on a similar close relationship between the neurochemical melatonin and the hallucinogenic drug harmine. Thus a simple examination of the chemical formulae of the hallucinogenic drugs and the neurohumours is sufficient to suggest four specific hypotheses (that need not be competitive but rather complementary) of the aetiology of the illness. All this, however, is at rather a low level of sophistication and these working hypotheses need to be extended by determining more facts about the mode of action of these hallucinogenic drugs. Their chemical formulae suggest useful hypotheses but no doubt better hypotheses will come from an understanding of the biochemical mechanisms in the brain on which these drugs exert their deleterious effects. How then can we discover how these agents produce their effects? This can be done,

NEUROBIOLOGY AND PSYCHIATRY

5

in part, as follows. The drug in question can be tested for its effects on various enzymes and chemical reactions known to occur in the brain, or on various neurophysiological phenomena. However, most of these drugs have more than one action and some of these actions are shared by non-psychotomimetic analogues, so it becomes necessary to distinguish between those actions that are relevant to its psychotomimetic properties and those that are merely fortuitous. This can be done by studies of structureactivity relationships (SAR) whereby close chemical relatives of the drug are synthesized and tested biochemically, physiologically and for their psychotomimetic properties. In this way it was shown that the peripheral anti-serotonin action of LSD is not relevant to its central psychotomimetic properties since this action is also shared by its psychiatrically inactive analogue Brom-LSD. Furthermore, more advanced SAR studies can, by deduction from the nature of the molecular structures involved, tell us something about the possible modes of action of the drug and the possible configuration of its effector sites. Information and hypotheses obtained and developed by these deductions can be tested by an experimental study of the actual chemical mechanism involved, and, if found to be valid, the presence of this type of aberration in this chemical mechanism can be sought in schizophrenic metabolism. The first compound that we tested on humans was trimethoxyphenyl-isopropylamine(Peretz et al., 1955) (Fig. 3, VII). This is of interest as it combines the molec-

Fig. 3. Formulae of TMA and other mescaline analogues.

ular configuration of amphetamine and mescaline. It proved to have about double the hallucinogenic potency of mescaline itself. This finding has been recently confirmed (Shulgin et al., 1961). Tests werealso made of adrenochrome but, as no double blind studies were done, the results were inconclusive. It appeared in some instances to induce psychotomimetic changes of a subtle kind but placebo reactions were not ruled References p . 38

J. R. S M Y T H I E S

6

out and some other workers were not able to confirm these claims. More recently, however, reports have appeared, using double blind controls, substantiating these claims (Groff et al., 1961). The marked instability of adrenochrome is probably in part responsible for this confusion. Then in 1959 a more extensive series was conducted at the Worcester Foundation i n collaboration with Dr. Koella and Dr. Levy. We used in the first instance the Winter and Flataker test. Ln this test rats are trained to climb a rope for food reward and the time they take is measured. The effect of drugs on this time can then be measured. The formulae of the drugs that we used are shown in Fig. 3. We compared their effect with that of mesraline (Smythies and Levy, 1960);the results are shown in TABLE 1 C O M P A R I S O N U r T W E E N T H E E F T E C T S OF MESCALINE A N D O I I I C R D R U G S

Each figure gives the mean increase in climbing time of the rats per cage in seconds over thc mean of the three initial pre-injection control runs. The effect of saline control runs is also shown. Each point is the mean of 15 readings. (Taken from Smythies and Levy, 1960) -

_

~

-

~

-

_

_

_

~

_

_

_

Minutes after injection

Experimetrt I Mescaline

TV V Experinreti/ I1

(a) Mescaline I1 Saline (b) Mescaline 11

Saline ( c ) Mescaline

IT

Saline

50 50 50

so 50

10 39 4 I92

20 130 1 248

30 I72 2 233

40 238 3 246

50 235 8 230

5

20 I73 85 2 144 232

3s 176 38

50 I86 20 8 123 I92 12 136 97 15

80 160 3 7

I20 90

1s 25 I00 __

30 60

84 203 8 70 87 18

15

I03 115 3

15

I34 228 8 120 I07 8

60 178 3 219

65

85 8 96 60 18

Table I. This table demonstrates that the effect of removing the 5-methoxy group from mescaline is to reduce activity, as measured by this test, by some 50%. The replacement of the 4-methoxy group by a hydroxyl group abolishes all activity and its replacement by the heavier phenoxy group increases activity. Our second investigation was based on developing a valid neuropharmacological test for the action of mescaline on the electrical activity of the brain (Smythies et nE., 1960). We chose the optic evoked potential in the visual cortex of the unanaesthetized rabbit and used a quantitative statistically controlled means of evaluation of the evoked potential. The form of the potential is shown in Fig. 4. Note the large primary response and the smaller following waves. We injected the mescaline intravenously at

_

_

~

NEUROBIOLOGY A N D PSYCHIATRY

7

Fig. 4. Twenty superimposed sweeps, control series. Note waves I, 11, 111 and I V as well as smaller subsequent waves. Calibration: 200 pV and 25 msec. In all figures negativity is recorded upwards. (Taken from Smythies et al., 1960.)

four doses 5, 10, 20, and 40 mg/kg and then measured the mean % change from the control (which consisted of the mean of the 20 potentials evoked just before the injections). The potentials were sampled at intervals in groups of 10 or 20 for the next two hours. The results are shown in Figs. 5-7. These figures demonstrate that a small

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0 1 2 3"1020304050

A 0 1 2 3 "10 20 3 0 4 0 50

Fig. 5. Ordinate: the percentage change in the amplitude of wave I with standard' deviations. Abscissa: time after injection, in min. Solid line: mean change with drug; dotted line: mean change with saline controls. Upper left graph, 40 mg/kg; upper right, 20 mg/kg; lower left, 10 mg/kg; lower right, 5 mg/kg. Mean of 6 experiments at each dose level. Note that the horizontal dotted line in each figure is the 100% reference line. The dotted saline control curve for 6 experiments is given in the same graph as the 40 mg/kg dose curve. (Taken from Smythies et al., 1960). ReJerences p . 38

8

J. R. S M Y T H I E S

dose of mescaline potentiates wave I and that a larger dose first inhibits it and then potentiates it - a biphasic effect that turns up quite frequently in studies of mescaline action. The effect on subsequent waves is qualitatively similar except that the inhibitory component becomes progressively more marked the later the wave, appearing 200 190 180.

no.

I

160. 150. lA0 130. 120. 110 100.

90. 80. 701

60.

A

A

0 1 2 3”10M3ILLI550d30120

Fig. 6. Coordinates same as Fig. 5 for wave 11. Upper lcft graph, mean of 4 experiments at 40 nig/kg (in 2 experiments the wave was below threshold amplitude in the initial control run); upper right, 6 experiments at 20 rng/kg; lower left, 6 experiments at 10 ingikg, and 3 experiments with saline control (dotted curve in upper left graph). (Taken from Smythies et al., 1960). 130

120. 110. 100-

90807060.

140

A 0 1 2 360 20 30 4 0 5 0

Fig. 7 . Coordinates same as Fig. 5 for waves 111 and IV combined. Upper left graph, mean of 10 experiments a t 40 mg/kg, upper right, 9 experiments at 20 mg/kg; lower left, 9 experiments at 10 mg/kg: lower right 10 experiments at 5 nig/kg, and 1 I with saline control (dotted curve in upper left graph). (Taken from Smythies et al., 1960.)

NEUROBIOLOGY A N D PSYCHIATRY

9

at smaller dosage levels and in the case of waves 111 and IV practically swamping the potentiating effect which, at the 3 higher dosage levels, is reduced to a small hump on the downward curve. Fig. 8 gives examples of the actual wave forms obtained.

Fig. 8. Early and late effects of mescaline on evoked response. (A.) Control, the 6 sweeps immediately before injection of the drug. Note the 4 waves: a large primary response, small wave 11, large and constant wave 111 and small wave IV. Read from bottom up. (B) The same experiment as (A). Sweeps 2 to 7 following the end of the i.v. injection of mescaline (20 mg/kg). Note the diminution in amplitude of all waves and the increase in latency. (C) Another animal. Control sweeps 6 to 1 1 . Note the variable form of the primary response and of waves 11, I11 and IV. (D) The same experiment as (C). Three minutes following the end of the injection (20 mg/kg). Note the potentiation of waves I and I1 and the diminution of waves 111 and IV. Note also the great increase in stability of the wave form. Calibration: 300 pV and 25 msec. (From Smythies et al., 1960.)

Fig. 9 shows that a good dose response curve can be obtained on a basis of the degree of potentiation of mescaline on the amplitude of wave I. Thus a suitable measure of one effect of mescaline has been obtained. The next step is to determine how specific this is for mescaline, or to what extent it is shared by other drugs such as amphetamine and other hallucinogens such as LSD. Comparative studies of this kind are absolutely necessary before we can assign any significance to results of any such investigation. A further stage is to study the interaction of drugs (such as giving mescaline after reserpine to see if its effects depend in any way upon brain levels of amines, and so on). I conducted one such experiment at Galesburg (Smythies. 1959a). R#frrrnrrr n 7R

10

J. R. S M Y T H I E S

Fig, 9. Dose-response curve for the potentiating effect of mescaline on amplitude of wave I. Ordinate: mean "/o change during the 3rd min after injection. Abscissa: dose of mescaline in nig/kg. Standard deviations shown as vertical bars. Straight line fitted by method of least squares. (Taken froin Smythies et a!., 1960.)

A simple behavioural test was used. The time that a mouse took to travel a certain distance on a pole was measured. LSD (1-2 mg s.c.) increased this time as is shown in Fig. 10. Adrenolutin produced little effect by itself but it significantly reduced

T

140 T

P FOR DIFFERLNCE

ELTWEEN ( L S D )

( L S D t ADRENDLUTIN).

AND

anw.mN

0 . 0 5 AND 0.02

u 0

5

10

15

20

26

30

Time after injection (min )

Fig. 10. Showing the effect of lysergic acid diethylamide (LSD), adrenolutin and their interaction. Cages A-D, each point representing 40 mice: 0 = saline; ++ = LSD (1 mg/kg); A = adrenolutin (20 mg/kg); 0 LSD adrenolutin. (Taken from Smythies, 1959a.) 1

+

the effect of LSD when both were given together ( p < 0.05). This effect was confirmed in another experiment where the mice climbed the pole under the influence of hunger drive ( p < 0.05). There are a number of possible mechanisms for this effect. BOL also slowed down the satiated mice (it appeared to have a sedative effect) but it had no effect on hungry mice. The interaction between BOL and adrenolutin was not studied. Whilst studying under Prof, Zangwill at Cambridge previous to this, I collaborated with Dr. Harley-Mason and Dr. Laird in a study of the metabolism of mescaline in

11

NEUROBIOLOGY A N D PSYCHIATRY

the normal human subject (Harley-Mason et al., 1958). It was determined that some 35 % of ingested mescaline is excreted unchanged, a small proportion is converted to 3-methoxy-4,5-dihydroxyphenylethylamine (Fig. 3, 111) (a close relative to metanephrine) and the rest was unaccounted for. Currently at Edinburgh we are engaged in a programme using automated behavioural techniques (Smythies and Sykes, 1964). The method used in this study was a conditioned avoidance situation in a Levine shuttle box. The rats were trained t o avoid shock by crossing to the other half of the shuttle box on hearing the buzzer. Reports in the literature on the effect of mescaline on the conditioned-avoidance response (CAR) were conflicting, so our first aim was to determine what this effect in fact is. We used one dose (25 mg/kg) and our experimental design was to give a saline control run, followed the next day by a mescaline trial and the day after by a second saline run. Two weeks later the same cycle was repeated. Each run consisted of 7 sets of 20 stimulus presentations randomly spaced and occupying eight minutes. Five minutes time-out separated each set. The drug was given by intraperitoneal injection:between the second and third set. The number of shocks received and the reaction times (between the onset of the buzzer stimulus and the crossing of the rat to the other half of the shuttle box) were measured. In each case the results are expressed as the differences between the mean mescaline and the mean saline scores. In this design, series, etc. effects are compensated for. Each point on the graphs represents 1080 presentations of the stimulus. The statistical method used was, Wilcoxon’s non-parametric ranking method for paired replicates. Fig. 11 shows that the overall effect of the mescaline is to increase the reaction time and the number of shocks received, followed by a period of decreased reaction time. MESCALINE

M-S

25 M G

/

KG

SHOCKS

9

,

0.8

-SHOCKS

.--t..RT 06

04

* * * *

*

*P
P < 0.05

0.2

0

*

-0.2

-4 3

1 -0.4

2

3

4

s

6

7

TIME

Fig. 11. Graphs showing the effect of 25 mg/kg mescaline i.p. upon the reaction times and number of shocks for 9 rats in the first series, expressed in mescaline-saline (M-S) score. Ordinate (left): number of shocks. Ordinate (right): reaction time in mms (recording speed : 25 mms per min). Abscissa (bottom): time in runs of 20 trials. Line through 0: mean saline score. References p . 38

12

J. R. S M Y T H I E S

Qualitatively the animals under mescaline appeared confused at first and this was followed by a period of increased excitability. There were not at this dosage any signs of paralysis. However there were marked individual differences between different animal$. Figs. 12 and 13 show three rats with widely different reactions to mescaline. Insome the confusional effect is dominant and in others the 'excitatory' effect. Fig. 14 shows a typical record from rat 9. The kymograph ran only during the stimulus response period. The shock trace shows the number of shocks received. The trial trace shows the number of stimulus presentations and the reaction times. Comparison of the mescaline run with the saline controls shows that the number of shocks

NUMBER

M-5

OF SHOCKS

MESCALINE

25 M G I K G

14 12 10

0 6

4 2

0 2

, 1

2

3

4

5

6

_ 7

TIME

Fig. 12. Graphs showing individual differences, expressed in M -S scores, i n the number of shocks received by three animals at a dose of 25 mg/kg mescaline i.p. Ordinate: number of shocks. Abscissa (bottom): time in runs of 20 trials. Line through 0: mean saline scores.

REACTION TIME

M-5

MESCALINE 2 5 M G I I G

I T

2221 08-

I6

RAT

-

-16

LS

-1s

LS

o--bo 22

14 2 -'

1008-

Ob0402-

0-

0 20406. 00'

.

1

,

2

1

3

4

5

.

7

TIME

Fig. 13. Graphs showing individual differences, expressed in M-S scores, in the reaction times of three animals at a dose of 25 mg/kg mescaline i.p. Ordinate: reaction time in mms (recording speed = 25 m m s per min). Abscissa (bottom): time in runs of 20 trials. Line through 0: mean saline scores.

13

NEUROBIOLOGY A N D PSYCHIATRY

received is increased. The reaction time is also increased for a short period after the injection but this gives way to a decreased reaction time. SHOCK TRIAL

MESCALINE HCL ANIMAL 9 0

Fig. 14. A sample record showing the effect of 25 mg/kg mescaline upon one animal’s response. Saline I: saline control on Day 1. Mescaline HCI: mescaline hydrochloride (25 mg/kg i.p.) on Day 2. Saline 11: saline control on Day 4. Inj.: time of injection. Shock: number of shocks. Trial: period of time during which buzzer sounded. SHOCKS

MESCALINE

RT

2 5 M G / K G (M-5)

X-4-4

M I SHOCKS

C--+-O . .

M 2 SHOCKS

%--X---XMI

RT

O--Q--QMZ

RT

P < 0-01(SHOCKS) P.M.

-4

(RT)

I

J 1

2

3

4

5

6

7

Fig. 15 Comparison between the first injection of mescaline and the second, for animals in Series I (N = 9) expressed in M-S scores; interval between injections (25 mg/kg i.p.) is two weeks. Ordinate (left): number of shocks. Ordinate (right): reaction time in mnis (recording speed = 25 mms per min). Abscissa (bottom): time in runs of 20 trials. Line through 0: mean saline scores. References p . 38

J . R. S M Y T H I E S

14

L 51

X

AL

K TRIAL SH(

: TRIAL

TI

+ INJ 4

SALINE

+

50MGIKG

SALINE

IOOMGIKG

TRIMETHOXYPHENYLALANI"

I

ANIMAL 9 0

Fig. 16. A sainplc record showing the effect o f m-trimethoxyphenylalanine (50 and 100 mg/kg) upon one animal's response (cf. Fig. 2). Saline I: saline control on Day 1. Trimethoxyphenylalanine: 50 mg/kg on Day 2 and 100 nig/kg i p. on Day I I . Saline 11: saline control on Day 12. Inj.: time of injection. Shock: number of shocks. Trial: period of timc during which b u u e r sounded (i.e. reaction time).

Fig. 15 shows that a tolerance effect is to be found even if two weeks are left between thc dose$ of mescaline. The rats received fewer shocks (compared with their respective saline controls) during the second mescaline run than during the first. Thus we have shown that mescaline i n a dose of 25 mg/kg definitely depresses the CAR as measured both by the number of shocks received and the reaction time. The effect is howcvcr biphasic as it is followed by a period of decreased reaction time. At present we are studying the effect of different doses of mescaline and using other behavioural techniques. We will then use these methods to effect structure-activity studies of mescaline analogues. We have already tested one such compound - the amino acid derivativc of mescaline (trimethoxyphenylalanine, Fig. 3, VI). Amines in general do not pass the blood-brain barrier easily whereas their amino acid derivatives do (cf. 5HTP and 5HT; DOPA and dopamine). So one would expect that the corresponding mescaline analogue would be more active than mescaline itself. However, Fig. 16 shows that even i n a dose of 100 mg/kg it has no effect on rat behaviour. Therefore mescaline is clearly different from the other amines in this regard. Work along these lines is proceeding. N E U KO A N A T O M Y

For anyone -,;hing to study the mode of action of the nervous system it sound knowledge of the anatomy of the brain is of first importance. 1 therefore devoted some 18 months to this end at the University of British Columbia.

NEUKOBlOLOGY A N D PSYCHIATRY

15

The problem was to determine if silver staining methods can tell us anything about the form of the synapses in the human brain. These methods had been used extensively in the cord, where various types of synapse had been described, but little work had been done in the brain and what work there was took the form of a few haphazard observations. The main difficulties here are ( i ) the silver stain (we used the Rio-Hortega method) is necessarily selective and will not stain all synapses but possibly only certain kinds ; (ii)human brains subjected to anatomical study have necessarily undergone various periods of post-mortem autolysis and this might be expected to affect such delicate structures as synapses. In particular it is well known that degenerating axon terminals (following axonal section) are easier to stain with silver than normal ones. Tn our paper (Smythies et al., 1957) we described small objects that we called ‘boutons’ in the human brain. This term now requires qualification, particularly in the light of work carried out later at Galesburg (Smythies and Tnman, 1960). We observed two types of ‘bouton’ in the human cerebrum ( I ) a free globular or ringshaped form without any axon visible going to it, widely distributed in the brain but without any apparent connection with nerve cell bodies (as seen in Fig. 18) and (2) bodies closely applied in large numbers to the large motor cells in the globus pallidus. These were quite unlike the former having a peculiar fibrillary and granular appearance (Fig. 24). They were very like the terminals seen around the anterior horn cells in the spinal cord and certainly seem to be synaptic in function since preterminal axons can be seen going to them. They were, however, seen nowhere else in the brain besides the globus pallidus. Type ( I ) ‘boutons’ may represent a number of different things: (a) axon terminals whose preterminal fibres lack the necessary constituents to stain with silver; or (b) products of degeneration of axon terminals: i.e. the actual terminals may not stain with silver but following autolysis they may undergo biochemical changes that enable them or their breakdown fragments to stain with silver; or (c) some structures in the brain not connected with axon terminals although they just happen to look very much like them; or (d) some breakdown products of (c). Such bodies cannot be seen at all in dog brain that is fixed by perfusion during life (Smythies and Inman, 1960). If postmortem autolysis is allowed to occur such bodies appear progressively the longer the period of autolysis (from 1 to 48 h : as described in more detail later). In the following account the term ‘bouton’ will refer to these small darkly stained globular or ring-shaped objects. Their size varied from 0.8-4 ,u. They may represent particular types of axon terminal or possibly some form of degenerate axon terminal (or even of some other brain structure). However, as will be apparent, the pattern of form and distribution in different parts of the brain are quite regular and constant and thus we can with certainty conclude the following : different regions of the brain are specified by diflerent biochemical (or some such) specificities such that, when autolysis occurs, small objects are either produced or rendered visible with the properties outlined above (or it is of course also possible in human brain that some of these would have been visible in any case) - and these become observable in greater quantities in some parts of the brain than in others and also their morphology is difReferences p . 38

16

J. R. S M Y T H I E S

ferent in an orderly way in different parts of the brain. Hence these patterns of ‘bouton’ distribution reported here may reflect biochemical and/or structural differences between the regions of the brain concerned. The term ‘bouton’ is used in this report with these qualifications and is not meant to imply that these are necessarily boutons terminaux. The further significance of these patterns of distri bution and morphology can only be ascertained (if at all) by such means as the electron microscope T A B L E I1 T H E DISTRIBUTION OF BOUTONS TERMINAUX I N TIIE M O T O R A N D

SENSORY

ISOCORTEX

(Taken from Smythies, 1955)

_

_

~

_

_ ~~

Cvtoar cliitecfuta1 area Cortical layer

I

2

3

4

5

6

7

8

0

2 4 29 4 0 0

0 I 41 3 2

I 8 43 10

0

25 I

0 5 25 6

0 4 70 3

0 I80

200

0 6 84 0 6 2 210

4 208

26

28

35

36

~

1 2 3 4 5

6 Fields counted Bouton per unit volume

1

1

1

0 158

I 190

158

2 180

13

15

18

23

4

1

46 9 7 I

10

I = Visual koniocortex (area 17), NC2. 2 = Visual parakoniocortex (area 18), NC2. 3 = Parietal koniocortex, NC2.4 = Parietal parakoniocortex, NC2. 5 = Temporal koniocortex, NC2.6 = Temporal parakoniocortex, NC2. 7 Agranular cortex (area 6), NCI. 8 = Agranular cortex var. gigantopyramidalis (area 4), NC2. :

or histochemistry. These may show some histochemical differences or differences in ultrastructure that can be correlated with our findings using silver stains. However, as we studied three human brains and found essentially the same pattern of bouton distribution and morphology in each, it is improbable that these are artefacts of any kind or have no structural or functional significance. The boutons were counted by quantitative methods suited to particular regions which are described in detail in our original publication. Our findings may be summarized as follows: (Z) In isocortex the boutons are concentrated almost exclusively in layer 3. Tables I1 and I11 give details for sensory, motor, eulaminate and dysgranular isocortex. (Each field has a n approximate volume of fixed brain of 0.00008 mm3: each ‘unit volume’ is 100 fields.) Furthermore there is a distinct gradient with the primary sensory cortex containing the least number, then motor cortex, and then association cortex. (2) In allocortex the boutons are concentrated in layer 5 (Table IV) and in juxtallocortex, intermediate between isocortex and allocortex the main concentration is in layer 3 with a second smaller concentration in layer 5 (Table V).

17

NEUROBIOLOGY A N D PSYCHIATRY

T A B L E 111 T H E D I S T R I B U T I O N OF B O U T O N S T E R M I N A U X I N T H E

EULAMLNATE A N D D Y S G R A N U L A R I S O C O R T E X

(Taken from Smythies, 1955) Cytoarchitectural area Cortical layer -

-

9 _~ ~ _ _ ~_

-

~

1 2 3 4

3 78

5

6

6

~~

1

6

10 ____

0 2

11

12

13

0

0 6 196 2

15 20 1

____ _

14 ~

0 12 22 1

15 ~

16

0

7 430 24 5 2

11

5

2

11 4

14 10

0 12 259 6 29 17

176

192

182

190

209

202

85

91

96

100

131

169

4 182

101 2

0

_

5

2

4 0

5 0

Fields counicd

170

I 60

Bouton per unit volumc

43

51

5

9 = Parietal eulaminate cortex (area 7), NC2. 10 = Temporal eulaminate cortex (area 21), NC2. 11 = Eulaminate cortex from occipito-temporal gyrus (area 36), NCI. 12 = Dysgranular cortex (area 8), NC1. 13 Frontal eulaminate cortex (area lo), NC2. 14 = Eulaminate cortex from lateral orbital gyrus, NC2. 15 = Temporal eulaminate cortex (area 38), NC2. 16 = Frontal eulaminate 1

cortex (area 9), NCl.

T A B L E IV P H E DISTRIBUTION OF B O U T O N S T E R M I N A U X I N THE ALLOCORTEX,

NCI

(Taken from Smythies, 1955) -__

Location

Total

Cortical layer 1 2 3 _________~________

0 1

0 0

2 0 2 1

Fields counted Bouton per unit volume

1 2 3 4 5

6

4

Bouton per 100 fields

0 4

%

3 5 101 7

0 2 12

0 0 19

3 I I6 16

2 140 14

13 290 18

0 1 4 4 86 5

164

21 8

222

238

-

-

3

41

53

62

-

-

14

I = Allocortex from posterior entorhinal area ( 3 mm from iso-allocortical junction [i-aj)). 2 = Allocortex from intermediate entorhinal area (1 mm from i-aj). 3 = Allocortex from intermediate entorhinal area (5 mm from i-aj). 4 = Allocortex from anterior entorhinal area (3 mm from i-aj). Rqterrncrs p . 38

I. R. S M Y T H I E S

18

TABLE V T H F D I S T R I B U T I O Nor B O U T O N S T E I C M I N A U XI N T H E

JUXTALLOCORTtX AND MESOCORTFX

(Tdken from Smythies, 1955) -_-__-_-__--________ __ C~vtoarchitecturatarea _______________ Cortical layer ____ 17 18 I9 20 21 22 - __ _____ ___ ~

1 2 3 4 5

0 11 1415

0

0

8 I30 7

IS 1292 22 31 4

24 I898 8 29 * 5

Fields counted

21 9

154

168

Routon per unit volume

548

765

1 I42

6

0 8 3568 12 96

0 5 432 3 33

10

1

240

236

168

460

1397

24 3

0 17 1183 4

150 20

17 -- Juxtallocortex from posterior part of hippocampal gyrus (6 n m from iso-allocortical junction (i-aj)), NCI. 18 - Juxtallocortex from antcrior part of hippocampal gyrus (4 mm from i-aj), NCl. 19 - Mesocortex from area 24 just above the genu of corpus callosum in dcpths of callosal sulcus, NCI. 20 = As 19, from crest of gyrus, NCI. 21 = Parolfactory area, NC2. 22 = Mcsocortex from posterior part of area 24, N C I . * Layer 5 here is very thin.

The juxtallocortex had a high concentration of boutons, particularly in the parolfactory area where there were more than 100 times as many boutons as in the corresponding layer of the primary visual cortex. The form of the boutons in all these regions was mainly small, clear rings. In the cinguiate gyrus however they tended to be larger and darker. Figs. 17 and I8 show typical fields from isocortex and the cingulate gyrus respectively. We also examined various subcortical structures amongst which were the following: ( 3 ) Amygdala: This nucleus contains a complex pattern of bouton distribution illustrated i n Figs. 19-21. Each number gives the number of boutons in 10 fields (using the oil immersion lens) counted on a grid at intervals of I mm. The lateral nucleus contains large numbers of boutons particularly in the small-celled region and the posterior part. Morphologically these resembled those in the cingulate gyrus. The basal nucleus contained very few except for a moderate accumulation at the extreme anterior part of the small-celled region. The other nuclei contained moderate numbers. ( 4 ) Hypothalamus: In this the ventro-medial nucleus contained the most (up to 30 per 10 fields) followed by the lateral nucleus and medial mammillary nucleus. The paraventricular and supraoptic nuclei contained very few. The ventro-medial nucleus contained a high proportion of a peculiar fibrillary type of bouton. (5) Hippocampus: This also shows a complex and consistent pattern of bouton distribution (Figs. 22, 23). The main features of interest are: The anterior part of the

NEUROBIOLOGY AND PSYCHIATRY

19

Fig. 17. Three boutons teriiiinaux in the parietal eulaniinate cortex (layer 3) of brain NC2. x 2600. (Taken from Smythies, 1955.)

presubiculum contains very many small boutons as does the stratum radiatum of the cornu ammonis and the molecular layer of the dentate gyrus. The latter are remarkable for being the smallest and most delicate seen anywhere by us in the cerebrum. The References p . 38

20

J. K. S M Y T H I E S

subiculum and stratum lucidum of the lateral part of the cornu ammonis contain, in contrast, many large and densely staining boutoris of the ‘cingulate’ type.

Fig. 18. Boutons terminaux around a nerve cell in the anterior part of the cingulate gyrus (layer 3) of the brain NCI. x 1760. (Taken from Smythies, 1955.)

Fig. 19. For legend

see p. 21.

NEUROBIOLOGY A N D PSYCHIATRY

21

Fig. 20.

Fig. 21. Figs. 19, 20, 21. Maps of the amygdaloid complex of the brain NC1 showing the complex patterns of bouton distribution. Fig. 19 (13 p ) shows an anterior section; Fig. 20 (12 p) an intermediate section; and Fig. 21 (17,u) a posterior section. Abbreviations: A.A.A. =- anterior amygdaloid area; A.B.N. = accessory basal nucleus; A.C. = allocortex; B.N. = basal nucleus; C.A. = cortico-amygdaloid transition area; C.N. = cortical nucleus; Ce.N. = central nucleus; d.c. = densocellular part; g. = glomerular cell portion (of superficial part of basal nucleus); g.c. = gracile cell part; L.N. = lateral nucleus; L.P. = pyriform lobe; L.V. = lateral ventricle; M. = medial nucleus; mag. c. = large-celled portion; med. c. = medium-celled portion; p.c. = small-celled portion; PPC. = prepyriform cortex; py = pyramidal-celled portion (of superficial part of basal nucleus); S. = striated area; S.B. = superficial part of basal nucleus. (Taken from Smythies, 1955.)

(6) Thalamus: There are very few boutons in most of the thalamic nuclei. The only exceptions are the parafascicular nucleus, which contains a moderate number of large, dark and irregular boutons, and the lateral geniculate body which contains many, large, dark and regularly-shaped boutons evenly distributed throughout the six layers. References p . 38

22

J . R. S M Y T H I E S

Fig. 22. Map of the anterior part of the hippocampus showing the patterns of bouton distribution (13 p ) . Abbreviations: A. = alveus; Ac. - allocortex; C.A. = cornu ammonis; CAUD. = tail of caudate nucleus; D. = dentate gyrus; g. - granular layer; L.V. - lateral ventricle; m. = molecular layer; P.C. = layer of polymorph cells; PARAS. - parasubiculum; PRES. = presubiculum; S. = subiculum; S1.= prosubiculum; S.L. = stratum lucidum and layer of polymorph cells; S.R. = stratum radiatum; a. = closely packed cells of S.L.; b. = loosely packed cells of S.L.; c. = polymorph cells. (Taken from Srnythies, 1955.)

No boutons were ever seen by us in the medial pulvinar nucleus. (7)Basal nuclei: No boutons were seen by us in the caudate nucleus and only very few in the putamen. The internal division of the globus pallidus, on the other hand, shows an interesting collection of different types of bouton: the first type is concentrated in large clear rings near the distal portion of the thick apical dendrites of the large pallidal motor neurones. The second type of bouton (Figs. 24, 25) is of a peculiar fibrillary and granular appearance and is clustered thickly on the surface of the bodies of the large pallidal motor neurones and the proximal portions of their apical dendrites, where the boutons look very like those seen on the motorneurones of the spinal cord. The third type is very rare. These boutons present a club-shaped appearance with their base closely applied to the surface of the apical dendrite (Fig. 26). In the globus pallidus it was also possible to see and photograph bodies looking exactly like the large dark ‘cingulate-type’ boutons, that we have demonstrated elsewhere in the brain, in visible continuity with preterminal axons (Figs. 27-30). This supports the hypothesis that similar bodies seen elsewhere in the brain arealso axon terminals

N E U R O B I O L O G Y AND P S Y C H I A T R Y

23

or some derivatives of axon terminals, whose preterminal fibres, however, cannot be stained by this method. In the substantia nigra the boutons are numerous and often very large (up to 3 x 4 p ) . They seem to be concentrated in areas away from the pigmented cells and in cell free regions or around the large non-pigmented cells. Thus to summarize, the following features characterize different brain regions: (1) ‘Boutons’, as described and interpreted above, are concentrated in layer 3 ofthe isocortex, layer 5 of the allocortex and in both layers (although 3 > 5) of the juxtallocortex. The juxtallocortex contains many more boutons than other cortical regions. (2) Within the isocortex there is a distinct gradient in bouton concentration, namely primary sensory cortex < motor cortex < ‘association’ cortex.

Fig. 23. Map of the posterior part of the hippocampus (16 p). Abbreviations: C.A. = cornu ammonk; D. = dentate gyrus; J.A. = juxtallocortex; m. = molecular 1ayer;O.T. = occipito-tempora gyrus; P.C. = layer of polymorph cells; PARAS. = parasubiculum; PRES. = presubiculum; S. = subiculum; S . l , prosubiculum; S.L. = stratum lucidum and layer of polymorph cells; S.R. = stratum radiatum. (Taken from Smythies, 1955.) 2

(3) The boutons in the cingulate gyrus have a characteristic morphology. (4) The amygdala and other nuclear masses contain constant and varied patterns of bouton distribution and morphology. In this connection it is of interest that the anterior part of the cingulate gyrus, the lateral nucleus of the amygdala and the presubiculum each present very similar pictures with numerous, large and darkly staining boutons. This suggests that these areas of the rhinencephalon may have some factor in common. This is contrasted with the paucity of boutons inthe basalamygdaReferences p 38

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Fig. 24. Boutons terininaux of the second type around a large motor cell and its apical dendrite in the internal division of the globus pallidus of the brain NC I . X 21 00. (Taken froin Smythies, 1955.)

loid nucleus, most of the thalamus and the corpus striatum. The globus pallidus and substantia nigra present their own characteristic appearances. More recently 1 collaborated in a study at Galesburg with Dr. Inman to see if we could determine whether post-mortem autolysis played any role in this as we suspected (Smythies and Inman, 1960). The brains of six dogs were used in this study.

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In one the whole brain of the living anaesthetized dog was perfused with saline, followed by formol saline and removed. In the second one half of the brain was

I

Fig. 25. Types of boutons from the internal division of the globus pallidus of the brain NCl. The second drawing from the right in the top row shows a large dendrite cut transversely surrounded with nine granular fibrillary boutons of the second type. (Taken from Smythies, 1955.)

removed at operation and the remainder perfused. The portion removed was fixed after 1 h. In three other dogs the brains were fixed at varying intervals after death (I, 24,48 and 72 h); storage was at room temperature. Sections were cut from different parts of the brain and stained by the same methods that had been used in our human studies. In the case of all sections fixed during life there was a good neuropil but no boutons

Fig. 26. Two adjacent dendrites in the internal division of the globus pallidus of the brain NCl with a variety of boutons. Note particularly the primitive bell-shaped bouton. (Taken from Smythies, 1955.)

at all were seen (Fig. 3 I )After 1 h the fibres in the neuropil had become somewhat irregular and a few ‘boutons’ were visible, clearly connected to preterminal fibres arounda few of the Betz cells but nowhere else (Fig. 32). After 24 h (Fig. 33) the neuropil had almost completely fragmented or was not stainable and many ‘boutons’ were seen closely packed around the large pyramidal cells of the motor cortex, a few in the sensory cortex but none at the frontal or occipital poles. After 48 h (Fig. 34) this process had References p . 38

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Fig. 27. Boutons terminaux of the second type on an apical dendrife of a similar cell as is shown in Fig. 24. The fibrillary and granular structure is well seen. Brain NC1. x 2100. (Taken from Smythies,

1955.)

extended even further. Boutons were now plentiful even in the sensory regions and a few were seen at the poles. After 72 h the brain was too softened for adequate sections to be cut. In the three human brains that we studied, autolysis had proceeded in 4 h in one

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case and for about 24 h in the other two. There were no discernible differences between the three brains but the 24 h cadavers had of course been stored under chilled

Fig. 28. A bouton terminal in continuity with a preterniinal fibre in the internal division of the globus pallidus of the brain NCI. x 2000. (Taken from Smythies, 1955.)

conditions. So one can conclude that our findings in the human brains probably reflect differences in bouton structure and function (in terms of stainability by silver and to some extent liability to autolytic changes and details thereof) as well possibly of other structures. At least these studies suggest that correlations between our findings and electron microscopic studies and histochemical studies of these same regions be sought. The regularity and constancy of these patterns of ‘bouton’ distribution and morphology suggest that they can be used to give specific information about brain structure and function particularly if they can be so correlated with data obtained with other techniques. Further silver staining is not of much use to this end, as one is unable to determine to what degree the stain is selective in its action and so to what extent a genuine morphology is revealed. As a start electron microscopy of the cingulate gyrus or the parolfactory area and the primary sensory cortex might reveal similar differences to the gross ones that we have described here. PSYCHOLOGY

Before I went to Dr. Himwich’s laboratory I spent two years at Cambridge in the References p. 38

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Fig. 29. As Fig. 28. x 2000. Other typical boutons (of the first type) can be seen in Figs. 29 and 30. The middle part of the preterminal fibre is out of focus. (Taken from Smythies, 1955.)

Psychology Laboratory with Prof. Zangwill. Some years previous to this, whilst working at the National Hospital, Queen Square with Dr. Cobb I became interested in the peculiar visual hallucinations or images induced by looking at a stroboscopic lamp.

Fig. 30. A bouton terminal in continuity with a preterniinal fibre (the niiddle part of which is out of focus) in the internal division of the globus pallidus of the brain NCI. x 2000.

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Very little attention had been paid to these patterns and there was no detailed or systematic description of them in the literature. Grey Walter had produced the interesting hypothesis (1 950) that they represented interference by the intermittent stimulus with a scanning television-like mechanism. Brown and Gebhard (1948), using monocular stimulation, were the first to observe that this stimulus will evoke two quite different kinds of visual phenomenon, which they called the ‘bright phase’ and the ‘dark phase’. The former consists of mainly geometrical patterns; the latter consisted in their case of ‘a writhiqg mass of violet flecks against a yellow-green background’. They brought forward the hypothesis that the dark phase patterns are a function of the neurones related to the closed eye. In my investigation (Smythies 1959b, c, 1960) I used 35 normal subjects, all skilled observers, who were given the task of describing the phenomena evoked by the stimulus, which was provided by an electronic stroboscope and also by a beam of light interrupted by a rotating episcotister under certain conditions set out in detail in these papers. The results were essentially as follows, dealing first with the dark phase patterns. These differed from the bright phase patterns in that they were only seen with single eye stimulation and the form and movement of the patterns were quite different: the bright phase patterns were predominantly geometrical and flickered whereas the dark phase patterns showed no trace of geometrical form and swirled around rather than flickering. Various types of dark phase patterns were described as follows: Amorphous. This consisted of two colours usually a red and a green, which swirled around in a very characteristic fashion described as like ‘oil on the surface of water’ or ‘boiling mud or porridge’. Particulate. Here the same oily swirling motion obtained but small objects swirled instead of sheets of colour. They were variously described as ‘bacteria’, ‘pond-life’, ‘powder on a liquid surface’, etc. Stationary patterns. These were of various kinds - paisley patterns, marble, inkblots, a map, a leaf pattern, etc. These were usually themselves stationary but were characteristically covered with a vivid appearance described as clear rippling water. Designs. A curious variety seen by 3 subjects were of an ornamental quality and were described as being like Victorian wall paper or modern abstract designs. Scenes. Four subjects reported scenes or visual hallucinations proper in which fully formed objects were observed. In some the same scene repeated itself and in some there was a constant stream of different images. In the former group one subject reported a shoal of fish in an aquarium and another clumps of grass and leaves as if the observer were looking through a gap in a hedge. The latter group reported a constant stream of common-place scenes - as one subject described it, like a number of scenes in a badly cut film. The dark phase patterns were in general much more clear-cut and definite than the bright phase ones. The subjects found them very interesting and even fascinating. The dark phase alternated with the bright phase in a manner very reminiscent of retinal rivalry. In some further quantitative experiments I noted that the dark phase patterns are potentiated (as expressed by the amount of time they were observable) References p . 38

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Fig. 31. A Betz cell in the cortex of a brain perfused during life ( x 3000). (Taken from Smythies and Inman, 1960.) Fig. 32. A Betz cell in a brain that has undergone 1 h post-mortem autolysis ( X 3000). (Taken from Smythies and Inman, 1960.) Fig. 33. The apical dendrites of two Betz cells in a brain after 24 h post-mortem autolysis. Portions of other cells and a fragmenting neuropil are also seen. Some putrefactive bacteria are seen in the lower part of the figure ( x 3000). (Taken from Smythies and Inman, 1960.) Fig. 34. A pyramidal cell together with a fragmenting neuropil in layer 5 of the motor cortex of brain after 48 h post-mortem autolysis ( x 3000). (Taken from Smythies and Inman, 1960.)

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by shining a dim light into the eye that was not subjected to the stroboscopic srimulation and they were inhibited by shining a bright light into this eye. This inhibition was still in evidence some 30-90 sec after the stroboscopic illumination had been switched off. This provides further evidence that the dark phase patterns arise in the neurones connected to the shut eye. The bright phase patterns also showed a great variety of form. For any one subject the general features of the patterns that he experiences will remain quite constant for years although there are wide differences between different subjects. One’s stroboscopic patterns are as individual as one’s EEG pattern. Some subjects see only one or two major patterns, some a large number of constantly changing ones and other people are intermediate between these two. Patterns following light and dark adaptation of the eyes will also be different. The patterns themselves fall into various categories. The most interesting fact was that in the vast majority of cases each pattern was composed entirely of straight lines or of curved lines. A pattern made up of straight and curved lines together was hardly ever seen. The common straight line patterns were grouped as follows : (1) a cross, star figure, cobweb and snow-flake; (2) a grid, square(s) or rectangle(s), diamond(s), chess board and many parallel lines; ( 3 ) herringbone pattern, zig-zag lines, honeycomb and a mosaic. The common curved-line patterns were : (1) circle(s), ellipse(s), parabola(s), a spiral, a vortex, hyperbola(s); (2) sine waves, a ‘magnetic field’; (3) a flower pattern, a finger-print pattern. Some of these patterns are shown in Figs. 35-46. This fact suggests that the difference between a straight line and a curved line might be fundamental to the cerebral patterns of pattern analysis. These patterns moved in various ways characteristic of each pattern. For example wavy lines were subject to linear translations; concentric circles showed centrifugal or centripetal motions; a star figure would rock from side t o side; a point would oscillate violently to and fro; elements of a mosaic pattern would creep relative to each other; various topological stretchings might occur, as if the whole pattern were on a piece of rubber, as well as three-dimensional rotations of the whole pattern. One type of pattern, namely many thin dark vertical lines, was only obtained with uniocular stimulation. There were often different patterns in the periphery of the field to those in the centre. A common arrangement was to have a vortex rotating in the centre of the field with a mosaic or herringbone pattern in the periphery. There were also clear-cut differences at different frequencies of stimulation (range 4-24 flashes per sec). The pattern at higher frequencies were finer and made up of smaller elements than those obtained with slower frequencies. There were no consistent differences between the patterns obtained with the electronic stroboscope and the episcotister. Indeed the same patterns can be obtained merely by looking up at the sun with eyes closed and passing the extended fingers of one hand rapidly to and fro in front of one’s face. Any intermittent stimulus will evoke patterns that appear to be characteristic for that individual. This mode of stimulation also resulted in interesting after-images. With binocular stimulation the after-image was usually different from the bright phase pattern immediately preceding. Some of these after-images were remarkable for their reguRefererrces p . 38

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Figs. 35-40 are photographs of pastel drawings made by the subjccts immediately after observation. (Taken from Smythies, 1958.) Fig. 35. Star figure made of herringbone. T.P. subject 3. Colour: black and white. Fig. 36. Banded circles figurc. D.P. subject 3. Colour: black and white. Fig. 37. Star figure. T.P.subject 9. Colours: yellow, red and black on grey. Fig. 38. Diffraction pattern. Subject 1 1 . Colours: brown on yellow. Fig. 39. Spiral with peripheral mosaic. D.P. from subject 8. Colours: blue on yellow. h g . 40. Scallop pattern. D.P. subject 14. Colours: red and blue on yellow.

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Figs. 41-46 are photographs of pastel drawings made by the subjects immediately after observation. (Taken from Smythies, 1958.) Fig. 41. Thin blue line figure. I.P. subject 1 . Colours: blue on white. Fig. 42. Diamond spiral. D.P. subject 13. Colours: spiral, yellow; dots, blue o n Lery pale grey. Fig. 43. Mazeimosaic. D.P. subject 35. Colours: blue, yellow (at center), black, olive-green and grey on pale grey. Fig. 44. Vertical line pattern. I.P. with OR, subject 32. Colours: black and purple on white. Fig. 45. Wedge pattern. 1.P. with OR. subject 14. Colours: black on white. Fig. 46. A family of parabolas superimposed on a grid from a subject outside the series. Colours: parabolas, yellow and black; grid, brown and purple on pale grey. References p . 38

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48

49

51

Figs. 47-52. Line drawings copied by the author from line drawings by the subjects made immediately after observation. Types of binocular after images. Subjects 26, 18, 26, 14,1,17, respectively. Colours: Fig. 48, red and green in pairs on black; Fig. 49, crimson, yellow and black; Fig. 51, Various: black and green, purple and green, black and white, brown and green, black, white and yellow; Fig. 52: green on black. (Taken from Smythies, 1958.)

larity and complexity. Examples are illustrated in Figs. 47-52. After uniocular stimulation, however, another type of after-image was common, described as ‘spaghetti’, ‘filigree’, or ‘worms’ and illustrated in Figs. 53-58. These usually occurred in those subjects in whom dark phase patterns occurred (i.e. about half of our subjects).

The bright phase patterns A number of hypotheses have been put forward to account for the stroboscopic patterns. The problem that we have to solve is why, when we look at a uniform field under intermittent illumination, one does not merely see the stimulus but sees instead these complex and interesting patterns. Thejirst hypothesis. The explanation usually put forward by early workers is that these patterns represent entoptically visualised retinal structures. Any radial system of lines could be ascribed to the retinal blood vessels; hexagonal patterns to the pigment cells of the retina; central diamonds to the macula pigment, etc. However,

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these patterns are mainly too regular and geometrical to be biological structures and moreover they may be observed to rotate in a regular fashion and to execute other gross movements which precludes their being retinal structures. The only pattern met with in this investigation that merited suchanexplanation was the central blob or disc. This was particularly prominent in blue light and might have been the entoptic visualisation of the yellow macular pigment. One could not be certain about this as, on occasion, a bright yellow central disc could be obtained in yellow light. Nevertheless such an origin for this pattern could not be eliminated and thus all central blobs or discs were excluded from the pattern counts. The second hypothesis. This was first put forward at some length by Grey Walter (1950). He represents the patterns as interference phenomena produced in a scanning mechanism attempting to deal with an intermittent signal and equates them with the patterns that can be produced on the screen of a television set by illuminating the studio with an intermittent light. He makes Lhe significant point that the form of any such patterns will be determined by the particular form of the scanning raster (the arrangement of scanning beams used to build up the final picture) employed. Now clearly, if this hypothesis is true, the physiologically important information of the form of the actual raster employed by the physiological ‘scanning’ mechanism

53

54

56

Figs. 53-58. Types of uniocular after-images of the ‘spaghetti’ kind. Same subjects as Figs. 47-52, except for Fig. 57 (subject 15) and Fig. 55, which corresponds to the verbal description of subject 2 and not to a drawing. (Taken from Smythies, 1958.) References p . 38

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can be obtained by the study of these patterns. For example, a linear scan might be expected to give rise to patterns of segments of straight lines, horizontally oriented in the case of a horizontal scan or vertically oriented in the case of a vertical scan. Similary, a polar scan would give rise to patterns compounded of segments of curved lines and a radial scan (of the type commonly used in radar) would give rise to patterns compounded of radially arranged lines. One observation and two series of my experiments are relevant to this hypothesis. The observation is that the types of pattern found do seem to fall into the three classes suggested above from a consideration of the commonly used rasters i.e. patterns of class 3, 4 and 6 lend themselves readily to a derivation from a linear, radial and polar scan respectively. Class 5 patterns could possibly arise from a linear scan also as, for example, herringbone patterns are a common aberration seen on ordinary television screens. This correlation may, of course, merely be a coincidence but it does suggest that a study of such interference patterns obtainable with the various types of scan might be of interest to a communication engineer in this context. The / h i d hypothesis. This was put forward by Barlow (personal communication) as follows: ‘the flickering field evokes very vigorous activity in the visual pathways, but the stimulus is almost totally unfamiliar to the subject. I n communicating his sensations the subject has to refer to causes of his sensations which are ordinary and familiar, since these are what his vocabulary is adapted to. The prevalence of moving lines and contours in the subject’s descriptions might be explained along these lines, since these are the commonly occurring, simple stimuli which will cause vigorous and sustained activity in the ‘on’ and ‘off’ units of the retina, but it must be admitted that there are other, specific features of the descriptions which are not accounted for’. Thus the functional ‘on’ and ‘off’ elements of the retina are normally stimulated by contours of darkness and light - by pattern - crossing the retina during the ceaseless movements of the eyeball and environment. Therefore it may be supposed that the brain interprets activity in the fibres coming from these retinal elements in terms of pattern. The intermittent means of illumination here employed may be supposed to generate a good deal of fairly regular activity in the on-off cells, fibres and their associated analysing mechanisms, Thus the aberrant form of the perceptions induced by this mode of stimulation might give us information about the manner of functioning of these mechanisms. Normally the retinal mechanisms code, and to some extent analyse, the information received a t the retinal surface. This coded information is transmitted to the cortex and is there decoded and analysed to yield the final perception of the object. Thus the aberrant perceptions represented by the stroboscopic patterns might arise from faulty coding, transmission or analysis (or some combination of these) of information presented in this case in a form with which the perceptual mechanisms are not designed to handle, A regular grid, a whirlpool, a family of parabolas, arising out of a simple stimulus of a spatially uniform field under conditions of temporally intermittent illumination, may reflect some fundamental specification of the visual mechanism. One way of thinking about this might be as follows. The pattern analysing mechanisms of the brain are here presented with information - with coded signals - of a

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totally unfamiliar kind. They receive a powerful intimation of pattern but without any particular pattern being specified. The brain then makes ‘hypotheses’ and the everchanging play of patterns may represent the various ‘hypotheses’ tried and discarded in turn. Against this, however, is the fact that the mechanisms never seem tolearn their error. The unsatisfactory ‘hypotheses’ continue to be produced in spite of this continued failure and in spite of the conscious knowledge of thesubject that he is really seeing only a flickering light. The analysis may, however, be conducted at levels of the nervous system not amenable to conscious control. But this hypothesis does not explain why some people’s patterns should be so stable and unchanging, unless we suppose that the selection of pattern to fit the stimulus is not entirely random but is modified to some extent by the functional characteristics of the analysing mechanisms to give a bias, to a greater or lesser extent in different people, to certain kinds of pattern. I t may then be possible to deduce these functional characteristics from the form and behaviour of the patterns. But the particular deductions can only be made by someone familiar with the detailed operation of telecommunication mechanisms. However, it is possible at this stage to draw attention to what may be important clues for this. These are ( i ) the fact that the patterns are not compounded of straight and curved lines as connected elements. This links up to some degree with one feature of one of Deutsch‘s (1955) models of cortical mechanisms, which he states would experience some difficulty in distinguishing straight and curved lines. This feature of the stroboscopic patterns suggests that the brain handles straight and curved lines differently. ( i i ) The effect on the patterns of dark and light adaptation; (iii) the effect of suddenly doubling and halving the flash frequency; ( i v ) the difference between the initial and the developed patterns; (v) the fact that the parallel vertical line pattern is obtained only with uniocular stimulation and the other constant features of the phenomenon described. Thefourth hypotlzesis. This was suggested by Donaldson (personal communication) in the form that these patterns represent the formation of corresponding domains among retinal and/or cortical neurones along the lines suggested by Cragg and Temperley (1954). Barlow notes that the lateral inhibition in the retina makes it likely that the conditions are right for domain formation, and the differential spread of excitation and inhibition around neurones may lead to complex pattern formation. One can imagine that each successive wave of excitation from the retina subjected to this intermittent stimulation may interact in the cortex to form complex ‘wave’ patterns much as the vibrations of a bow drawn across the edge of a tray leads to complex patterns in sand laid on the tray. Lastly we can note that these stroboscopic patterns are very similar to the form constants described by Kliiver (1942) as typical of the initial stage of the mescaline hallucinosis. Thus we can end this essay, as we began, with mescaline. In addition the effect of the stroboscopic stimulation potentiates the effect of mescaline and hallucinations of the mescaline type may be induced by its use with a dose of mescaline (200 mg) that would not ordinarily induce them.

References p . 38

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REFERENCES BROWN,C. R.,AND GEBHARD, J. W., (1948); Visual field articulation in the absence of spatial stimulus gradients. J . exp. Psychol., 38, 188-200. BRUNE, G. G., A N D HIMWICH, H. E., (1963); Biogenic amines and behavior in schizophrenic patients. Recent Adv. biol. Psychiat. In the press. CRAGG,B. G., AND TEMPERLEY, H.N.V., (1954); The organisation of neurones: a co-operative analogy. J. Electroenceph. clin. Neurophysiol., 6,85-92. DEUTSCH, J. A., (1955); A theory of shape recognition. Brit. J . Psychol., 46, 30-37. FRIEDHOFF, A. J., AND VANWINKLE, E., (1962); The characteristics of amines found in the urine of schizophrenic patients. J . nerv. ment. Dis., 135, 550-555. GADDUM, J. H., (1954); Drugs antagonistic to 5-hydroxytryptamine. Ciba Foundation Symposium on Hypertension. G. E. W. Wolstenholme and M. P. Cameron, Editors. London, Churchill (p. 75). GREYWALTER, W., (1950); Features in the electrophysiology of mental mechanisms. Perspectives in Neuropsychiatry. D. Richter, Editor. London, Lewis (pp. 67-94). GROFF,s., VOJTECHOVSKY, M.,VITEK, v . , AND FRANKOVA, s., (1961); Clinical and experimental study of central effects of adrenochrome. Abstracts 111. World Congress of Psychiatry. Montreal, (p. 210). HARLEY-MASON, J., LAIRD,A., AND SMYTHIES, J. R., (1958); The metabolism of mescaline in the human. Confin. Neurol. (Basel), 18,152-155. KLUVEK, H., (1942); Mechanisms of Hallucinations. Stua'ies in Personality. New York, McGraw-Hill. MCISAAC, W. M., (1961); A histochemical concept of mental disease. Postgrad. Med., 30, 111-118. OSMOND, H., AND SMYTHIES, J.R.,(1952); Schizophrenia. A new approach. J , ment. Sci., 98,309-315. PBRETZ, D., SMYTHIES, J. R., AND GIBSON, W. C., (1955); A new hallucinogen. Trimethoxyphenylb-aminopropane. J. ment. Sci., 101, 317-329. POLLIN, W., CARDON, P. V. JR., AND KETY,S. S . , (1961); EKects of amino acid feedings in schizophrenic patients treated with iproniazid. Science, 133, 104-105. SHULGIN, A. T., BUNNELL, S., AND SARGENT, T., (1961); The psychotoniimetic properties of 3, 4, 5-trimethoxyamphetaminc. Nature, 189, 1011-1012. SMYTHIES, J . R., (1955); The Morphology of the Synapses in the Human Cerebrum. Thesis, University of Cambridge. SMYTHIES, J. R., (1958); A Study of the Subjective Stroboscopic Patterns. Thesis, University of Cambridge. SMYTHIES, J. R., (1959a); Quantitative measurement of the effect of lysergic acid diethylamide on mice and its interactions with othcr drugs. Nature, 183, 545-546. SMYTHIES, J. R., (1959b); The stroboscopic patterns. I. The dark phase. Brit. J. Psychol., 50,106-1 16. SMYTHIES, J. R.,(1959c); The stroboscopic patterns. 11. The phenomenology of the bright phase and after-images. Brit. J . Psychol., 50,305-324. SMYTHIES, J. R., (1960); The stroboscopic patterns. 111. Further experiments and discussion. Brit. J . Psychol., 51, 247-255. SMYTHIES, J. R., (1963); Schizophrenia. Chemistry, Metabolism and Treatment. Springfield, Thomas. SMYTHIES, J. R., GIBSON, W. C., AND PURKIS, V. A., (1957); The distribution and morphology of boutons terminaux. J . comp. Neurol., 108, 173-224. SMYTHIES, J. R., AND INMAN, 0. R., (1960); The effect of post-mortem autolysis on synaptic terminals in cerebral cortex of dog. J. Anat., 94, 241-243. SMYTHIES, J. R., KOELLA, W. P., A N D LEVY,C. K., (1960); The effect of mescaline on the optic evoked potentials in the unanesthetized rabbit. J. Pharmacol. Exp. Ther., 129,462-470. SMYTHIES, J. R., AND LEVY,C. K., (1960); The comparative psychopharmacology of some mescaline analogues. J . ment. Sci., 106,531-536. SMYTHIES, J. R., AND SYKES,E., (1964); The effect of mescaline upon the conditioned avoidance response in the rat. Psychopharmacologia, 6 , 163-172. TAKESADA, M., KAKIMOTO, Y., SANO,I., and KANEKO, Z., (1963); 3,4-Dimethoxyphenylethylamine and other amines in the urine of schizophrenic patients. Nature, 199,203-204. WOLBACH, JR.,A . B., ISBELL, H., and MINER, E. J., (1962); Cross tolerance between mescaline and LSD25 with a comparison of the mescaline and LSD reactions. Psychopharmacologia (Bed.), 3, 1-14. WOOLLEY, D. W., AND SHAW,E., (1954); A biochemical and pharmacological suggestion about certain mental disorders. Proc. nut. Acad. Sci., 40,228-231.

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Development of the Experimental Psychiatry Program at the Thudichum Psychiatric Research Laboratory CHRISTOPHER B U L L Thudichum Psychiatric Research Laboratory, Galesburg State Research Hospital, Galesburg, Ill. ( U . S . A . )

The clinical service under the direction of the laboratory consists of 4 wards. Two of these house selected chronic or semichronic patients, 30 male and 30 female, mostly with a diagnosis of schizophrenia. No special dietary control is provided for these patients, since the wards were organized mainly for evaluating the effects of various medications on behavior. Control of diet and provision for collection of urine specimens is available on another ward with a population varying from 10 to 20 schizophrenic patients. Of these patients 12 are subjects for a longitudinal aging study which has now been in progress for 9 years. The fourth ward houses 13 patients with a moderate degree of mental deficiency who are not psychotic and who frequently serve as ‘controls’ for the schizophrenic patients on the metabolic ward, and are also subjects of the longitudinal study of the aging process. The patient population of the research wards has been fairly stable so that their usual behavior is well known and any changes can be readily recognized. New patients can be easily transferred from the rest of the hospital wards if circumstances require it, or studies can be organized on other types of wards in cooperation with regular clinical staff of the hospital. The medical personnel that take part in most of the studies are also involved in laboratory research; there having been no full-time clinical staff employed by the laboratory during most of the program. The experimental psychiatry program as an integral part of the laboratory service has developed rather recently, mainly since 1956. From 1955 until 1957 several of the tranquilizers, stimulants and antidepressants and similar psychoactive drugs were tested or compared on wards assigned to the research service. The drugs evaluated had passed the preliminary testing stage and most of them had been introduced on the commercial market. The approach was a conventional clinical one regarding effectiveness. In 1956 there was a change in emphasis to determining the excretion of urinary indoleamines in schizophrenic patients. This direction t o the studies developed from an interest in animal experiments on the role of serotonin and of catecholamines in brain function and on the effect of drugs such as the Rauwolfia alkaloids and the monoamine oxidase inhibitors on the physiology and behavior of animals. In 1956 and again in 1959 studies were made of the excretion of 5-hydroxyindoleacetic acid (5-HIAA) and of the effect of reserpine on urinary tryptamine and indole-3-acetic ReJerencm p. 46/47

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acid excretion in mental defectives and schizophrenics. The release of serotonin from body sites by reserpine had been evidenced by a drop in serotonin content in brain, blood and intestine in animals and in blood of man. It had also been demonstrated by an increase in the urinary excretion of 5-hydroxyindoleacetic acid in animals, using massive doses of reserpine. However, up to the time of the study initiated by this laboratory, no experiments with humans had been reported dealing with the effect of reserpine on urinary excretion of 5-HIAA. I n this study reserpine, 2 mg twice daily, was administered to a group composed of schizophrenic patients and highgrade mentally defective patients (Valcourt, 1959). The excretion of 5-HIAA was increased on the first day of medication in both groups of patients, but fell after the first day of medication and, after the cessation of reserpine medication, was reduced to prernedication levels or lower. These data gave further evidence in human beings that serotonin was released by the action of reserpine. This investigation was followed by others correlating the behavior of schizophrenic patients and urinary indoleamines. As an example a study (Brune and Himwich, 1960b) was undertaken on the metabolic ward to ascertain whether or not reserpine because of its obvious chemical similarity to 5-hydroxytryptamine had any effect on the urinary excretion of tryptamine and, the tryptamine derivative, indole-3-acetic acid. During the period of reserpine administration and collection of specimens, changes in mental condition of the patients were scored for comparison with the biochemical findings, The subjects consisted of 4 male mental defectives, 1 of whom was also psychotic, 9 schizophrenics and 2 boys with phenylpyruvic oligophrenia. All patients except the 2 boys received a constant protein diet, and were interviewed every day. As long as the patients did not show any symptoms of activation of their psychosis, all values for tryptamine were at normal levels. In those patients who experienced activation of psychotic behavior an elevation of urinary tryptamine was found. In most patients a slight increase in the excretion of total indole-3-acetic acid also occurred. ln 5 of the 7 episodes the exacerbations occurred under the influence of reserpine, but this relationship also occurred in two patients during a placebo period. It was felt that the reserpine acted by facilitating intrinsic abnormal metabolic changes. Since results of this investigation indicated a close association between tryptamine excretion and behavior, further studies were undertaken (Brune and Himwich, 1961a, b, 1962a) to ascertain whether or not such a correlation held between behavior and 5-hydroxyindoleacetic acid and indole-3-acetic acid. Another objective of these studies was to determine the effects of the combined administration of reserpine and isocarboxazid on both behavior and the urinary elimination of indoles in man. In all instances observed, disturbances of behavior were associated with urinary increases of the indole metabolites. It was suggested then that both drugs, reserpine and isocarboxazid have biphasic effects, inducing either tranquilization or behavioral disturbances, the first being associated with a moderate increase of free serotonin and the latter with a larger increase in that indoleamine in the brain. Attempts were then made (Brune and Pscheidt, 1961; Brune et al., 1963c) to confirm with more patients the effects found previously. It again became clear that

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in the individual patients, there were increased excretion levels with increase in psychotic symptomatology. Sometimes 5-hydroxyindoleacetic acid was increased more than indole-3-acetic acid and sometimes the reverse was true. Tryptamine, however, was consistently elevated in these episodes and therefore was considered the most sensitive and reliable indicator of variations of urinary indole excretion. These individual differences suggested that different pathways of indole metabolism were affected to a differing degree in individual patients. The described alterations of indole metabolism during states of increased psychotic activity were observed, whether the psychosis was associated with either elevated or reduced motor activity. For this reason the observed increases in the excretion of these indole derivatives could not be related to the state of motor activity. No correlation could be established between anxiety and urinary excretion of indoles. In some patients, who suffered a sudden flare-up of the psychosis an increase in urinary indoles occurred before clinical symptoms became overt. This pattern suggested that the alterations were not a secondary function of the psychosis. If activation of the psychosis was accompanied by marked anxiety, there was also an increase in urinary total catechols. This rise, however, in contrast to that seen in the indoles, occurred simultaneously with the appearance of the clinical symptoms. There were several clinical drug evaluations during this period but except for studies of the effectiveness of 2 new phenothiazines, triflupromazine and trifluoperazine (Himwich et al., 1959; Rinaldi et al., 1959), they also reflected the interest of the group in the brain indoleamines. These investigations included a study of a serotonin antagonist, a comparison of 2 monoamine oxidase inhibitors, and an evaluation of a serotonin analog. The serotonin antagonist, Pathcol, synthesized by Wooley and Shaw was given to acutely disturbed schizophrenic patients (Vassiliou et al., 1961). The drug had been synthesized in hopes of obtaining information regarding the effects of altering the serotonin level in the brain on behavior in man, Wooley having postulated that an excess of serotonin was present in the brain in acute psychotic states. The drug had exerted tranquilizing effects in laboratory animals and had been well tolerated in preliminary trials with non-psychotic human beings. Vassiliou et al. noted moderate, temporary improvements in the form of better emotional control and better organization of thought processes. Since there was no direct evidence that Pathcol enters the brain, it was recognized that the results of the study could not be used to either substantiate or refute the hypothesis that serotonin in the brain contributed to acute psychotic states. In the work involving monoamine oxidase inhibitors (Vassiliou and Himwich, 1961) 30 clinically depressed patients were given a battery of psychological tests while half received isocarboxazid and the other half, nialamide. The psychologist was unaware of the expected action of the drug and consequently selected a variety of tests in contrast to the usual situation where only depressive symptoms are studied. It was found that there were no significant differences between the two groups and that both drugs lowered the performance of the subjects i n tests involving functions referable to higher mental processes and psychomotor coordination. References p. 46/47

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A benzyl analog of serotonin, BAS, had been tried clinically as a treatment for hypertension. It had been synthesized by Wooley to counteract the effect of serotonin on the blood vessels. The rationale for its use was the possibility that reserpine as a somewhat similar indole was effective in hypertension by displacing serotonin from its receptor sites, and that a serotonin analog might have similar action. BAS was effective in reducing hypertension, but showed in addition a strong sedative or tranquilizing action. Because of this action and because of the interest in the possible role of serotonin in mental disease, a clinical trial with moderately disturbed psychotic patients was arranged with BAS (Rudy et al., 1958). Some striking improvements in thinking, speech and behavior occurred in some of the patients, and 22 out of the 24 were tranquillized. However, the side effects which included a feeling of weakness and fatigue or ataxia made the drug impractical for clinical use although of interest from a theoretical point of view. In addition, JB 8035, a piperazinoglycolate, was tested for therapeutic effectiveness (Tourlentes et al., 1960), since it appeared to antagonize some of the psychotomimetic properties of a series of anticholinergic agents, the piperidinoglycolates. This substance was found in studies with rats to reduce muscle tension, increase motor activity and increase sensitivity to external stimuli, and to decrease performance time in maze learning and conditioned avoidance tasks. Psychological tests detected improved performance in a group of 32 chronic schizophrenic patients although clinical observation failed to show parallel improvement, the patients appearing more disturbed and withdrawn. The observation on the metabolic ward of correlations between behavior and the level of urinary indoleamines as influenced by reserpine, which also produces clinical extrapyramidal symptoms led to a study (Brune et al., 1962) to determine the effect on the mental state of drug-induced extrapyramidal reactions. Trifluoperazine instead of reserpine was used. Other workers had reported that extrapyramidal reactions were necessary in order to achieve maximal therapeutic success, but there was lack of agreement as to what degree of change extrapyramidal functioning was associated with the most clinical improvement. The behavior of the patients was recorded as extrapyramidal reactions developed under administration of trifluoperazine and also as these reactions were ameliorated by the antiparkinson agent trihexyphenidyl. It was concluded that, in general, the optimal improvement of behavior during neuroleptic treatment was associated with very slight extrapyramidal symptoms especially those that were manifested only in handwriting changes. The study was repeated in similar fashion (Brune et al., 1962) using a trifluoperazine amobarbital combination. Similar conclusions were reached regarding the optimal amount of extrapyramidal involvement. The conclusion, concurring with that of other workers, was regarded as a satisfactory goal in itself, with little interest being taken in the particular drug’s effectiveness in treating schizophrenia. The shift in interest from particular drugs to general principles that tended to coincide with the interests of the non-clinical work of the laboratory was also demonstrated in a study, in 1962 and 1963 (Bull and Berlet, 1964) of the effectiveness of a monoamine oxidase inhibitor in altering the extrapyramidal symptoms of patients

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who had been treated with a phenothiazine or reserpine. Since postmortem studies of brains of patients with Parkinsonism had shown a depletion of dopamine, noradrenaline and serotonin, it was reasoned that there might be a causal relationship between these levels and the occurrence of the symptoms of Parkinsonism. Naturally occurring cases of Parkinsonism and cases produced by the treatment with trifluoperazine or with reserpine were given the monoamine oxidase inhibitor, isocarboxazid, in an attempt to reverse the depletion of the brain amines and so change the severity of the extrapyramidal symptoms. The organic patients improved, the reserpine-induced symptoms showed a trend toward improvement but the trifluoperazine-induced symptoms did not improve. Again, the hypothetical relationship between a response and the level of biogenic amines as influenced by the reserpine and the M A 0 inhibitor was considered to be of greater interest than any immediate practical clinical application. Similarly, a study (unpublished) on one volunteer of the effect on depressive symptoms of supplementing the supposed rise in brain biogenic amines due to M A 0 inhibitor by the administration of the serotonin precursor tryptophan, was also approached from the point of view of a metabolic rather than strictly therapeutic study. A detailed study of the one patient was considered of greater interest to the laboratory at that time than a larger clinical evaluation of the use of tryptophan as a treatment method. A study in 1961 and 1962 of the effect of methionine loading on the behavior of schizophrenic patients, conducted on the metabolic ward was another example of the shift of interest to combining biochemical work of the laboratory with the psychiatric studies on the wards, and of studying problems and compounds of theoretical interest. Laboratory staff members had become interested in the view that aberrations of indole metabolism might serve as an associated pathogenic factor in schizophrenic psychosis. This interest was based on the discovery of hallucinogenic indole derivatives, on the reports that urinary excretions of indoles in schizophrenics differ quantitatively or qualitatively from those of normal subjects, and on the observation that psychotropic drugs used in the treatment of mentally ill patients affect indoleamines in the brain of animals. In one experiment (Brune and Himwich, 1962b) 9 schizophrenic patients were given 20-g or 40-g (70/kg of body weight) doses of DL-methionine after prernedication with isocarboxazid. The results of this study confirmed findings by other workers that a majority of the patients treated with a combination of methionine and isocarboxazid showed symptoms of intoxication with euphoria, somnolence, disorientation and confusion, and with larger doses had symptoms of a recurrence or accentuation of their individual psychopathological symptoms. The interest in the effect of indoles on behavior continued with a subsequent study in ‘loading’ with tryptophan, in which an accentuation of pre-existing psychopathology was noted in 2 patients but not in 5 remaining patients who were in either full or partial remission (Brune and Himwich, 1963). It was felt that since loading experiments with amino acids other than tryptophan and methionine did not produce similar behavioral changes, these two might be involved together in a common mechanism. Perhaps indoles derived from tryptophan and an elevated level of methyl groups donated by methionine might facilitate the References p. 46/47

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formation of N,N-dimethylated indoleamines which are potent psychotogenic substances. Therefore a clinical study (Brune and Himwich, 1963) on the possible role of the methyl group of methionine in producing the behavioral changes wasundertaken utilizing the methyl group donor, betaine, as a substitute for methionine in loading experiments. Administration of betaine evoked behavioral changes similar to those observed during the lower dose of methionine (20 g/70 kg body weight) although they appeared more slowly, and were associated with an elevation of mood. To obtain further information on the question of the formation of psychoactive methylated compounds in schizophrenic patients, urinary indoleamines were studied by paper chromatographic techniques. It had been reported (Fischer ef al., 1961) that a bufotenin-like spot was found in some hallucinating schizophrenic patients which did not occur in non-schizophrenics. In our study (Brune and Himwich. 1963) urinary excretions of indoleamines were determined, using two-dimensional paper chromatography, in schizophrenic patients receiving betaine in combination with a monoamine oxidase inhibitor. In 3 of 4 patients, spots resembling those of urea, serotonin, tryptamine and bufotenin were noted. It was felt that results of these studies were compatible with the view that endogenously formed psychoactive amines may be more than casually involved in the psychotic behavior of schizophrenics, but it was recognized that further investigation was needed in many areas before a conclusion would be reached. In 1962 as it became even more evident how important dietary factors were in the study of the metabolism of indoles, a research dietician was added to the laboratory staff to plan and administer more controlled diets for the patients on the metabolic ward. At the present time the studies of the urinary indoles, catechols and other substances continue under improved nutritional conditions with measured intake, analysis of the left over food and so forth. In a current investigation the dietary intake of indoles is being limited producing a diminished output of indoles, to determine whether behavioral changes result and whether variations in tryptamine excretion accompany variations in psychotic activity (Berlet et al., 1964, 1965). In order to perhaps get a clearer understanding ofwhether the variation oftryptamine excretion merely accompanies the behavioral change or whether alterations of indole metabolism are more basically related to the cause of the behavioral change, the intake of methionine and tryptophan is being systematically varied and the concurrent behavior of the patients noted. Another continuing clinical project has been a longitudinal study of the aging process, with various measurements being made repeatedly at least once a year for a prolonged period, hopefully for the life of the individual. The study involving 25 patients (ages 40 to 64), schizophrenic and high grade mental defectives, was started in 1954. Liver function tests, blood cholesterol, nitrogenous substances, blood sugar and A/G ratios, urinalysis, hematological studies, EKG, and bone densities are done. In addition physical measurements such as body dimensions, strength, blood pressure, heart rate, vital capacity, visual acuity and visual fields, hearing, and psychological tests are obtained at regular intervals. As yet, clear cut trends have not been established. A fairly frequent ‘turnover’ among the medical personnel who take part in the

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clinical programs of the laboratory has been the result of the reputation of the laboratory for providing basic training for people working there for a limited period. This frequent change of personnel has had the effect perhaps of stimulating a wide variety of projects while at the same time it has made long-term studies and follow-up studies less practical or impossible. Because of the laboratory’s interest in the biological, chemical and pharmacological aspects of mental disorder, the personnel taking part in clinical work have stressed this approach in their observations of patients. To conclude, there has been a gradual evolution of the present program over a period of 8 years with the clinical program steadily reflecting more closely the program of the laboratory as a whole. SUMMARY

This laboratory has four wards under its direction, one of which is organized with its own kitchen, for metabolic studies and one of which houses moderate grade mental defectives to serve as controls for metabolic studies regarding schizophrenia. From 1955 to 1957 the major clinical research concerned the testing of various commercial psychoactive drugs. Since 1956 there has been persistent interest in the study of urinary indoleamines and catecholamines and their relationship to variations in schizophrenic behavior, whether spontaneous or in response to monoamine oxidase inhibitors or reserpine. Exacerbations of schizophrenic behavior, whether spontaneous or following administration of these psychoactive drugs, were found to result in an increased excretion of the indole derivatives 5-hydroxyindoleacetic acid, indole-3-acetic acid, and, most consistently, tryptamine. These elevations did not seem to be directly related to changes in motor activity or to the degree of anxiety, nor to be merely a secondary function of the psychotic activity. Sometimes they even preceded the onset of activation of symptoms. An increase in urinary catechols followed such activation if it was accompanied by marked anxiety. Further studies of indole metabolism and behavior have involved methionine and tryptophan loading with resulting increase in psychotic symptomatology. This approach led to a hypothesis that indoles derived from tryptophan and an elevated level of methyl groups donated by methionine might facilitate the formation of N,N-dimethylated indoleamines which are potent psychotogenic substances, and that such mechanism might be involved in the production of schizophrenic symptoms. Studies were undertaken which appear to support this hypothesis. Other interests have involved evaluating the effect of drug-induced extrapyramidal symptoms on schizophrenic symptomatology, the effect of monoamine oxidase inhibitions on extrapyramidal symptoms, the clinical effect of a serotonin antagonist, a serotonin analog, and some other psychoactive drugs. Since 1954 there has been a longitudinal study of the physical and psychological effects of aging in schizophrenics and controls. The following bibliography is a complete compilation of the clinical publications to date from the Thudichum Psychiatric Research Laboratory most of which are referred to specifically inIthe text. References p . 46/47

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REFERENCES BERLET, H. H., BULL,C., HIMWICH, H. E., KOHL,H., MATSUMOTO, K., PSCHEIDT, G. R., SPAIDE, J., TOURLENTES, T. T., AND VALVERDE, J. M., (1964); An endogenous metabolic factor in schizophrenic behavior. Science, 141, 311-313. BERLET, H. H., BULL,C., HIMWICH, H. E., KOHL,H., MATSUMOTO, K., PSCHEIDT, G. R., SPAIDE, J., TOURLENTES, T. T., AND VALVERDE, J. M., (1965); Effect of diet on schizophrenic behavior. Psychopathology of Schizophrenia. P. H. HOCHAND J. ZUBIN,Editors. Proceedings of the 54th Annual Meeting of the American Psychopathological Association. New York, Grune and Stratton. In press. BRUNE, G. G., (1961); Correlations between psychotropic drug effects, water and mineral metabolism in schizophrenic patients. Fed. Proc., 20, 306d. BRUNE,G . G., A N D HIXWICH,H. E., (1960a); The effect of reserpine on urinary tryptamine and indole-3-acetic acid in mcntal deficiency, schizophrenia and phenylpyruvic oligophrenia. Fed. Proc., 19, 194. BRUNE, G., AND HIMWICH, H. E., (1960b); Effects of reserpine on urinary tryptamine and indole-3acetic acid excretion in mental deficiency, schizophrenia and phenylpyruvic oligophrenia. Acta of the International Meeting on the Techniquesfor the Study ofPsychotropic Drugs, Bologna, June 26-27. BRUNE,G. G., AND HIMWICH, H. E., (1961a); Biphasic action of reserpine and isocarboxazid on behavior and serotonin metabolism. Science, 133, 190-192. BRUNE, G. G., AND HIMWICH, H. E., (1961b); Correlations between behavior and urinary indole aniincs during treatment with reserpine and isocarboxazid, separately and together. NeuroPsychophannacology. E. Rothlin, Editor. Amsterdam, Elsevier (Vol. 2, 465-474). BRUNE,G . G . , AND HIMWICH,H. E., (1962a); Tndole metabolites in schizophrenic patients. Arch. gen. Psychiat., 6, 324-328. BRUNE,G. G., AND HIMWICH, H. E., (1962b); Effects of methionine loading on the behavior of schizophrenic patients. J. nerv. ment. Dis., 134, 447450. B I ~ U NG. E ,G., AND HIMWICH, H. E., (1963); Biogenic aniiiies and behavior in schizophrenic patients. Recent Advances in Biological Psychiatry. Joseph Wortis, Editor. New York, Plenum Press (Vol. 5, 144-1 60). BRUNE,G. G., KOBAYASHI, T., BULL,C., TOURLENTES, T. T., AND HIMWICH, H. E., (1962); Relevance of drug-induced extrapyramidal reactions to behavioral changes during neuroleptic treatment. 11. Combined treatment with trifluoperazine-amobarbital. Comprehens. Psychiat., 3, 292-296. BRUNE,G . G., MORPURGO, C., BIELKUS,A., KOBAYASHI, T., TOURLENTES, T. T., AND HIMWICH, H. E., (1 962); Relevance of drug-induced extrapyramidal reactions to behavioral changes during neuroleptic treatment. 1. Treatment with trifluoperazine singly and in combination with trihexyphenidyl. Comprehens. Psychiat., 3, 227-234. BRUNF,G . G., AND PSCHEIDT, G. R., (1961); Correlations between behavior and urinary excretion of indole amines and catecholamines in schizophrenic patients as affected by drugs. Fed. Proc., 20, 889-893. BRUNE, G. G., PSCHEIDT, G. R., AND HIMWICH, H. E., (1961a); Effects of reserpine and isocarboxazid on behavior of mental patients and on some urinary products. Presented at: Symposium on the Biology of Schizophrenia, Battle Creek, March 16-17. BRUNE, G . G., PSCHEIDT, C.R., AND HIMWICH, H. E., (1961b); Correlations between the behaviour of patients with mental disturbances and effects of psychoactive drugs on some urinary products. Presented at: The Third World Congress of Psychiatry, Montreal, June 4-10 (Vol. 1, 111-1 17). BRUNE, G. G., PSCHEIDT, G.R., AND HIMwIcti, H. E., (1963~);Different responses of urinary tryptamine and of total catecholamines during treatment with reserpine and isocarboxazid in schizophrenic patients. Int. J . Neuropharmacol., 2, 17-23. BULL,C., A N D BERLET, H. H., (1964); Effects of isocarboxazid on spontaneous and drug-induced extrapyramidal alterations. Biogenic Amines. H. E. Himwich and W. A. Himwich, Editors. Progress in Bruin Research, Vol. 8. New York-Amsterdam, Elsevier (pp. 21 1-214). FISCHER, E., LAGRAVERE, T. A. F., VASQUEZ, A. J., AND DI STFFANO, A. O., (1961); A bufotenin-like substance in the urine of schizophrenics. J . nerv. ment. Dis., 133, 441. HAGENAUER, F., RUDY,L. H., AND HIMWICH, H. E., (1957a); A comparative study of two central nervous system simulants, Mer-22 and SKF No. 5 , on chronic blocked and withdrawn psychotic patients. Amer. J. Psychiat., 113, 840. HAGENAUER, F., RUDY,L. H., AND HIMWICH, H. E., (1957b): A comparative evaluation of two new central nervous system stimulants in severe psychoses. J. clin. exp. Psychopath., 18, 248-257.

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HAYNES, E. E., (1960); Urinary excretion of chlorpromazine in man. J . lab. clin. Med., 56, 570-575. HIMWICH, H. E., AND BRUNE,G. G., (1961); Correlations between amine metabolism and activity of psychosis in schizophrenic patients. Presented at : The Third World Congress of Psychiatry, Montreal, June 4-10 (Part 1 , p. 124). HIMWICH, H. E., COSTA,E., RINALDI,F., A N D RUDY,L. H., (1959); Triflupromazine and trifluoperazine in the treatment of disturbed mentally defective patients. Amer. J. ment. Defic., 64, 711-712. HIMWICH, H. E., WOLFF,K., HUNSICKER, A. L., AND HIMWICH, W. A., (1955); Some behaviord effects associated with feeding sodium glutamate to patients with psychiatric disorders. J. nerv. ment. Dis., 121, 40-49. PSCHEIDT,G. R., BRUNE,G. G., AND HIMWICH, H. E., (1960); Effect of t'herapeutic doses of psychotropic drugs on clinical syrnptomatology and urinary amines. Physiologist, 3, 126. PSCHEIDT, G. R., BRUNE,G . G., AND HIMWICH, H. E., (1961); Uniform response of biogenic amines to psychotropic drugs in selected schizophrenic patients. Fed. Proc., 20, 305c. RINALDI,F., COSTA,E., RUDY,L. H., HIMWICH, H. E., TUTEUR,W., AND GLOTZER, J., (1959); Triflupromazine (vesprin) in the treatment of psychotic patients. Biological Psychiatry. Jules H. Masserman, Editor. Vol. 1. New York, Grune and Stratton (pp. 292-305). RINALDI, F., HAYNES, E. E., RUDY,L. H., AND HIMWICH, H. E., (1956); Therapeutic effects of azacyclonol in psychotic patients. Psychiut. Res. Rep. Amer. psychiut. Ass., 4, 115-123. RINALDI, F., HAYNES,E. E., RUDY,L. H., AND HIMWICH, H. E., (1957); Therapeutic effects of azacyclonol in psychotic patients. Tranquilizing Drugs. Harold E. Himwich, Editor. Washington, D. C., American Association for Advancement of Science (pp. 115-123). RINALDI, F., RUDY,L. H., AND HIMWICH, H. E., (1955); The use of Frenquel in the treatment of disturbed patients with psychoses of long duration. Amer. J. Psychiat., 112, 343-348. RINALDI, F., RUDY,L. H., AND HIMWICH, H. E., (1956); Clinical evaluation of azacyclonol, chlorpromazine and reserpine on a group of chronic psychotic patients. Amer. J. Psychiut., 112, 678-683. RUDY,L. H., COSTA,E., RINALDT, F., AND HIMWICH, H. E., (1958); Clinical evaluation of BAS (benzyl analog of serotonin): a tranquilizing drug. J. new. menf. Dis., 126, 284-288. RUDY,L. H., HIMWICH, H. E., AND RINALDI, F., (1958); A clinical evaluation of psychopharmacological agents in the management of disturbed mentally defective patients. Amer. J. ment. Defic., 62, 8 55-8 60. RUDY,L. H., HIMWICH, H. E., AND TASHER, D. C., (1957); Clinical evaluation of two phenothiazine compounds - promazine and mepazine. Amer. J. Psychiut., 113, 979-983. RUDY,L. H., RINALDI,F., COSTA,E., HIMWICH, H. E., TUTEUR,W., AND GLOTZER, J., (1958); Triflupromazine and trifluoperazine: two new tranquilizers. Amer. J. Psychiut., 114, 747-748. RUDY,L. H., RINALDI, F., A N D HIMWICH, H. E., (1956); Comparativeeffects of azacyclonol, reserpine and chlorpromazine on moderately disturbed psychotic patients with long histories of hospitalization. Psychiat. Res. Rep. Amer. psychiat. Ass., 4, 49-63. SCHUT,J. W., AND HIMWICH, H. E., (1955); The effect of Meratran on twenty-five institutionalized mental patients. Amer. J . Psychiat., 3, 837-840. TOURLENTES, T., AXIOTIS, A., HUNSICKER, A., HURD,D., VASSILIOU, G., AND ABOOD,L. G., (1960); Effects of a new piperazinoglycolate on chronic schizophrenics. J. Neuropsychiut., 2, 49-53. TOURLENTES, T. T., HIMWICH, H. E., AND HUCKINS, D. S., (1957); A clinical evaluation of L-glutavite in the treatment of elderly chronic deteriorated mental patients. Illinois med. J., 112, 121-124. VALCOURT, A. J., (1959); Study of excretion of 5-hydroxyindoleacetic acid in mental patients. Arch. Neurol. Psychiut., 81, 292-298. VASSILIOU, G., COSTA,E., BRUNE,G., MORPURGO, c . , AYALA,G., HIMWICH, H. E., AND VASSILIOU, V., (1961); A pilot study of the effects of Pathcol, a serotonin antimetabolite, on schizophrenic patients. Amer. J. Psychiat., 117, 1121-1 122. VASSILIOU, V., AND HIMWICH, H. E., (1961); Psychological effects of isocarboxazid and nialamide on a group of depressed patients. J. clin. Psychol., 17, 319-320. WHITE,R. P., (1957); Face-hand test responses of psychotic and mentally defective patients. Arch. Neurol. Psychiut., 77, 120-125. WHITE,R. P., RINALDI, F., AND HIMWICH, H. E., (1956); Central and peripheral nervous effects of atropine sulfate and mepiperphenidol bromide (Darstine) on human subjects. J . appl. Physiol., 8, 635-642.

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Research Approaches to Problems in Mental Illness : Brain Neurohumor- Enzyme Systems and Behavior M. H.APRISON The Instifufeof Psychiatric Research, and the ~ e p u r f ~ofe n Biochemistry ~~ and Psycl&ztvy, Indiana University, Indianapolis, Ind. (U.S.A.)

Conducting a research program in the field of mental health is a very difficult task. There are very few hints in the literature which give the investigator any leads on which to base an active research program. However, in the last decade the biochemical approach seemed to gain favor. With the advent of micro and semimicro methods for the determination of(a) enzymes, (b) biochemically important compounds involved in energy transfer processes, carbohydrate, lipid and protein metabolism, (c) electrolytes, and (d) neurohormones or neurohumoral agents (transmitter substances, modulators, etc.) in specific areas of the central nervous system (CNS), much data have accumulated which are valuable. However, we still do not know the cause of the socalled functional mental illnesses nor have the neurochemists implicated any specific biochemical pathway or pathways in these conditions. Tf one stops to consider how patients in a mental hospital differ from individuals on the outside, one obvious fact comes to the forefront. The patients within the mental hospital have been designated as individuals who exhibit abnormal behavioral patterns; that is, they exhibit behavior that is neither typical of nor acceptable to their peers on the outside. Why is their behavior different and how does the organism change so as to emit atypical behavioral patterns? If one agrees that the brain is the source of the biochemical and biophysical events which finally govern the behavior of an organism, one can then think along lines which ultimately lead to experiments within the laboratory. There are systems involved in transmission of impulses (information) in the brain which are extremely sensitive both pharmacologically and physiologically. 1s it possible to correlate biochemical changes in these important and delicately balanced systems with concomitant changes in behavior which may occur? The author, along with many investigators, feels that the neurohumoral agents in brain - acetylcholine (ACh), 5-hydroxytryptamine (serotonin or 5-HT), norepinephrine (NE), etc. - and their associated enzyme systems are very important in the mechanisms which ultimately determine the behavior of an organism. Such an assumption forces the investigator to analyze CNS tissue rather than peripheral fluids. This position immediately limits the type of experimental subject to be used in such experimentation for unfortunately human CNS tissue is ruled out. Human brain samples are almost impossible to get fresh under strictly controlled but varying

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49

behavioral conditions, a necessity for this type of investigation. However, using animal tissue presents the investigator immediately with different problems. Within the framework of psychiatric definitions it is not generally agreed whether calling an animal mentally ill has meaning since there is no way to communicate with the animal to determine if any component or variation of ‘mental illness’ is present. However, what one can do is quantitatively measure the behavioral responses of an animal and establish limits or criteria for typical and atypical behavior. The advantage of whole animal research (with an intact nervous system) is becoming obvious since it is only in such conditions that behavior exists as we wish to study. A major problem is to find a reliable and sensitive measure of behavior with a reproducibility approaching that of our chemical techniques. Once this is accomplished, neurochemical analyses can then be made on tissue obtained from animals whose behavioral changes have also been quantitatively measured. Two techniques are described which have been used by the author and his collaborators to measure behavioral changes in such animals which appear, one more than the other, to approach these criteria. These studies began at the Thudichum Psychiatric Research Laboratory and were continued at The Institute of Psychiatric Research, Indiana University Medical Center. The author is indebted to Dr. Harold E. Himwich for introducing him to brain research in general and encouraging him in the earlier neurochemical-behavioral studies now to be reviewed in the following section. It is therefore an extreme pleasure to be able to contribute this paper to Dr. Harold E. Himwich’s Festschrift on the occasion of his 70th birthday.

The acetylcholine-acetylcholinesterase system and behavior In 1949 Freedman and Himwich, while studying the lethality of di-isopropylfluorophosphate (DFP) with different sites of injection, reported the brain to be the part of the body most sensitive to this enzyme inhibitor. They noted that when DFP is injected into one common carotid artery, the animal exhibits forced circling movements or compulsive turning towards the side opposite the injection. Though forced circling movements are the most dramatic change observed in the animal, they are accompanied by other alterations in behavior such as tremors, constriction of the right pupil, chewing movements, eye and head nystagmus. The entire phenomenon has been named by these authors the ‘adversive syndrome’. It was found that it could be produced in rabbits, rats, dogs and monkeys, the latter being the best preparation. From 1949 to 1953, Himwich and co-workers studied the adversive syndrome from the biochemical, neurophysiological, and neuropharmacological approach (Hampson et al., 1950; Essig et al., 1950a, b, 1953; Himwich et al., 1950; Bouzarth and Himwich, 1952; Hirnwich, 1953). The data obtained by these research workers were not sufficient to explain the mechanism involved in the production of the adversive syndrome. However, it was suggested that DFP inhibited the brain enzyme, acetylcholinesterase (AChE), which is the enzyme that destroys the neurohumor acetylcholine (ACh). With this enzyme inhibited, ACh was thought to accumulate on the Rrferences p. 78-80

50

M. H. A P R I S O N

side of the brain in which the injection was made resulting in stimulation of that side and hence production of the effect noted. An observation reported by Essig et al. (1953), namely, that in rabbits the turning response is sometimes reversed in direction, led the author to design a series of experiments in which the behavioral changes would be observed, recorded, and correlated with any concomitant biochemical changes in the cholinergic system of the brain that occurred in these animals. Albino rabbits weighing approximately 2 kg were used in these experiments. The right common carotid artery was isolated after the administration of a local anesthetic (0.5% pontocaine), and all arterial injections were made into it. DFP (0.05 mg/kg) was injected slowly over a period of approximately 1 min into the unoccluded artery in order to minimize the interference with the normal blood flow. The rabbit was then allowed to move freely on a large rubber mat. After a 20-min interval, during which a single behavioral pattern was observed, an injection of air was made into a marginal ear vein. Immediately after death the brain was removed. It then was freed of blood and the tissue to be analyzed for AChE activity was taken from the following left and right areas : frontal cortex (grey), caudate nucleus, thalamus, superior colliculus and inferior colliculus and immediately frozen on dry ice. In Fig. 1, a rabbit brain is

Fig. 1. Brain of adult rabbit. Left cerebral hemisphere cut and retracted back exhibiting the lateral ventricle, choroid plexus, and caudate nucleus, as well as thalamus, superior and inferior corpora quadrigemina. The cerebellum and medulla also are shown.

shown. The left cerebral hemisphere has been cut and retracted back exposing the lateral ventricle and showing the caudate nucleus, choroid plexus, thalamus, superior and inferior corpora quadrigemina, cerebellum, and medulla. The samples to be

NEUROHUMOR-ENZYME

SYSTEMS A N D B E H A V I O R

51

Fig. 2. Albino rabbit displaying circus movement to its left after receiving a unilateral (right) intracarotid injection of 0.05 mg/kg DFP.

analyzed were weighed while frozen and then homogenized. The tissue suspension was diluted with 0.5 % NaCl solution until its final concentration was approximately 1.8 mg/ml. The latter suspension was used as the enzyme source in all the analyses for AChE activity. In the two (0.10 nig/kg) and three (0.15 mg/kg) dose series, repeated injections of 0.05 mg/kg DFP were made into the right common carotid artery of the rabbits. If, after the final injection of DFP, the animal displayed a single behavioral pattern for 20 minutes, the brain was removed and analyzed as described above. When DFP was injected into the right common carotid artery of rabbits, one of three different behavioral states was noted (Aprison et al., 1954a, b). The majority of the animals circled away from the injected side (‘lefters’; see Fig. 2), fewer were unaffected by the drug and maintained their normal mode of progression, while some circled toward the injected side (‘righters’; see Fig. 3). Since these animals were subjected to the same experimental conditions, namely, the injection of DFP into the right common carotid artery, and three distinct behavioral patterns were observed at each dose, it was decided to study the effect of DFP on AChE in specific areas of the brain from rabbits in each group. References p . 7830

52

M. 11. A P R I S O N

Fig. 3. Albino rabbit displaying circus movement to its right aftcr receiving a unilateral (right) intracarotid injection of 0.05 mg/kg DFP.

The AChE data for the frontal cortex and caudate nucleus in those experiments in which the intracarotid dose of DFP was varied from 0.05-0.15 mg/kg are shown in Fig. 4. This figure contains the results of all the rabbits which circled either to the left or to the right. To illustrate better the effect of progressively increasing doses of the anticholinesterase, the data for the frontal cortex and caudate nucleus on the right and left sides of the brain are shown separately in the figure. The level of AChE in these two structures is thought to be important in determining whether forced circling movements will be produced in the rabbits (Aprison et a/., 1956). The DFP is injected on the right side and therefore the AChE activities in these brain areas are all lower than the activities of the corresponding structures on the left side. In Fig. 4 (upper left), the AChE data are given for the left frontal cortex and left caudate nucleus of rabbits which were exhibiting forced circling movements to the left. The AChE activity diminished linearly with increasing doses of DFP in both structures, the fall being more rapid in the case of the caudate nucleus. The data for the AChE activities in the frontal cortex and caudate nucleus on the right side of the brain of ‘lefters’ are given in the upper right part of Fig. 4. The greatest absolute fall in the enzymatic activity of the caudate nucleus and frontal cortex occurred at the 0.05 mg/kg dose. At this point, the AChE activity of the caudate nucleus was reduced to 3.9 % of normal and remained approximately at that low level with the two higher

NEUKOHUMOR-ENZYME

SYSTEMS A N D BEHAVIOR

53

doses of injected DFP. The AChE activity of the frontal cortex decreased to 42.3 % of normal at the 0.05 mg/kg dose of DFP and then also leveled off, but at about 30% of normal. However, the absolute values of AChE in both structures at 0.05, 0.10, and 0.15 mg/kg of DFP are statistically the same. The results shown at the lower left part of Fig. 4 are for the AChE activities in the left frontal cortex and left caudate nucleus from rabbits exhibiting circus movements to the right. The AChE activity in the left caudate nucleus fell linearly with increasing dose of DFP. The slope of this curve was almost the same as that for the left caudate nucleus from ‘lefters’, a decrease of9.2 x 10-6units ofAChEactivity per 0.05 mg/kgDFP. When the data for this brain structure in ‘lefters’ and ‘righters’ at each of the three dosesofDFP were subjected to the Student ‘t’ test, no statistical significant difference was found. On the other hand, the data for the left frontal cortex of ‘righters’ did not display a uniform fall in enzymatic activity as was true in the case of the ‘lefters’. In this case, a significant decrease in enzymatic activity was noted at 0.05 mg/kg dose of DFP followed by a leveling off at the higher concentrations of the anticholinesterase. The three mean AChE values for this tissue were all lower in the case of the ‘righter’ than in the ‘lefter’. The curves indicating the relationship between the DFP dose injected and the AChE activities for these same structures on the right side of the brain taken from ‘righters’ are shown at the lower right part of Fig. 4. I n the case of the right frontal cortex, the mean AChE activities i n ‘righters’ are lower than for ‘lefters’ at each of the three doses of DF P LEFTERS

CAUOATE NUCLEUS

40

Y

u

4

LEFT FRONTAL CORTEX

30t\ 20

RIGHT

0.05 0.10 0.15 D F P DOSE m g / k g

D F P DOSE m g / k g

6ot

RIGHTERS

RIGHTERS

CAUDATE NUCLEUS

u 30

30 w S

20

‘0

RIGHT FRONTAL CORTEX

0.05 0.10 OFP DOSE m g / k g

0.15

‘0

0.05 0.10 0.15 DFP DOSE m g / k g

Fig. 4.Acetylcholiiiesterase activity of the right and left frontal cortex and caudate nucleus in rabbits exhibiting compulsive circling movements after receiving DFP. The AChE activity on the ordinate axes is expressed as mequiv. of ACh hydrolyzed per mg wet tissue per min x lo6. The dose of DFP (mg/kg) injected into the right common carotid artery is shown on the abscissa axes. Each point represents the mean value of the enzymatic activity found in specific brain areas of 7-10 rabbits. All AChE analyses of tissue from a given animal were done in triplicate. (By permission from the Amer. J . Physiol.) References p . 78-80

54

M. H. A P R I S O N

injected intracarotidly. On the other hand, the data for the right caudate nucleus are approximately the same for ‘righters’ and ‘lefters’. In order to illustrate the difference in AChE activities in the right and left cortex taken from rabbits which circled, the data are reproduced on an expanded ordinate scale in Fig. 5 . The mean enzymatic activity in corresponding tissues taken from these brain structures in ‘righters’ is lcwer in every case than from ‘lefters’. The differences in the means at all doses, and in both the left and right frontal cortex, are statistically significant when tested by the Student ‘t’ test. However, it should be pointed out that the extremely low levels of enzymatic activity in the right cortex of the ‘righters’ (approximately 1 x 10-6 mequiv./mg/min) is at the lower limit of the method. Additional AChE data from 6 other brain structures for ‘lefters’ and ‘righters’ are shown in Table I. The enzymatic activities for the right and left thalamus, superior colliculus and inferior colliculus are presented as the mean plus or minus the standard error. In Table 1 (‘lefters’), the AChE activities for the tissue on the right side of the brain were all lower than on the left at all three doses of DFP administered. Furthermore, with the greater doses of the anticholinesterase still lower AChE activities were found

I

0 05

I

0.10 DFP dose (rng/kg)

I

0.15

Fig. 5. Effect of the intracarotid dose of DFP on the AChE activity in the right and left frontal cortex in ‘lefters’ and ‘righters’. (By permission from the Amer. J . Physiol.)

in each tissue. When these results are compared to see if the differences in the mean values for similar right and left brain structures are significantly distinct, only the data for the thalamus at 0.15 mg/kg DFP were found to have a P value lower than 0.02. The AChE data for the ‘righters’ are slightly different. Although the mean enzymatic activity in each tissue fell with increasing dose of DFP, the asymmetry between right and left brain structures is less pronounced than in the case of the ‘lefters’. The Student ‘t’ test was applied to the mean AChE values of similar brain areas from Table I. The AChE data of ‘righters’ and ‘lefters’ werecompared at all three doses of DFP. Only the values for the superior colliculi were found to differ significantly at 0.10 mg/kg of DFP.

I COMPARISON OF BRAIN

(Mean

AChE

ACTIVITIES I N RABBITS EXHIBITING FORCED TURNING MOVEMENTS

S.E. mean) x 106. All analyses were made in triplicate. The results were calculated as mequiv. ACh hydrolyzed/mg wet tissue/min.

Tissue

Right thalamus Left thalamus Right superior colliculus Left superior colliculus Right inferior colliculus Left inferior colliculus

* Mean of 8 animals.

** Mean of 6 animals. 5 Mean of 5 animals.

Controls ‘Lefters’ 0.1 ml HzOjkg injected 0.05 mglkg DFP 0.10 mglkg DFP ( n = 10) (n = 8) (n = 6 )

13.34 i 0.59 13.04 ?C 0.79 28.32 & 1.19 27.21 f 1.33 8.98 f 1.19 10.59 f 1.27

6.07 f 1.36 9.02 & 1.41 20.16 & 1.25 22.32 & 1.52 5.13 f 0.66 5.77 f 1.00

4.60 f 1.03 7.93 i 0.89 9.79 & 1.905 14.46 f 0.79 5.23 i0.63 5.70 f 0.61

‘Righters’

0.15 mglkg DFP ( n = 7) 3.82 f 0.57* 7.14 5 1.03 9.29 i 1.57 11.61 i 1.53* 3.17 & 0.58 4.62 f 0.95

0.05 mglkg DFP 0.10 mg/k,nDFP 0.15 mg/kg DFP (n = 6) (n = 6) ( n = 7) 8.15 i 1.15 9.22 i 1.33 22.16 i 0.595 22.04 i 1.49 6.73 i0.96 6.32 & 1.49

6.45 i 0.56 9.27 i 0.59 18.04 f 1.10 19.33 & 1.56 5.03 i 0.63 5.30 & 0.61

4.78 i 0.46** 4.90 f 0.70 10.32 f 0 . 9 6 10.68 i 1.28 1.93 & 0.58 3.92 i0.68

56

M . 11. A P R I S O N

T A B L E I1 A C E 1 YLCHOLlNESTkltASE AC1 I V I T I F S IN POSTFKIOR B R A I N A K F A S I N RABBITS

Values arc mean 1 S.D. -__

~ _ _ _

Y

lo6.

____

~

-~

6) 0.1 tnl HzOjkg

Control ( n .~ ~

Right flocculonodular lobe Left flocculonodular lobe Right lingular lobe Left lingular lobe Right pons Left pons Right medulla oblongata Left medulla oblongata

.

-

-

+

9.36 1.91 9.50 & 2.1 1 12.23 t 2.50 13.02 1.02 13.36 3.31 13.33 & 3.08 16.61 i 3.08 15.06 3.5 I

+

‘Lefterr’ (n = 7) 0.05nigjkg D F P - _____ __

8.53 & 2.00 10.27 & 1.52 13.41 & 2.10 13.80 3.40 12.96 3.10 15.17 t 3.56 15.83 t 3.54 19.04 -1 3.38

In Table 11, the AChE activities of the posterior portions of the brain are given as the mean & CT for 6 controls and 7 ‘lefters’. Only 0.05 nig/kg DFP was injected into the right common carotid artery in these experiments; 0. I ml/kg water was used for the controls. The data for the latter series showed no asymmetry between posterior left and right brain parts. When the results for similar brain areas are compared statistically in the ‘lefter’ series, no significant differences were found. Although the data are not included, these posterior brain regions also were analyzed for AChE activity in one ‘righter’, and another rabbit that did not display any turning movements (‘neutral’). The results also were not significantly distinct from the control series, nor the data for the ‘lefters’. From the data presented, it is seen that the compulsive circling of rabbits produced by the unilateral intracarotid injection of DFP, under the experimental conditions described, is due chiefly to the effect of the anticholinesterase on the cortex, and secondarily to an action on the caudate nucleus. The observed changes in enzyme activity suggested a hypothesis to explain the direction of the forced turning in these animals. It is assumed in the following discussion that only the AChE-ACh system was affected significantly by the relatively low doses of DFP injected to produce the syndrome (Dixon and Needham, 1946). The enzymatic analyses of 5 bilateral areas of the brain revealed that forced circling, whether to the right or left, was associated with an asymmetric reduction of AChE activity. I n all animals that exhibited forced circling in either direction, the data for the right caudate nucleus revealed the most profound depression of enzyme activity. Thus, an asymmetry of AChE activity appears to be a prerequisite for the development of forced circling. The asymmetry in AChE activity between corresponding areas on the right and left side of the brain probably was due to the fact that with normal hemodynamics, the right common carotid artery is the main source of the blood irrigating the right side of the telencephalon (McDonald and Potter, 1951). This may be the reason why a statistical comparison of the data from particular areas on the right side of the brain at all doses of DFP used excluding the frontal cortex. of both ‘righters’ and ‘lefters’, revealed no

NEUROHUMOR-ENZYME

SYSTEMS A N D BEHAVIOR

57

significant differences between the two groups. However, at this stage of the research a distinction between ‘righters’ and ‘lefters’ was possible because although the means of the AChE activity for the right frontal cortex, left frontal cortex, and left caudate nucleus were lower at all three doses of DFP in the cases of the ‘righters’, only the AChE activities of the cortices were significantly lower. When the AChE activity in the brain is reduced, there is a concomitant increase of ACh. This was shown by Cortell et al. (1941), Stewart (1952), and Michaelis et al. (1954). Circling movements to the left probably are due to stimulation of the right cortex due to the greater accumulation of ACh in this brain area than on the left side. On the other hand, the compulsory movements to the right are difficult to explain because of the AChE data of the cortices. Since atropine has been shown to prevent as well as correct forced circling (Himwich et al., 1950; Nathan et al., 1955), additional evidence that ACh is the agent involved in the production of these compulsive movements is available. However, the role of ACh in the caudate nucleus is not clear. In this connection it should be pointed out that the AChE activities in the right and left caudate nucleus in both ‘lefters’ and ‘righters’ were not significantly different. The AChE activity of the right caudate nucleus was so low (4% of normal) that it is possible it was nonfunctional due to a biochemical lesion (Bullock et al., 1947). This may be equivalent to ablation of the right caudate nucleus which results in the production of forced turning to the right (Mettler, 1942). Therefore, the possible loss of this caudate could be an additional factor in producing animals that circle to the right. If this is the case, in ‘lefters’, the latter effect of the caudate nucleus must be overshadowed by the stimulating action of the right cortex.

RANGE OF lNHl0lTlON -

I 0

20

4

RANGE OF RANGE OF STIMULATION~PHISIOLOGICAL SAFETY

40

4

8

60

80

-

1

100

AChE ACTIVITY ( % OF NORMAL)

Fig. 6 . Correlation of AChE activity with the functional state of the cortical tissue in rabbit brain. (By permission from the Amev. J . Physiol.)

In Fig. 6, the mean AChE activities for the cortices in ‘lefters’ and ‘righters’ are presented as per cent of normal to show that the data fall into three distinct ranges. The AChE values in the lowest range through which curve A passes (right frontal cortex of ‘righters’) have decreased to such an extent that the ACh may have accumulated to a value where it depresses the function of the right frontal cortex. In Riqerences p . 78-80

M. H . A P R I S O N

58

the same animal, the ACh in the left cortex presumably accumulated to a level which resulted in stimulation (see curve B) resulting in an animal which circled to the right. On the other hand, the AChE activity in the right cortex of ‘lefters’ was at such a level that the ACh that accumulated can still stimulate this brain structure (curve B), and the rabbit circled to the left. Finally, the AChE activity in the left cortex approached normal values and the concentration of ACh in this area probably was normal, and hence did not affect the animal’s behavior. The vertical lines in Fig. 6 used to designate the three AChE ranges were drawn to indicate approximate levels. More extensive experiments may shift the exact values for these ranges. However, the purpose of this figure was to show that the enzyme data suggested that three functional levels could exist in the CNS, namely inhibition, stimulation and physiological safety (Aprison et al., 1956). To further check this theory, it was necessary to measure ACh in the right and left cerebral cortices and caudate nuclei of turning animals. However, before investing time to develop a method to determine ACh in small samples of brain tissue containing DFP, it was decided to see if it was possible to produce compulsive turning in rabbits by injecting ACh or Mecholyl, the ,!?-methylderivative of ACh, into the right common carotid artery. These experiments were successful (Aprison et al., 1956), although the duration of the turning phenomena was much less than that observed after DFP administration. This latter result was not surprising since in these experiments there was sufficient enzyme available in blood to hydrolyze most of the injected ACh within a short period of time. The implications of these data were discussed elsewhere (Aprison et al., 1956). TABLE 111 ‘ F R E E ’ ACh C O N T E N T (pg/g)I N R A B B I T B R A I N P A R T S Eserine-Reinecke Salt Variation (see Aprison and Nathan, 1957a).

Rabbit ~

Left cortex

Right cortex

Left caudate nucleus

Right caudate nucleus

0.84 0.74 4.06

0.85 I .76 2.54

5.70 5.64 10.07

6.41 15.47 15.09

~~

Normal (8) ‘Lefters’ (6) ‘Righters’ (3)

Encouraged by these results, a bioassay procedure was developed for assaying ‘free’ ACh in small brain samples taken from rabbits injected with DFP (Aprison and Nathan, 1957a). The method was patterned in part after the one reported by Bentley and Shaw (1952). Guinea-pig ileum (3-4 cm portions) served as test object. Brain samples as small as 40 mg of caudate nucleus and 155 mg of cerebral cortex were analyzed. The data for the ‘free’ ACh content (mean) in the left and right cerebral cortex and caudate nucleus from 17 rabbits are summarized in Table 111. The results for a particular area in the normal group were similar for each side of the brain. However,

NEUROHUMOR-ENZYME

SYSTEMS AND BEHAVIOR

59

the ACh concentration in the caudate nucleus is over 7 times higher than that in the cortex. In the ACh data for the 4 brain parts taken from ‘lefters’ (as a result of the injection of 0.1 mg/kg DFP into the right common carotid artery), the mean ACh concentration for the left cortex is 0.74 pg/g while for the right cortex it is 1.76 pg/g. This 1 pg/g difference is higher than the concentration found for the left side. In the caudate nuclei of the ‘lefters’ the absolute asymmetry in ACh concentration was even greater than that of the cerebral cortices, reaching approximately 10 pg/g, or 10 times that noted between the cortices. Furthermore, the right cortex and right caudate of the ‘lefters’ were approximately 2.5 times higher than the corresponding structures on the left side of the brain. In the case of the ‘righters’, a difference of 1.5 pg/g occurred between the cortices. However, in these animals the left cortex was higher than the right cortex, exactly reversed from the case of the ‘lefters’. This result supports our original concern with the AChE data for the left cortex in ‘righters’. The mean ACh levels for the caudate nuclei are 10 pg/g for the left and 15 pg/g for the right. The value for the right caudate nucleus in the ‘righters’ is essentially the same as that found for this same structure in the ‘lefters’. However, the mean ACh content for the left caudate nucleus in ‘righters’ was almost twice that found for the structure in ‘lefters’. TABLE I V , F R E E ’ ACh C O N C E N T R A T I O N S (pg/g) I N B R A I N P A R T S O F R A B B I T S N O T T U R N I N G O R M O V I N G S P O N T A N E O U S L Y AFTER A U N I L A T E R A L I N T R A C A R O T I D I N J E C T I O N OF D F P

no.

Left cortex

cortex

1 2 3 4

0.96 0.63 1.81 0.97

1.12 0.75 2.11 1.17

Rabbit

Left caudate nucleus

Right caudate nucleus

8.10 5.36 5.18 I .65

10.96 11.29 7.96 15.02

In Table IV, ACh data are shown from 4 experiments in which the animals did not display any spontaneous movements, although the same dose of DFP was injected under the same experimental conditions. The mean for the left and right cerebral cortex is 1.1 pg/g and 1.3 pg/g respectively, while that of the left caudate nucleus is 5.1 pg/g, and the right caudate nucleus is 11.3 pg/g. No significant asymmetry in ACh concentration was noted in the right and left cortices, while it was marked in the case of the caudate nuclei. As in all the other data, the right caudate nucleus had the higher ACh concentration. Extension of the proposed hypothesis to include the ACh concentration was desirable (Aprison and Nathan, 1957b). The ACh data presented substantiated the concept in the main. In ‘lefters’, the functional state or level of the left cortex and left caudate was one of physiological safety. The ACh concentration was found to be approximately normal, although the AChE activity dropped to approximately 60 % Rqferences p . 78-80

60

M.

11. A P R I S O N

of normal. On the other hand, the functional state of the right cortex was thought to be one of stimulation, since its ACh content rose over 200% of normal. In the case of ‘righters’, it was found that although the ACh content of the right cortex rose to 300% of normal, that in the left cortex rose to 480%. Therefore, the much higher concentration of ACh i n the left cortex suggested that greater stimulation of this tissue resulted in compulsive turning to the contralateral side. Finally, in injected animals in which asymmetry in ACh concentration between both sides of the cortex was lacking, spontaneous movements (turning or forward) were not observed. From the ACh data, the condition of inhibition in the right cortex of ‘righters’ i n the proposed hypothesis could no longer be deduced from the standpoint of excessive or high neuroliumor concentration. However, if inhibition of function did occur, it would have to be due to the direct action of DFP. Until it can be shown that inhibition did occur i n the right cortex, only the functional states of physiological safety and stimulation are now compatible with the data. Since the AChE data, as well as the ACh data, for the caudate nuclei in ‘righters’ and ‘lefters’ show little difference, it was difficult to implicate the caudate alone in the mechanism of compulsive turning. It was thought that the caudate nucleus may be involved in a secondary manner in the production of rabbits circling to the left. However, the biochemical evidence suggested that stiinulation due to the accumulated ACh on one side of the cerebral cortex may be the primary factor i n causing the auimal to exhibit compulsive circling toward the contralateral side. It occurred to the author that the number of observed turns per minute executed by the rabbits just prior to the removal of the brain parts for biochemical analysis might be a measure of the asymmetry i n ACh concentration between the right and left cerebral cortex. Furthermore, if the above idea was correct, this should be true irrespective of the direction of the compulsive turning. Unfortunately, the number of turns cxecuted by each of the rabbits were not recorded i n the earlier experiment. Therefore, to test this idea, circus movements were produced i n another series of 19 ‘lefters’ and 7 ‘righters’ by injecting 0. I mg/kg DFP into the right common carotid artery. The number of turns executed by the rabbits were noted at different times after the injection of DFP. In the case of the 16 control animals, 0.1 mg/kg water was injected instead of DFP. After the animal was observed for 20 min and had shown a single behavioral pattern, it was prepared for surgical excision of the desired brain areas. The tissue was processed and ACh determined (Aprison, 1958). T n a given animal thc free ACh content of the left cortex was subtracted from that found for the right cortex. This value in pg/g was expressed as A AChnc-Lc and was the unit plotted against the turns per minute made by the animal just before it was sacrificed. These data are shown in Fig. 7. In ‘lefters’ d AChnc-Lc was a positive value, because the right cortex had a higher ACh content than thc left. In the case of the ‘righters’, the left cortex had a higher ACh content, and the difference in these two numbers was negative. The mean of the difference data between the right and left cortices of the 16 controls was 0.03 pg/g. Therefore, two assumptions were made: ( I ) the curve to describe the data passes through the origin, and ( 2 ) the curve is linear in the region studied. When the method of least squares was applied to the

+

N E U R O H U M 0 R - E N Z Y M E S Y S T E M S A N D B EH A V 1 0 R

2.0

o

61

bL=+0.18

- 25 L

Fig. 7. Relationship between the asymmetric accumulation of ACh in the cerebral cortices of rabbits and the rate of compulsive turns they make after the injection of 0.1 mg/kg of DFP into the right common carotid artery. (By permission from the J. Nertrochern.)

data, it was found that the slope of the curve for the ‘lefter’ data was CO.18, while that for the ‘righters’ was -0.23. A general equation to describe the relationship between the rate of turning, direction of turning, and the amount of asymmetry of ACh in the right and left cortices of rabbits induced to exhibit circus movements as a result of the injection of 0.1 mg/kg DFP into the right common carotid artery is: f D = k R where D is 3 AChR(s I.C in ,ug/g, k is 0.2, R is the rate of turning 20 miii after the DFP injection ( R < 10 tjmin), and a plus (+) sign indicates ‘lefters’ while a minus (-) sign indicates ‘righters’. N o such correlation was found in the case of the caudate nuclei. Although it was possible to correlate the rate of turning by the rabbit with the amount asymmetry of ACh in its cerebral cortices after the unilateral intracarotid iiijectioii of DFP, this type of behavioral measure leaves much to be desired. Furthermore, compulsive turning or circling is not a behavioral condition which lends itself easily to further study. It was at this time that the author was introduced to the use of operant conditioning as a technique for measuring the behavior of an animal (Ferster and Skinner, 1957). By selecting a simple response or bit of behavior (e.g. a press of a lever by an animal) and giving reinforcement (e.g. food) at appropriate times, it is possible to get a consistent and reproducible baseline of behavior from hour to hour, or day to day. By varying the pattern in which reinforcement is received by the animal, schedules can be changed from very simple to the most complex. These baselines i n turn permit the measurements of the behavioral effects of injected compounds such as neurohumor precursors, enzyme inhibitors and other drugs (Dews, 1956, 1958; Verhave, 1959; Aprison and Ferster, 1961a; etc.). Rel>iettces p 78-80

62

M. H. A P R I S O N

The serotonin-monoamine oxidase system and behavior Since evidence has accumulated that neurohumoral agents such as 5-HT, norepinephrine (NE) as well as ACh have major effects on the behavior of living organisms by their action on the central nervous system, it was decided to continue the research program by studying the serotonin-monoamine oxidase (MAO) system and then the NE-MA0 system. In the last few years considerable data have been reported on brain serotonin levels. This interest initially came about when it was shown in pharmacological studies on smooth muscle that D-lysergic acid diethylamide (LSD-25), a compound which provokes schizophrenic-like states in man, is a serotonin antagonist at certain doses (Gadduin, 1954), but acts synergistically at lower doses (Costa, 1956). The work of Woolley and Shaw (1954a, b) on other compounds structurally

5-HYDROXYTRY PTAMINE: SEROTONIN S-Adenosyl Methionine

0

CH30 m

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mcHz

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5-HYDROXYINDOLEACETIC ACID

N-ACETYL- 5-METHOXYTRYPTAMINE: ME LATONIN

Fig. 8. Metabolism of serotonin.

N E U R O H U M O R - E N Z Y M E SYSTEMS A N D B E H A V I O R

63

related to serotonin also added impetus for work in this area. These studies suggested to many that serotonin may have a role in brain function. Other studies showed that the necessary enzymes for the synthesis and destruction of 5-HT are present in brain. In Fig. 8, the metabolic steps involved in the formation and destruction of 5-HT are shown. The level of 5-HT can be elevated by (a) injecting its precursor, 5-hydroxytryptophan (5-HTP) or (b) injecting an M A 0 inhibitor such as iproniazid (I-isopropyl-2-isonicotinyl hydrazine phosphate), or (c) injecting 5-HTP after iproniazid pretreatment. Animals with elevated brain 5-HT levels were reported to exhibit marked central (behavioral) disturbances (Udenfriend et al., 1957; Bogdanski et a/., 1958; Costa and Rinaldi, 1958). However, the resulting abnormal behavior in these studies was described only in subjective terms.

1 0 MIN

Fig. 9. Record of a portion of an experimental session showing a typical performance. The bird’s responses are recorded cumulatively against time giving a curve whose slope is the rate of pecking

A first step in this study was a specification of the behavior of the animal for which the biochemical correlate was sought. Pigeons (White Carneaux Cocks), 6 months old at the start of the experiment, were used because at this time, 1958, the experimental psychologists had accumulated much behavioral data on pigeons. The hungry birds were trained to perform in a ‘Skinner’ box. The performance recorded was that of pecking at a 1-inch disk commonly used in operant conditioning research with pigeons. The birds pecked because they were hungry and were rewarded with food according to a preset schedule. The experimental session was 6 h long or 55 reinforcements, whichever occurred first. When injected compounds disrupted the bird’s performance severely, the session was prolonged t o measure the recovery of the performance. A multiple fixed-ratio, fixed-interval schedule (multiple FR 50 FI 10) was used as the baseline to determine the behavioral effects of injected 5-HTP (Aprison andFerster, 1961a). Fig. 9 is an actual record of a portion of an experimental session. Every time the bird pecked at the disk, it operated an electrical contact which allowed the experiment to be automatically programmed and recorded. In the figure, the bird’s pecks were recorded cumulatively against time. The scale of the record is given in the grid and References p . 78-80

64

M. H. A P R l S O N

r

u)

w

Fig. 10. Cumulativc-response curve for bird 9 Y showing the details of the early effect of the 5-HTP injections. Record A, gives control and saline performances. Records B through F are for the doses as indicated in the subscript. (By permission from the J . Pharmacol. exp. Ther.)

the oblique lines indicate where the reinforcements occurred. The portion of the curve between the first two oblique lines show the fixed-ratio performance (FR) during which time the disk was illuminated red; the reinforcement occurred as a result of the 50th peck. The bird begins to peck immediately and continues at a rate of approximately 4 pecks per second until 50 responses are emitted. The color of the disk was then changed to white; in this color the reinforcement is delivered on the basis of elapsed time rather than number of responses. The first peck after 10 min operated the food magazine. This was the fixed-interval (FI) component of the multiple schedule. During this stimulus, the bird pauses for a considerable part of the 10-min interval and then begins pecking at a rate of 1 peck per second until a response is reinforced at the end of the fixed-interval. The two schedules of reinforcement alternate for the entire length of the experimental session producing a reliable repetition of the patterns of responding illustrated in the figure. Fig. 10 gives a detailed record of a portion of a pigeon’s performance (9 Y). The bird’s responses are accumulated against time in the curve, and each segment

N E U R O H U M O R - - E N Z Y M E SYSTEMS A N D DEHAVIOI<

65

contains the first 6 reinforcements immediately following injection. Record A, coiitains the segments from control and saline sessions and showsthe normal performance under the multiple schedule of reinforcement. In the fixed-ratio segments the bird responds rapidly at about 4 pecks per sec until reinforcement. In the fixed-interval segment, the bird pauses for more than half of the 10-min interval before it begins pecking. The number of pecks in the fixed-interval component gives the main data. Record B, showing the effects of the 10 mg/kg 5-HTP doses, does not differ from the control sessions except perhaps in a third session, where the number of responses in the three fixed-interval segments is somewhat less than the control values, and the terminal rate of responding during the second fixed-interval segment is lower than any of those occurring in control sessions. The 15 mg/kg injection of 5-HTP in C shows possible effects in the final fixed-interval segment in the first two curves and in the last two segments of the third curve where responding has all but ceased. In D, the 25 mg/kg 5-HTP produces an almost immediate marked decline in responding in the fixed-interval segment. Except for a pause of about 2 min in one of the fixed-ratio segments, the fixed ratio is unaffected in spite of the marked effect on the fixedinterval performance. This result is in accord with the previous results reported by Dews (1955) with the effect of sodium pentobarbital injected into animals trained on RECOVERY OF BEHAVIORAL PERFORMANCE AFTER 5-HTP ADMINISTRATION

Fig. 1 I . Continuation of cumulative-response curves for the records shown in Fig. 10. (By permission from the J . Pharmacol. e x p . Ther.\ References p. 78-80

66

M. H. A P R I S O N

the same baseline performance. Two injections at 50 mg/kg in E, as well as the one at 75 mg/kg, produce a more severe effect. At 25 mg/kg a brief period following the injection occurs during which the fixedinterval performance is disrupted while the performance under the fixed-ratio color is relatively unimpaired. At the highest doses, 50 mg/kg and 75 mg/kg, both the fixed-interval and fixed-ratio performances were disrupted. However, there is considerable variation from bird to bird on the onset of complete disruption of the fixedratio performance at the higher doses of 5-HTP. Fig. 1 1 shows details of the same bird’s performance as it recovered from the 5-HTP injection. Each segment is a continuation of the performances already shown in Fig. 10. The 8, 10, and 15 mg/kg curves fall within the normal range. At 25 mg/kg the rate of responding in 3 and 4 fixed intervals, respectively, is considerably below normal, but the intervening fixed-ratio performances are not affected. Thereafter, the performance is not different from the controls. The 50 mg/kg injections disrupted the performance for approximately 90 to I30 min. However, once responding begins, the form of recovery is similar to the ones described above for 25 mg/kg. After the 75 mg/kg injection the performance does not return to baseline, even after 230 min. During the recovery, normal performance occurs in the fixed-ratio while the rate of responding in the fixed-interval is still low. Additional studies provided several behavioral parameters which could be used in determining doses-response curves after the injection of 5-HTP. These were as follows : ( I ) the number of responses emitted in a given period of time, (2) the time necessary , the time necessary to make one-half the responses in the control session ( T R ~ )(3) to make one-quarter of the responses in the experimental session (T/4), and (4) the time of atypical or abnormal responding (‘T’) (Aprison and Ferster, 1961a, b, c ; Aprison et al., 1962). These different behavioral measures evolved from the need for more sensitive behavioral measures. The interrelationship between these behavioral measures will be the subject of a future publication. In Fig. 12 the behavioral effect (Tn12) produced as a result of an injection of 5-HTP i.m. into the breast muscle is plotted as a function of 25, 50 and75mg/kg doses in 8 birds. Each point is the median value while the vertical lines give the interquartile range. The behavioral effect of the injected 5-HTP increased linearly with dose except at the lower values. The first major effect of injected 5-HTP occurred at 25 mg/kg. The usual response was a cessation of pecking. The duration of the pause increased with the dose of 5-HTP. During this time, the birds were noted to be standing, facing in various directions, usually immobile, but exhibiting body tremors, head shaking and frequently shifting from one foot to the other. A more detailed account of the onset and recovery of one bird’s performance after injection of various doses of 5-HTP is presented in Fig. 13 where the raw data have been plotted. The number of pecks the bird emits is cumulated against time, and each curve represents a complete daily session. The range of the control sessions is given by the stippled area. The curves for the 8 and 15 mg/kg doses fall well within the range of the control injections, as was also shown in the dose-effect curve of Fig. 12 (Aprison and Ferster, 1961a).

NEUROHUMOR-ENZYME

SYSTEMS AND BEHAVIOR

67

350'-

300--

-

250--

-2

ks zoo--

DOSE OF DL-5-HYDROXYTRYPTOPHAN HYDRATE ( m d k g )

Fig. 12. Behavioral effects of intramuscularly injected D,L-S-hydroxytryptophan hydrate (5-HTP). The behavioral effect is given by the measurement TRIZ, which is defined in the text. Each point is the median value; the vertical bars represent the interquartile range. The number of animals used are given by the first number, and the total number of injections made at each dose is given by the second number. (By permission from the J . Pharmacol. exp. Ther.)

Since 5-HT crosses the blood-brain barrier with difficulty compared to 5-HTP (Costa and Aprison, 1958a), it was decided to study what effect, if any, did 5-HT have on pigeons working on a multiple F R 50 FI 10 schedule of reinforcement. Therefore, the same type of experiments were repeated with serotonin creatinine sulfate hydrate (5-HT). In Table V, data are presented to show the behavioral effect (TR,~)of intramuscularly injected 5-HT. A comparable behavioral effect was produced with a much smaller dose of 5-HT than 5-HTP. However, the behavioral effect in the 5-HT experiments may be due to its peripheral actions. In this connection it should be reported that two pigeons died, after receiving an intramuscular injection of 10 mg/kg of 5-HT (4.3 mg/kg free serotonin). To date, this has never happened while using 5-HTP in doses up to 75 mg/kg in the same manner. Larger amounts have not been injected. Since small doses of 5-HT had large behavioral effects, it might be supposed that the behavioral effects of 5-HTP were also due to 5-HT being formed peripherally. There is evidence, however, that in spite of the fact that 5-HTP acting peripherally may affect the animal's behavior its major influence is through the CNS. The results of Udenfriend et al. (1957) bear on this issue. They report that iproniazid is a poor References p . 78-80

68

M. H . A P R I S O N

14 000 13 000 12 000 v1

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7 000 6 000

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3 000 2 000

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50

1

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1

100

150

200

1 1

250

1

1

1

1

300

350

400

450

500

C U M U L A T I V E T I M E IN M I N

Fig. 13. Bird 9 Y, a reduced plot of responses cumulated against time. Each curve is a plot of responses cumulating against time for a single experimental session. The range of the control session is given by the stippled area and the doses of the experimental sessions are marked on the curve. (By permission from the J . Pharnracol. exp. Ther.)

T A B L E \.’ B E H A V I O R A L E r b E C T OF I N T R A M U S C U L A R L Y I N J E C T E D S E R O T O N I N I N P I G E O N S

Data obtained on 6 pigeons; two received I injection, the others received several injections spaced approximately 7 8 h apart. Dose of 5-HT

l n i ~ l k g*) 0 2 4 10

Behavioral nieasiire T R ~(min) Z

________________-

Individual valireA 132, 115, 112, 162,115,116, 132, 155,105 153, 172, 123, 145, 152 206,221, 142, 197 210,305,322

Mean 127 149 191 279

____________.__.___________

* Dose expressed as mg of serotonin creatinine sulfate hydrate/kg.

69

W E U R O H U M O R - E N Z Y M E SYSTEMS A N D B E H A V I O R

M A 0 inhibitor in vivo except in the brain. They have shown that in experiments in which 5-HTP is administered to mice pretreated with iproniazid, the increase in the whole animal’s 5-HT level did not differ from controls given 5-HTP alone. When experiments were repeated but the head separated f r o n the carcass, the results showed that brain 5-HT levels were markedly elevated in the iproniazid plus 5-HTP group. These studies would suggest that a behavioral experiment in which iproniazid and 5-HTP were used could supply information whether the behavioral effects were due to a central or peripheral mechanism. If iproniazid, in fact, enhanced the behavioral effects of injected 5-HTP, then it is likely to be due to elevated brain 5-HT levels resulting from the reduced M A 0 activity. This is in fact what occurs when the experiment involving quantitative behavioral measurements was carried out (Eerster and Aprison, 1959). Since this was true, it occurred to the author that if the same dose of 5-HTP was injected into iproniazid-treated pigeons at different times over a long

11 000

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150

200

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300

350

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Fig. 14. Performance curves of the pigeon showing the effect of a 5-HTP injection (50 mg/kg) at various times following iproniazid pretreatment at the beginning of the experiment. One hour after the iproniazid injection, a 50 mg/kg dose of 5-HTP was injected i.m.; then at different times over a 47-day period, the same dose of 5-HTP was given without any further iproniazid administration. The stippled band at the left represents the range of all the control data, including saline and iproniazid injections. The arrow indicates the point in the time sequence at which the 5-HTP injections were made on any specific day. The curves labelled A and B are the pre-iproniazid injections of 50 mg/kg of 5-HTP. (By permission from the J. Neurochem.) References p . 78-80

70

M. H. A P R I S O N

period of time, the fall in the original potentiation of the first 5-HTPLinjection might be correlated with the recovery or regeneration of the brain M A 0 activity. These ideas were checked by the experiments described below (Aprison and Ferster, 1961b). Saline as well as 50 mg/kg of iproniazid phosphate were found to have no effect on the baseline. Apparently, the injection of iproniazid alone does not result in a sufficiently high concentration of serotonin at specific brain sites to produce behavior effects. The procedure used was then to establish the pre-iproniazid behavioral effect of 50 mg/kg 5-HTP in trained pigeons performing on the multiple F R 50 FI 10 schedules of reinforcement in the Skinner boxes. After a week, the birds were given iproniazid. The M A 0 inhibitor was injected in three 50 mg/kg doses spaced equally over a period of approximately 40 h. In Fig. 14, data on an experiment are presented to show the enhanced behavioral effect of 50 mg/kg 5-HTP after pretreating with I

0 '

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0

I

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1

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10

20

30

40

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i

70

D A Y S

Fig. 15. Recovery of brain monoamine oxidase activity after iproniazid. Each of 32 pigeons was given the same iproniazid treatment as in the 5-HTP experiments and sacrificed at the indicated time. (By permission from the J . Neurochem.)

150 mg/kg of iproniazid. One hour after the third iproniazid injection, a 50 mg/kg dose of 5-HTP was given (curve 1). At different times over a 50-day period, the same 50 mg/kg 5-HTP dose was given without any further iproniazid administration; several typical daily performances are shown by curves 2, 3, 4, 5, 6, and 7. On days the pigeon was not injected with 5-HTP. its daily performance was that shown by the control data. The performance was recorded cumulatively as the number of pecks the pigeon makes plotted against time. The raw data were recorded on graphic cumulative recorders, digital display counters, and digital printing counters. The stippled band

NEUROHUMOR-ENZYME

SYSTEMS A N D BEHAVIOR

71

at the left of Fig. 14 represents the range of all the control data including saline and iproniazid injections. The arrow indicates the point at which injections were made in the time sequence. The curves labelled A and B are the pre-iproniazid injections of 50 mg/kg of 5-HTP. The data show the gradual return of the daily performance to the pre-iproniazid level. The identical experiment was repeated in the same as well as other pigeons with the only change being in the dose of 5-HTP injected (Aprison and Ferster, 1961b, c). A group of pigeons were then injected with three 50 mg/kg doses of iproniazid in the same way as described above and sacrificed at different times over the next 68 days. Brain and liver M A 0 activities were assayed as previously described (Aprison and Ferster, 1961b). The data in Fig. I5 show the recovery of brain M A 0 activity. Normal enzyme levels were reached in 35-42 days. The liver M A 0 activity in these same birds returned to normal in about 12 days, much sooner than the behavioral response returned to normal. The data from Fig. 14 are replotted in a simpler form in Fig. 16 to show the potentiation of behavioral effects of 5-HTP by iproniazid. This form is normally used for enzyme recovery curves (Fig. 15). The behavioral effect was expressed as the reciprocal of the atypical or depressed responding time (‘T’) during which the bird’s performance was disrupted by the injection of 5-HTP. After the first 5-HTP injection following the iproniazid treatment, the behavioral effects fell to about one-fourth of the control value. Thereafter, the bird’s performance gradually recovered, reaching maximal values of l/‘T’ at about 35 days. The l/‘T’ values larger than thecontrol occurring between the 35th and 47th day are probably due to the bird’s accommodation to repeated doses of 5-HTP rather than an above-normal increase of M A 0 activity during this period. The data presented in Fig. 17 for pigeon 13 Y, which is typical, correlate the behavioral effects of repeated 5-HTP doses (50 mg/kg) with brain M A 0 level present at that time. The ordinate values of each point are derived from behavioral measurements of the enhanced effect of injected 5-HTP after iproniazid pretreatment. The abscissa values were derived from the independent measurements of brain M A 0 activities described in Fig. 15. Even though the M A 0 data are from birds other than the one whose performance is being measured, the interbird variability is small enough for the M A 0 values to be reliably extrapolated. As the brain M A 0 activity increased, so did the behavioral measure (l/‘T’), the slope of the curve being greater at the higher enzyme levels. In similar experiments in which pigeons were pretreated with three 50 mg/kg doses of iproniazid, the effect of injecting 10 and 25 mg/kg doses of 5-HTP was also studied (Aprison and Ferster, 1961b, c). Therefore, it was possible to plot the behavioral effect against dose of 5-HTP, with brain M A 0 activity as a parameter. At each dose of 5-HTP, the greatest behavioral effect was obtained at the lowest brain M A 0 activity. The slopes of the curves decreased with a rise in brain M A 0 activity indicating clearly that a smaller behavioral effect of 5-HTP occurred as the brain M A 0 levels recovered (Aprison and Ferster, 1961b). Following the demonstration in pigeons that a quantitative relationship existed References p . 711-110

72

M. H. A P R I S O N

:I

1401

I

I

I

I

I

I

I

I

I

I

80

60

20

atypical responding

Fig. 16. Potentiation of the effect of 5-HTP by iproniazid pretreatment. The curve expresses the behavioral effect of a constant 5-HTP dose injected periodically during 47 days following iproniazid treatment. (By permission from the J . NeurocliePn.)

0.014

1

I

I

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Fig. 17. Correlation of the behavioral effect of 50 mg/kg of 5-HTP with the brain M A 0 activity current at the time of the injection. The range of the behavioral measurement and brain M A 0 activity are given in the upper left and lower right-hand part of the figure respectively. The behavioral measurement was expressed as I/'T', the rcciprocal of the time during which the bird's performance was depressed or atypical. Brain M A 0 activity was expressed as itmoles NHJ per g per h. (By permission from the J . Neuvocketrr.)

N E U R O H U M OR-.EN 2 Y M E S Y S TE M S A N D B E H A V I O R

73

between the change in its behavior and the change in its brain M A 0 activity when 5-HTP is injected after pretreatment with the enzyme inhibitor iproniazid, it became imperative to study the relationship between this behavioral change and brain 5-HT concentration. The original data (Aprison and Ferster, 1961b) had suggested that the behavioral change seen in the birds was due to the action of increased free 5-HT in brain, the latter’s concentration being controlled by the level of the activity of its catabolic enzyme, MAO. Therefore, in pigeons not pretreated with iproniazid, the length of the behavioral effect was thought to be determined by the length of time it took the enzyme to destroy the large excess of free 5-HT resulting from the injection of its precursor (50 mg/kg of 5-HTP). It was reasoned that the 5-HT concentration from the specific brain areas involved in the behavioral response should, therefore, pass through a maximum and then return to normal levels at the time the behavioral effect ceases and the pigeon’s performance becomes normal. The 5-HT concentration in brain areas and peripheral organs not involved directly or indirectly in the production of the behavioral effect should also pass through a maximum, but then return to normal either before or after the behavior returns to normal. The usual manner of handling the data in experiments of this type is to determine the 5-HT concentrations in various tissues from many animals as a function of time, and then plot the mean neurohumor level at each time along with its range or standard deviation. In the study to be described this was not done for the following reason. The technique mentioned above makes no attempt to account for the variation of the behavioral effect from bird to bird even though the same dose of 5-HTP was injected under identical conditions. The experiments described below permitted the application of an unusual technique, namely, using the predetermined maximal behavior of each bird as its own weighting factor. By dividing the duration (minutes) of the behavioral effect exhibited by the pigeon from injection until death by the total length of time (minutes) of the behavioral effect (determined during earlier sessions), a new number was found which, though dimensionless, was proportional to the time of the behavioral effect on the terminal day for the particular pigeon under consideration. This number was designated as a percentage of ‘T’. This new method of handling the raw data markedly reduced variability and permitted the use of fewer trained birds to complete the study. By using percentage ‘T’ as the independent variable and 5-HT concentration as the dependent variable, it was possible to compare and correlate the duration of change in 5-HT concentration with the duration of the behavioral effect produced by the injection of 5-HTP. Tissue preparation and 5-HT assay: at some predetermined time based on the behavioral data (see below), a pigeon was removed from the Skinner box and immediately decapitated. The brain was quickly removed from the skull and divided into 4 samples based on function and convenience : telencephalon, cerebellum, ponsmedulla oblongata, and diencephslon plus optic lobes. The parts were immediately frozen between pieces of dry ice as they were removed; each was wrapped with aluminum foil, labelled, and stored at -28”. Simultaneously, the liver, heart, and lungs were removed. The organs were cut into small pieces, freed of blood clots, and processed in the same manner as the brain parts. Serotonin analyses were carried out Refeiences p . 78-80

74

M. H. A P R I S O N

within 24 h. The method of extracting and assaying 5-HT from brain and the other tissue samples followed closely the analytical procedure of Bogdanksi et al. (1956). However, three minor modifications were necessary owing to the small tissue samples of the pons-medulla oblongata and cerebellum (Aprison et al., 1962). The birds were trained in a special multiple-unit pigeon elevator (Ferster and Aprison, 1960) prior to being placed in the standard Skinner box. This technique provided the study with a ready source of trained birds for 5-HT analysis. The total time of training each bird was approximately 9-12 weeks. The baseline used to determine the effects of the injected 5-HTP was again a multiple FR 50 FT 10 schedule of reinforcement. The bird’s performance on control and experimental sessions was typical of the data presented above. The major behavioral effect of 50 mg/kg 5-HTP was determined by measuring ‘T’. This period of time (in minutes) was determined quantitatively. The data from experimental and control sessions for each bird were plotted on the same graph, cumulative responses (pecks) on the ordinate and cumulative time in minutes on the abscissa. The duration of the period of atypical or depressed responding was then easily measured. After the raw data were plotted in this manner, the best straight lines were constructed through the data relating to the period after recovery and to the period during maximal behavioral effect. The lines were extended to their point of intersection, defined as point P. The behavioral measure, ‘T’, was taken as the time interval from injection to the extrapolated value on the time axis, defined by P (Aprison et al., 1962). Since it was assumed in the design of the experiment that abnormal behavior would result from the elevation of normal 5-HT levels in brain, it was decided that the period between the time of injection and time on the abccissa axis corresponding to point P is equal to ‘T’. In general, when a bird’s responding was not totally depressed but certainly atypical (i.e. a very small number of responses was made either in the fixed-ratio or fixed-interval) the X-coordinate of point P still determined its value. Clearly there is one unsupported assumption made in handling the data, namely, that the nature of the behavioral response is the same in all the pigeons. Since the environmental conditions of the experiment are so rigidly controlled, and the administered metabolite presumably acts in the same system, there is good justification for making this assumption. The mean ‘T’ for the 14 pigeons in the experimental group was 154 min varying in range from 82 min to 308 min. After the ‘T’ value was determined for each trained pigeon from the mean of at least 3 runs (the measurement of ‘T’ was reproducible within f 15 %), it was decided at what percentage of ‘T’ the bird should be killed. At a subsequent experimental session (at least 60 h later), a 4th injection of 50 mg/kg of 5-HTP was made, the bird was killed at the determined time, and specimens were taken for analysis. The 5-HT concentration (,ug/g) of the telencephalon, diencephalon plus optic lobes, cerebellum, and pons-medulla oblongata of the brain, as well as liver, heart, and lung, was measured in 9 control pigeons. These data are shown in Table VI. Each bird was injected intramuscularly with 0.5 ml of saline instead of 5-BTP. The mean 5-HT values of the above-mentioned parts were 1.04, 0.91, 0.25, 1.17, 0.32, 0.22, and 0.17

NEUROHUMOR-ENZYME

75

SYSTEMS A N D BEHAVIOR

TABLE VI S E R O T O N I N C O N C E N T R A T I O N ( / l g / g ) I N S E V E R A L B R A I N ARE AS A N D P E R I P H E R A L O R G A N S

Pigeon no.

State Of training

Telencephalon

1 2 3 4 5 6 7 8T YT

None None None None None None None Trained Trained

1.00 1.08 0.95 0.78 1.35 0.97 1.24 0.95 1.03

DiencephCerealon bellum optic lobes

+

0.60 0.66 1.07 0.74 1.09

1.19 0.78 1.12

0.35 0.17 0.16 0.44 0.12 0.34 0.19 0.31 0.16

Pornmedulla

Liver

Heart

Lung

1.41 0.96 0.93 1.41 0.93 1.04 1.31 1.50 0.99

0.48 0.39 0.22 0.33 0.31 0.27 0.23 0.43 0.25

0.30 0.21 0.28 0.13 0.18 0.30 0.11 0.21 0.23

0.26 0.18 0.08 0.11 0.22 0.18 0.20 0.12

*

pglg, respectively. Included in the controls were two trained pigeons; they had been working on the multiple FR 50 FI 10 schedule of reinforcement as long as the experimental birds. In addition, these pigeons were given the same series of intramuscular injections of 5-HTP. However, saline was injected on the day they were killed. Neither the 5-HT data nor the weight of the brain parts for these two pigeons differed significantly from those of the 7 untrained pigeons. The peripheral parts did decrease in weight (liver > heart > lung). Apparently, the serotonin levels decreased proportionally; this resulted in 5-HT concentrations of the order found in comparable organs in the other 7 pigeons. In Fig. 18, the 5-HT concentrations are shown in 4 different brain areas during the complete period of atypical responding as well as after the behavior has returned to normal. After an intramuscular injection of 50 mg/kg 5-HTP, the brain 5-HT level reached its maximum at 30% ‘T’. This occurred in all 4 brain areas studied. There appeared to be a slight lag before the 5-HT levels began to rise rapidly. The 5-HT concentration of the pons-medulla oblongata and cerebellum samples decreased rapidly after reaching the maximum; at 60 % ‘T’, well within the period during which the pigeons were still not responding normally, the 5-HT concentrations returned to normal. The 5-HT concentration in the telencephalon and diencephalon (plus optic lobes) declined more gradually after reaching the maximum, attaining normal values at approximately 100% ‘T’. At this point the pigeons were beginning to respond normally to the presentation of the stimuli of the schedule of reinforcement. The maximal 5-HT concentration in each brain part was about 4 times higher than the mean normal value of the respective part. The highest 5-HT concentration reached was 4.75 ,ug/g, which occurred in the pons-medulla oblongata. The maximal concentration in the telencephalon was 4.2 ,ug/g, in the diencephalon it was 3.5 pglg, and in the cerebellum, 1.O ,ug/g. The rate of change in 5-HT concentrations of the three peripheral tissues in the experimental birds receiving 50 mg/kg of 5-HTP followed in general the same pattern Referenres p . 78-80

M. H. A P R I S O N

76 5 .O 4 .O 3 .O 2 .o

1.o M

2.0

2

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ua

CEREBELLUM

4.0

3.0 2.0

1.0 0

5.0 4.0

3 .O 2 .o

1 .o 0 0

60 70 80 90 100 110 120 130 140 P E R C E N T ”T”

10 20 30 40 50

ATYPICAL BEHAVIOR or DEPRESSED

-+t-;:z:::

-b

Fig. 18. Serotonin concentrations in 4 different parts of the pigeon brain during the complete period of atypical responding as well as approximately 45 min after behavior had returned to normal. Each point represents a single determination of a part taken from a trained pigeon that received 50 mg/kg 5-HTP intramuscularly. (By permission from the J . Neurochem.)

as that noted in the brain parts. These data are shown in Fig. 19. The maxima appeared at approximately 25% ‘T’, a little earlier than in brain tissue. The heart and lung 5-HT concentrations returned to normal values at 60% ‘T’. The maximal heart 5-HT concentration was approximately 1.5 ,ug/g, a value 7.5 times higher than the mean control value. In the lung, the maximum was about 4 times higher than the mean control value (0. I7 ,ug/g). In the liver, the 5-HT concentration levelled off at 1.5 pg/g after passing through a maximum at 3.1 ,/ig/g (or 10 times higher than normal). The 5-HT level to which the liver returned was 5 times higher than normal. The exact length of time for the liver 5-HTconcentration to return to normal was not determined; it is certainly longer than 128% ‘T’. Apparently the decline in liver 5-HT concentration is much more gradual than in the brain, heart, and lung. Recent experiments in this laboratory have shown that M A 0 activity is much higher in pigeon liver than

NEUROHUMOR-ENZYME

77

SYSTEMS A N D BEHAVIOR

3.0 M

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90

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LUNG

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110 120 130 140

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Fig. 19. Serotonin concentrations in liber, heart, and lung during the period described in Fig. 18. Each point represents the average of triplicate determinations of the part taken from a trained pigeon receiving 50 mp/kg of 5-HTP intramuscularly. (By permission from the J . Neurocheni.)

in the brain when assayed against tyramine as substrate, but the difference was much less when 5-HT was used as substrate. It therefore appears that the liver enzyme is less specific in its activity towards 5-HT than the brain enzyme, and that there may be several types of monoamine oxidase as in the case of the cholinesterases. Of the brain and peripheral tissues studied, the telencephalon and diencephalon were the only parts in which the 5-HT concentration passed through a maximum and then returned to normal at the same time as the behavior returned to normal. The author feels that the biochemical data presented provide evidence that the behavioral effect is due mainly to a central mechanism rather than a peripheral one. Furthermore since the posterior parts of the brain returned to their normal 5-HT concentration at 60% 'T', the data show that all portions of the brain are probably not involved in the production of the behavioral changes noted. Finally, it appears that the location of the area or areas affected by the increased levels of free 5-HT which appear to be responsible for the atypical or depressed behavior exhibited by the whole organism is to be found in the telencephalon and the diencephalon, a finding that has also been suggested in the case of man. Included in these structures is the limbic system which contains the limbic lobe ('old' cortex) along with its associated nuclear structures (MacLean, 1952). The role of the limbic system in emotional behavior has been suggested many times, the first time probably by Papez (1937). In 5-HT studies on human brain, Costa and Aprison (1958b) found that the allocortex or Rcferrric es p . 78-80

78

M . H. A P R I S O N

‘old’ cortex had a higher 5-HT concentration than the isocortex. In addition the structures of the diencephalon and mesencephalon contained the largest amounts of 5-HT. Paasonen et al. (1957) using dog brain, also found that the cortical areas of the limbic system (medial and lateral pyriform cortex, entorhinal cortex, and hippocampus) showed relatively higher 5-HT values than the neocortex. In addition, the amygdala, septum, hypothalamus, and caudate nucleus also had high 5-HT values. The 5-HT behavioral experiments presented in this review of the author’s work strikingly confirm the hypothesis suggested earlier for the explanation of the behavioral effect. Although the exact mechanism of action of serotonin in brain is still unknown, several hypotheses or theories have been suggested during the last few years (Marrazzi and Hart, 1955; Woolley and Shaw, 1957; Woolley, 1958; Brodie et af., 1959; Costa, 1960; Costa e ta]., 1961 ; Aprison, 1962). However, onlythetheory suggested by Aprison (1 962) attempts to explain the biphasic action of 5-HT reported in the literature (Bogdanski et al., 1958; Costa and Rinaldi, 1958; Himwich and Costa, 1960; Costa et al., 1960). This was done by suggesting that 5-HT is a chemical modulator which exerts its action via the acetylcholine-cholinesterase system by competing with ACh molecules at the receptor proteins sites and the AChE sites at specific locations within the CNS. At the present time, sufficient data are not available to confirm any of the above suggestions. SUMMARY

The line of research reviewed above provides a basis for further study of which biochemical systems can affect the total behavior of an intact animal. In addition, it provides a technique for localizing the area within the brain which may be involved in the final control of the behavior. Although only two biochemical systems have been studied to date by the author and his co-workers, it also permits the testing of other systems suspected of being involved in the production of abnormal or atypical behavior. Finally, as the biochemical techniques of measuring these important labile compounds are developed to the point of assaying still smaller quantities of brain tissue containing neurohumoral agents, at levels such as are probably present in the free form at synapses, our knowledge in this area will advance rapidly. ACKNOWLEDGEMENT

The latter portion of the work reported in this paper was supported with funds from Research Grant MH-03225-05 from the National Institute of Mental Health, U.S. Public Health Service. REFERENCES APRISON, M. H., (1958); Rate of compulsive circling in relation to accumulation of cerebral acetylcholine. J. Neurochem., 2, 197-200. APRISON, M. H., (1962); On a proposed theory for the mechanism of action of serotonin in brain. Recent Adv. biol. Psychiat., 4, 133-146. APRISON, M. H., AND FERSTER, C. B., (1961a); Neurochemical correlates of behavior. 1. Quantitative measurement of the behavioral effects of the serotonin precursor, 5-hydroxytryptophan. J. Pharmacol. exp. Ther., 131, 100-107.

NEUROHUMOR-ENZYME

SYSTEMS AND BEHAVIOR

79

APRISON,M. H., AND FERSTER, c . B., (1961b); Neurochemical correlates of behavior. 11. Correlation of brain monoamine oxidase activity with behavioral changes after iproniazid and fi-hydroxytryptophan administration. J . Neurochem., 6, 350-357. APRISON, M. H., AND FERSTER, C. B., (1961~);Serotonin and behavior. Recent Adv. biol. Psychiat., 3, 151-162. APRISON,M. H., A N D NATHAN, P., (1957a); Determination of acetylcholine in small samples of fresh brain tissue. Arch. Biochem. Biophys., 66, 388-395. APRISON, M. H., AND NATHAN, P., (1957b); Acetylcholine concentrations in the brain of rabbits exhibiting forced turning movements. Amer. J . Physiol., 189, 389-394. APRISON, M. H., NATHAN, P., AND HIMWICH,H. E., (1954a); A study of the relationship between asymmetric acetylcholinesterase activities in rabbit brain and three behavioral patterns. Science, 119, 158-159. APRISON, M. H., NATHAN, P., AND HIMWICH, H. E., (1954b); Brain acetylcholinesterase activities in rabbits exhibiting three behavioral patterns following the intracarotid injection of di-isopropyl fluorophosphate. Amer. J . Physiol., 177, 175-1 78. APRISON, M. H., NATHAN, P., AND HIMWICH, H. E., (1956); Cholinergicrnechanism of brain involved in compulsive circling. Amer. J . Physiol., 184, 244-252. APRISON, M. H., WOLF,M. A., POULOS, G. J., AND FOLKERTH, T. L., (1962); Neurochemical correlates of behavior. 111. Variation of serotonin content in several brain areas and peripheral tissues of the pigeon following 5-hydroxytryptophan administration. J . Neurochem., 9, 575-584. BENTLEY, G. A., AND SHAW,F. H., (1952); The separation and assay of acetylcholine in tissue extracts. J . Pharmacol. exp. Ther., 106, 193-199. BOGDANSKI, D. F., PLETSCHER, A., BRODIE,B. B., A N D UDENFRIEND, S., (1956); Identification and assay of serotonin in brain. J . Pharmacol. exp. Ther., 117, 82-88. BOGDANSKI, D. F., WEISSBACH, H., AND UDENFRIEND, S., (1958); Pharmacological studies with the serotonin precursor, 5-Hydroxytryptophan. J . PharmacoI. exp. Ther., 122, 182-194. BOUZARTH, W. F., AND HIMWICH, H. E., (1952); Mechanism of seizures induced by di-isopropyl fluorophosphate (DFP). Amer. J. Psychiat., 108, 847-855. BRODIE, B. B., SPECTOR, S., AND SHORE,P. A., (1959); Interaction of drugs with norepinephrine in the brain. Symposium on Catecholamines. 0 . Krayer, Editor. Baltimore, Williams and Wilkins (pp. 548-564). BULLOCK, T. H., GRUNDFEST, H., NACHMANSOHN, D., AND ROTHENBERG, M. A., (1947); Effect of di-isopropyl fluorophosphate (DFP) on action potential and cholinesterase of nerve 11. J . Neurophysiol., 10, 63-78. CORTELL, R., FELDMAN, J., AND GELLHORN, E., (1941); Studies on cholinesterase activity and acetylcholine content of the central nervous system. Amer. J . Physiol., 132, 588-593. COSTA,E., (1956); Effects of hallucinogenic and tranquilizing drugs on serotonin evoked uterine contractions. Proc. Soc. exp. Biol. ( N .Y.), 91, 3941. COSTA,E., (1960); Analysis of a proposed mechanism of serotonin action in neurotransmission. Studies of Function in Health and Disease. A. S. Marrazzi and M. H. Aprison, Editors. Galesburg, Galesburg State Research Hospital Press (pp. 103-1 19). COSTA,E., AND APRISON, M. H., (1958a); Distribution of intracarotidly injected serotonin in brain. Amer. J. Physiol., 192, 95-100. COSTA,E., AND APRISON,M. H., (1958b); Studies on the 5-hydroxytryptamine (serotonin) content in human brain. J . nerv. ment. Dis., 126, 289-293. COSTA,E., GESSA,G. L., HIRSCH,C., KUNTZMAN, R., AND BRODIE, B. B., (1961); On current status of serotonin as a brain neurohormone and in action of reserpinelike drugs. Ann. N . Y. Acad. Sci., 96, 118-133. COSTA, E., PSCHEIDT, G. R., VANMETER, W. G., AND HIMWICH, H. E., (1960); Brain concentrations of biogenic amines and EEG patterns of rabbits. J . Pharmacol. exp. Ther., 130, 81-88. COSTA,E., AND RINALDI,F., (1958); Biochemical and electroencephalographic changes in the brain of rabbits injected with 5-hydroxytryptophan (influence of chlorpromazine premedication). Amer. J. Physiol., 194, 214-220. DEWS,P. B., (1955); Studies on behavior. I. Differential sensitivity to pentobarbital of pecking performance in pigeons depending on the schedule of reward. J . Pharmacol. exp. Ther., 113, 393-401. DEWS,P. B., (1956); Modification by drugs of performance on simple schedules of positive reinforcement. Ann. N . Y. Acad. Sci., 65, 268-281. DEWS,P. B., (1958); Studies on behavior. IV. Stimulant actions of methamphetamine. J. Pharmacol. exp. Ther., 122, 137-147.

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M. H. A P R I S O N

DIXON,M., A N D NEEDHAM, D. M., (1946); Biochemical research on chcmical warfare agents. Nature, 158, 432-438. Essic;, C. F., HAMPSON, J . L., B A L ~ SP., D., WILLIS,A,, AND HIMWICH, H . E., (1950a); Effect of panparnit on Lrain wave changes induced by DFP. Science, 111, 38-39. Essir;, C. F., HAMPSON,J. L., A N D HIMWICH, 1-1. E., (1953): Biochemically induced circling behavior. Confin. Neitrol., 13, 65-70. Essrc, C. F., HAMPSON,J . L., MCCAULEY, A., A N D HIMWICH,H. E., (1950b): An experimental analysis of biochemically induced circling behavior. 3. Neiirophysiol., 13, 269-275. FERSTER, C. B., A N D APRISON, M. H., (1959); Increased behavioral effects of 5-hydroxytryptophan by ipraniazid pretreatment. Pharti:nco/ogist, 1,75. FERSTER, C. B., A N D APRISON, M . H . , (1960); A multiple-unit pigeon apparatus. J . e x p . Anal. Behav., 3, 165-166. FERSTER, C . B., AND SKINNER, B. F., (1957); Schedules of Reinforcenzenl. New York, AppletonCen tury-Crofts. FREEDMAN, A . M., AND HIMWICH, H . E., (1949); D F P : Site of injection and variation i n response. Anier. J . Physiol., 156, 125-128. GAI)UUM, J. H., (1954); Drugs antagonistic to 5-hydroxytryptamine. Ciha Found. Sytnp. Hypertension-Humoral Neurogenic Facfnis. G . E. W. Wolstenholme and M. P. Cameron, Editors. Boston, Little, Brown (pp. 75-77). HAMPSON, J. L., Essic, C. F., MCCAULEY, A,, A N U HIMWICH, H . E., (1950); Effects of di-isopropyl fluorophosphate (DFP) on electroencephalogram and cholinesterase activity. Electroenceph. clin. Neurophysiol., 2,41-48. HIMWICH, H. E., ( I 953); Some effects of D F P and atropine on behavior. Arzneintittel-Forsch., 3, 228-23 I . HIMWICH, H . E., Essrc, C. F., HAMPSON, J . L., BALES,P. D., A N V FREEDMAN, A. M., (1950); Effect of trimethadione (Tridione) and other drugs on convulsions caused by di-isopropyl fluorophosphate (DFP). Airier. J . Psycliiat., 106, 816-820. H I M W I C W. H , A., AND COSTA,E., (1960); Behavioral changes associated with changes in concentration of brain serotonin. Fed. Proc., 19, 838-845. MACL~AN P., D., (1952); Some psychiatric implications of physiological studies on frontotemporal portion of limbic system (visceral brain). Elecfroenceph.elin. Neurophysiol., 4,407-418. MARRAZZI, A. S., A N D HART,E. R., (1955); The possible role of inhibition at adrenergic synapses in the mechanism of hallucinogenic and related drug reactions. J . nerv. ment. Dis., 122, 453457. MCDONALD, D. A., A N D POTTER, J . M., (1951); Distribution of blood to brain. 1.Physiol., 114,356. METTLER, F. A., ( I 942): Relation between pyramidal and extrapyramidal function. Ass. Res. ncrv. nwnt. Dis., 21, 150-227. MICHAELIS, M., FINESINGER, J. E., DEBALBIAN VERSTER, F., A N D ERICKSON, R. W., (1954); The erect of the intravenous injection of D F P and atropine on the level of free acetylcholine in the cerebral cortex of the rabbit. J . Pharniacol. exp. Ther., lil, 169-175. N A T H A N , P., APRISON, M . H., A N D H I M W I C H ., E., (1955); A comparison of the effects of atropine with those of several central nervous system stimulants on rabbits exhibiting forced circling following the intracarotid injection of di-isopropyl fluorophosphatc. Confin. Neurol. 15, 1-10. PAASONEN, M. K., MACLEAN,P. D., AND GIARMAN, N . J . , (1957); 5-Hydroxytryptamine (serotonin, enterainine) content on structures of the limbic system. J . Nenrochcm, 1, 326-333. PAPEZ,J. W., (1937); A proposed mechanism of emotion. Arch. Neurol. Psychiat., 38, 725-743. STEWART, W. C., (1952); Accumulation of acetylcholine in brain and blood of animals poisoned with cholinesterase inhibitors. Brit. J . Pharniacol., 7 , 270-276. UDENFRIEND, s., SHORE,P. A., BOGDANSKI, D. F., WEISSBACH, H., AND BRODIE,B. B., (1956); Biochemical physiological and pharmacological aspects of serotonin. Recent Progr. Horrn. Rm.13, I . UIXNFRIEND, S., WEISSBACH, H., A N D ROGDANSKI, D. F., (1957); Effect of iproniazid on serotonin metabolism in vivo. J . Pharmacol. exp. Tlier., 120, 255-260. VERHAVE, T., (1959); The effect of secobarbital on a multiple schedule in the monkey. J . exp. Anal. Behav., 2, 117-120. WOOLLPY, D. W., (1958):A probable mechanism of action of serotonin. Proc. N a t . Acad. Sci., 44,197. W~OLI.FY, D. W., A N D SHAW,E., (1954a); A biochemical and pharmacological suggestion about certain mental disorders. Proc. Nat. Acad. Sci., 40,228-23 I . WOOLLEY, D. W., A N U SHAW, E., (1954b); Some neurophysiological aspects of serotonin. Brit. ~7fd J., 2, 122-132. WOOLLEY, D. W., AND SHAW,E. N., (1957); Evidence for the participation of serotonin in mental processes. Ann. N. Y . Acari. Sci.. 66, 649-667.

81

Metabolism of Biogenic Amines and Psychotropic Drug Effects in Schizophrenic Patients G. G . B R U N E Neurologische Universitatsklinik und Poliklinik, Hamburg 20 (Germany)

During the last decade biochemical research in the field of mental illness was greatly stimulated by observations indicating that biogenic amines such as indoleamines and catecholamines may serve as neurotransmitters in brain function. This hypothesis was supported by the fact that many psychotropic agents such as psychotogenics, tranquilizers and antidepressants interfere with the metabolism or the actions of biogenic amines. I n the light of these investigations it was further speculated that these amines may play a role in schizophrenia. As yet, however, experimental results as well as their interpretations remain controversial. These controversies may find their explanation at least in part in differcnces of the methodological approaches to the problem of schizophrenia. Any attempt at a biological concept of ‘schizophrznia’ is confronted with the fact that the diagnosis is poorly defined and that this important mental illness may not represent a nosological entity but a variety of pathogenic conditions. I n addition, it is typical for the ‘schizophrenic’ disease to have a tendency to reversibility and severe psychotic states may alter with full or partial remissions. Therefore, it is conceivable that even in an individual patient various biochemical patterns may prevail at different times according to the actual state of the psychopathology. Our work was primarily concerned with the question of a rzlationship between amine metabolism and psychotic behavior. We studied this problem in patients belonging to the diagnostic category of ‘schizophrenia’, and especially in those with paranoid-hallucinatory psychoses. This paper will review some of our work on this subject, in particular thc investigations on urinary excrztions of indoles and catecholes in relation to psychotic behavior, and on the effects of indoleamine precursors, methyl donors and psychotropic drugs on the psychopathology of schizophrenic patients. All indoles derive from thc essential amino acid tryptophan. Decarboxylation of this amino acid yields tryptamine which in turn can be hydroxylated in 6-position to form 6-hydroxytryptamine. Hydroxylation of tryptophan in 5-position forms 5-hydroxytryptophan which is the precursor of 5-hydroxytryptamine or serotonin (Mitoma et al., 1956). Recently, Axelrod (1961) could show that the mammalian organism is able to produce N,N-methylated tryptamines such as N,N-dimethylR&ences

p . 95/96

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G. G . B R U N E

tryptamine and N,N-dimethyl-5-hydroxytryptamine.Only a very small fraction of the indoleamines formed in the body are found in the urine. The far greater portion is metabolized by monoamine oxidase to indole acids which are then excreted into the urine either in bound or free form (Weissbach et a]., 1959; Udenfriend et al., 1956; Erspamer, 1955). The catecholamines are derived from the amino acid tyrosine. Formation and fate of noradrenaline and adrenaline were reviewed by Schumann (1960) and Axelrod (1960). As is the case for the indoleamines, only small amounts of catecholamines are found in the urine the greater fraction undergoing metabolic changes before excretion. I n our studies on urinary indoles and catecholamines in relation to psychotic behavior we determined the indole derivatives, tryptamine, total indole-3-acetic acid and 5-hydroxyindoleacetic acid according to the methods reported by Sjoerdsma et al. (1959), Weissbach et al. (1959) and Udenfriend et al. (1955) respectively in 24-h urinary samples. Urinary total catecholamines (noradrenaline and adrenaline) were assayed by methods described by Von Euler and Floding (1956), and by Von Euler and Lishajko (1959). The importance of factors unrelated to, or not directly related to, the subject of the study but able to influence urinary excretion of the biogenic amines was considered in this investigation. It has been reported that the amount of urinary indoles is influenced by dietary factors, by medication, by metabolism of intestinal bacteria as well as by the function of the intestine itself. In addition, various somatic diseases are associated with abnormal excretion patterns. These factors have been previously reviewed by Brune and Himwich (1962). We tried to exclude these interfering factors as far as possible. The patients selected were free of somatic diseases and did not present feeding problems; before the investigation was begun and also throughout its entire course the patients received a constant protein diet of 100 g protein/day. Observations were made over a period of at least 20 days and some of the patients were studied repeatedly. In order to compare the level of the urinary amines with the actual state of the psychopathology, the patients were given daily psychiatric interviews and their behavior on the ward was also observed. The complexity of the psychopathology of individual patients is well-known and reduction to some clinically observable criteria, which, of course, means a simplification, is a methodological necessity. In our attempts to evaluate the intensity of the psychosis, particular regard was paid to such symptoms as the level of consciousness, affect, disorganization of thought, and disturbance of perception as well as to motor activity and ward adjustment. The intensity of the symptoms of the psychosis was divided into 5 degrees of severity: ( I ) no apparent psychotic activity; (2) slight; (3) moderate; (4) marked; and (5) severe activity of the psychosis. In addition, the degree of anxiety was evaluated according to criteria established previously (Brune et af., 1963; Brune and Pscheidt, 1961) and was divided into 5 different degrees of severity. In our patients when the psychosis was apparently inactive, the average tryptamine excretion was 9 I .6 pglday which is within the normal range (36-1 20 pglday, Sjoerdsma

A M I N E METABOLISM A N D P S Y C H O T I C B E H A V I O R

83

et al., 1959) while the average for urinary total indole-3-acetic acid was with 16.3 mg/day slightly above the range of normal (5.2-13.8 mg/day, Weissbach et al., 1959).

A correlation can be made between the degree of severity of the psychosis, the urinary levels of tryptamine and its metabolic product indole-3-acetic acid (Fig. 1). Associated with the intensification of the psychosis a continuous and significant increase in the urinary excretion of both indole derivatives occurred which was more marked for urinary tryptamine than for total indole-3-acetic acid. Protein intake continued at about the same magnitude during all degrees of psychotic activity with the exception of degree 5, during which protein intake dropped markedly. The data obtained during periods of placebo and reserpine administration (4 mg/day) have been combined because the same excretion patterns were found during both medication regimes (Brune and Pscheidt, 1961). Additional observations on 9 patients who werc included in the previous group showed similar excretion patterns and demonstrated that the described results are

~

.=PC0.01

.= p,D. o,

= =

Upper limit of normal range. ( Total I A A = 100 % in .lightly above the upper limit of normal rmse ) Probability of diiference from d e g r e e 1 .

Fig. 1 . Urinary tryptamine and total indole-3-acetic acid as related to the degree of psychotic activity. The values are calculated on a basis of 100 g protein intake/day. (Taken from Brune and Himwich, 1963). References p . 95!96

84

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.

No. of Patients

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I

9

T o t a l d a y s of investigation

295

11-Moderately or markedly active

mn U p p e r l i r n ~ t o f n o r m a l .

Fig. 2. Urinary indoles as related to the degree of psychotic activity. The data represent the averages for 295 and 65 days of investigation respectively. The values are calculated on a basis of 100 g protein intake/day. (Taken from Brune and Himwich, 1963).

rzproducible (Brune and Himwich, 1963). In this group with relatively few patients we contrasted degrees 1 and 2 of psychotic activity with degrees3 and 4 (Fig. 2). In the latter group with the more severe symptoms of psychosis excretion levels of tryptamine and total indole-3-acetic acid were significantly higher than when the psychosis was either apparently inactive or slightly active (tryptamine: p 0.001 : ; total indole-3-acetic acid: p < 0.01). A similar correlation to that observed for tryptamine and total indole-3-acetic acid was also found for the serotonin metabolite, 5-hydroxyindoleacetic acid (Fig. 3) i n a total of 20 patients (Brune and Pscheidt, 1961; Himwich and Brune, 1964). During a period of relatively inactive psychosis (degrce 1) the urinary 5-hydroxyindoleacetic acid was 5.9 nig/day, which i s within the normal range reported by Haverback et al. (1956). With intensification of the psychosis excretion values became significantly different from those obtained when thz patients showed no psychotic activity (p < 0.01). It is of interest that tryptamine which reflects chiefly tissue metabolism (Sjoerdsma et al., 1959) shows the most pronounced correlation to the intensity of the psychosis while urinary total indolc-3-acetic acid and 5-hydroxyindoleacetic acid which might be influenced either by the intestinal bacteria, the function of the intestine or both factors shows this correlation to a lesser extent. As was mentioned above, the described correlations between urinary levels of indoles

AMlNE METABOLISM A N D P S Y C H O T I C BEHAVIOR

85

PlTTTl = Upper l i m i t of n o r m a l range. o=PO. 01

Fig. 3. Urinary 5-hydroxyindoleacetic acid as related to the degree of psychotic activity. The values are calculated on a basis of 100 g protein intake/day.

and intensity of the psychopathology could be observed during placebo as well as during reserpine administration. During treatment with monoamine oxidase inhibitors (MAOI), however, different patterns became evident. With this medication urinary tryptamine rose in all patients whether there was an exacerbation of the psychosis or not. This discrepancy may be explained on the basis of observations of Hess et al. (1959), who found that in guinea-pigs administration of M A 0 1 induced a sharp rise in urinary tryptamine but left brain levels unaltered. Administration of L-tryptophan on the other hand increased brain tryptamine although at the same time it has less effect on urinary tryptamine than the MAOI. Combined administration of a monoamine oxidase inhibitor and L-tryptophan evoked the greatest increase in urinary tryptamine and simultaneously increased brain tryptamine although less was found than with L-tryptophan alone. In contrast, brain serotonin rises (Hess et al., 1959; Spector et al., 1959) while its urinary metabolite 5-hydroxyindoleacetic acid decreases during treatment with monoamine oxidase inhibitors (Sjoerdsma et al., 1958; Brune and Himwich, 1961). Urinary total indole-3-acetic acid is affected very slightly by the administration of a MA01 (Brune and Himwich, unpublished observations). Thus it appears that if monoamine oxidase is inhibited urinary tryptamine and total indole3-acetic acid represent a poor indicator of the tryptamine level in the brain. On the other hand reduction of urinary 5-hydroxyindoleacetic acid appears to be associated with an increase of brain serotonin during treatment with MAOI. References p . 95/96

86

G. C ; . B R U N E

TABLE I T O T A L C A T E C H O L S I N U R I N E A S R E L A T E D TO T H E D E G R E E O F P S Y C H O T I C ACTIVITY A N D t i R A D E OF ANXIETY

Patient

B.M. A.S.

F.A. G.F.

Degree of psychotic activity

1 1 1 1 2 4

Grade of anxiety

Average toral catechob in wlday*

74 45

34 24 56 70

3

15

4

70

* The values represent averages of 10 tot 25 consecutive days during which the patients were given placebo. (Taken from Brune and Pscheidt, 1961). Differentiating between the severity of the psychosis and degree of anxiety (Brune and Pscheidt, 1961), we observed that urinary total catecholamines correlated with the degree of anxiety and not to the intensity of the psychosis (Table 1). With an intensification of the symptoms of anxiety there was an increase in the excretion of urinary total catecholamines. Additional studies (Pscheidt et al., 1960, 1961 ; Brune et al., 1963) have shown that during treatment with reserpine and MA01 urinary excretion levels of catecholamines do not follow drug-induced changes in brain catecholamines observed in animals (Spector et al., 1958). Urinary excretion of total catecholamines wa5 found to be little influenced by therapeutic doses of these drugs unless drug-induced alteration of anxiety, blood pressure and heart rate also occurred. In all instances decreases or increases of sympathetic activity were accompanied by corresponding changes in the levels of total urinary catecholamines. In summary these studies show a correlation between excretion of the indole derivatives determined in urine and the intensity of the psychosis on one hand, and between the level of total urinary catecholamines and degree of anxiety and sympathetic activity on the other. No correlation was found between the diagnostic category of schizophrenia and urinary excretion of either indoles or catechols. These observations imply that the correlations found between the intensity of the psychosis and urinary indoles may not be specific for the ‘schizophrenic’ psychosis but may also occur in psychoses of a different nature. Furthermore, the descIibed correlations are valid only on a statistical basis which takes into account the fact that individual patients may show differing excretion patterns (Brune and Himwich, 1963). These findings may offer some explanation for contradictions in the results on urinary indoles in schizophrenia reported in the literature. In a second approach to the question of a relationship between indole metabolism and psychosis we used paper chromatography methods to establish whether or not N,N-rnethylated tryptamine derivatives could be detected in the urine of schizophre-

A M I N E M E T A B O L I S M AND PSYCHOTIC BEHAVIOR

87

nic patients. These N,N-methylated tryptamines are known to induce psychosis-like states in human beings (Boszormenyi and Szara, 1958; Fabing and Hawkins, 1956). Our studies (Brune and Himwich, unpublished observations; Brune et al., 1964) were carried out on 5 male schizophrenic patients with active psychosis and on 3 nonpsychotic mental defectives as a control group. Three of the schizophrenic patients were also studied during administration of therapeutic doses of the MAOI, isocarboxazid, in combination with betaine (isocarboxazid, 30 mg/day ; betaine, 0.226 g/kg/day). In determining urinary indoleamines either a 24-h urinary sample or the average daily urinary volume from a 5-day period were analyzed. The samples were made alkaline by addition of 10% sodium hydroxide and finally were adjusted to pH 10 with 1 M borate buffer. Subsequently, they were extracted three times with one half of their volumes of ferrous sulfate washed ether. After evaporation of the ether under vacuum, the residue was taken up into acetone and spotted on Whatman No. I paper. For the two-dimensional paper chromatography we used the solvent systems n-propanol-ammonia (1 N) (5 : 1) and n-butanol-acetic acid-water (4 : 1 : 1) and dimethylaminocinnamaldehyde as spray. The paper-chromatographic characteristics of test compounds such as urea and the indoleamines tryptamine, serotonin, N,Ndimethyltryptamine, N,N-dimethyl-4-hydroxytryptamjne,N,N-dimethyl-5-hydroxytryptamine (bufotenin) and N,N-dimethyl-6-hydroxytryptamine were determined by spotting 6 ,ug of the individual substances on Whatman No. 1 paper. A good separation of these compounds was observed with the exception of bufotenin and N,Ndimethyl-6-hydroxytryptamine. Despite the fact that these substances showed different colors after spraying with dimethylaminocinnamaldehyde the intensive blue color of bufotenin was seen to easily camouflage the light grey-greenish shade of the other amine. Samples of control compounds were also extracted from 1 1 of aqueous solution. It was found that these substances could be readily detected by our method when their concentrations were 20 pg/lOOO ml or higher. In the first group of patients consisting of 3 schizophrenics and 3 mental defectives we found in each case indoleamines resembling those of tryptamine and serotonin. T A B L E 11 U R I N A R Y E X C R E T I O N OF I N D O L E A M I N E S I N S C H I Z O P H R E N I C A N D M E N T A L L Y D E F E C T I V E P A TIE N TS D U R I N G P L A C E B 0 A D M I N I S T R AT I 0 N

The numerator denotes the number of 24-h urinary specimens analyzed, and the denominator signifies the number of samples found to contain the particular indole amine. (Taken from Brune et al., 1964). Serotonin

Bufotenin

313

313

212 212 212 212 414

212 212 212 212 414

311 211 21 I 210 210 410

Patient

Diagnosis

Tryptamine

A

Schizophrenia Schizophrenia Schizophrenia Mental deficiency Mental deficiency Mental deficiency

B D F G H Rgferences p . 95/96

88

G. G. B K U N E

In addition, in the schizophrenic group we also observed a compound with the paperchromatographic characteristics of bufotenin (Table 11). In a second group of 5 schizophienic patients receiving the combination of isocarboxazid and betaine (Table 111) tryptamine and serotonin were found on all occasions, but the bufoteninlike compound was observed only in the same 3 patients who had excreted this compound during placebo administration. In general, all spots were larger in size and more intensive in color during the combined medication than during placebo periods. T A B L E 111 U R I N A R Y E X C R E T I O N O F I N D O L E A M I N E S 1N S C H I Z O P H R E N I C P A T I E N T S D U R I N G COMBINED ADMINISTRATION OF ISOCARBOXAZID A N D BETAINE

The numerator denotes the number of 24-h urinary specimens analyzed, and the denominator signifies the number of samples found to contain the particular indoleamine. (Taken from Brune e t a / . , 1964). Patient

Tryptantine

Serotonin

Bnfotenin

.

When the test compounds were added to an average 24-h urinary sample the spots were enlarged and when they were added to about 1/10 of the final urinary extract the spots occurred in the same positions as the unknown compounds in the remaining extracts (Fig. 4). It should be mentioned that RF-values differed somewhat from cxperiment to experiment, probably due to differences in the ionic strength of the various extracts. The relative position of the individual spots to each other, the shade of color as well as speed of color development, however, remained constant in all experiments. Irvine (1961) reported the occurrence of a non-indolic mauve factor with a R p value approximately that of bufotenin in the urine of schizophrenics. This ‘mauke factor’, however, yields a rapid transient rose with dimethylcinnamaldehyde followed by a brownish purple shade and, therefore does not appear to be identical with the bufotenin-like spot we found in our experiments. On the whole, our observations are in agreement with the findings of Fischer et al. (1961) that schizophrenic patients with active psychosis excrete a substance with the paper-chromatographic characteristics of bufotenin. Fischer did not find this spot in schizophrenic patients whose psychosis was apparently inactive nor did we detect it in non-psychotic mental defective patients. Failure to find this substance would not necessarily mean that this substance is not excreted by non-psychotic human beings. Bumpus and Page (I955) using bioassay and paper-chromatographic prozedures found the bufotenin-like compound in normal human beings and it might be that, as is the case for tryptamine, total indole-3-acetic acid and 5-hydroxyindoleacetic acid, the bufotenin-like substance is excreted in lower amounts in non-psychotic persons and thus was not detected by our method.

AMINE METABOLISM A N D PSYCHOTIC BEHAVIOR N-PROPANOL - A M M O N I A

(IN1 (5

I1

-

89

I1

-_

-

AUTHENTIC COMPOUNDS ADDED TO 1 8 0 TOTAL VOLUME OF URINARY EXTRACT --RECOVERED COMPOUNDS FROM TOTAL VOLbME OF EXTRACT OF 3100nl OF URlhf

SPRAY. DIMETHYLAMINOCINNAMALOEHYDE -REAGENT

Fig. 4. Representative chromatographic identification of urinary indoleamines in schizophrenic patients. The numbers represent the following compounds: 1 = urea; 2 = serotonin; 3 = tryptamine; 4 = bufotenin. (Taken from Brune and Himwich, 1963).

The above-reported correlations between the amount of excreted urinary indoles and the degree of severity of the psychosis in schizophrenic patients may suggest that alterations in indole metabolism might be involved in the observed changes in the psychopathology. From a correlation alone, however, no statement can be made in terms of a cause and effect relationship. In order to obtain further information on the question of a role of biogenic amines in the psychosis of schizophrenic patients additional parameters must be tested. One way to accomplish this end is by the administration of agents capable of altering the level of indoles in the body. Among such agents are the tranquilizer, reserpine. the antidepressant monoamine oxidase inhibitors as well as the indolesmine precursors, tryptophan and 5-hydroxytryptophan. Reserpine releases serotonin from its stores and thus lowers the level of total serotonin in the body. Thus this drug may induce high levels of free serotonin in the brain during its initial phase of administration. On the other hand, the MA01 drugs elevate the level of serotonin in the brain by blocking its catabolism. Clinical observations show relationships to these biochemical patterns. Reserpine as well as the monoamine oxidase inhibitors can induce recurrences and exacerbations of the psychosis in schizophrenic patients. This reaction is known to occur mainly during the first phase of reserpine administration and during the latter phase of treatment with monoamine oxidase inhibitors, i.e. in those instances when the level of drug-induced free serotonin is highest (Brune and Himwich, 1961). In interpreting drug-induced behavioral changes in biochemical terms it should be observed that the mentioned drugs do not only affect the metabolism of indoles but also that of catecholamines as well as other References p . 95/96

90

G . G. RRUNE

biochemical parameters, and that the relative amount of the various biogenic amines, their rate of formation and destruction may represent important factors in determining brain function (Costa et al., 1960). From recent evidence obtained in animal experiments it appears, however, that indole metabolism represents an important factor in determining drug-induced behavioral alterations. The synthetic amino acid, a-methylnz-tyrosine, depletes brain norepinephrine in the brain of mice with no change of serotonin and the animals treated with this compound show no signs of sedation. If reserpine is now given, serotonin decreases and the animals become sedated (Hess et al., 1961 ; Rrodie et al., 1961). The concept that changes in indole metabolism correspond to behavioral alterations i s supported by the fact that indoleamine precursors accentuate behavioral changes evoked by MAOI. Behavioral effects were observed after administration of indoleamine precursors singly and in combination with MA01 in animals and man. Animal experiments carried out by various authors have shown that administration of moderate doses of 5-hydroxytryptophan had a calming action on the animals while higher doses induced excitement and disturbed behavior (Bogdanski et a / . , 1958; Costa et al., 1959; Himwich and Costa, 1960). Similar behavioral changes were also observed after administration of tryptamine (Brown, 1960; Tedeschi et al., 1959). 5-Hydroxytryptophan was also used in humans, but on account of severe peripheral side effects only relatively small doses could be used (Klee et a]., 1960; Pollin et al., 1961). Klee and his colleagues (1960) reported a recurrence of the psychosis in one schizophrenic patient during infusion of 5-hydroxytryptophan. Sjoerdsma and others (1 959) gave L-tryptophan to non-schizophrenic patients in combination with a MA01 and observed behavioral changes best described as drunkness. Tryptophan alone did not evoke any behavioral effects. Lauer et al. (1958) and Pollin et al. (1961) described recurrences and exacerbations of the psychosis in schi~ophrenicpatients after combined treatment with MA01 and tryptophan. Others (Shaw et al., 1959 and Sutton, 1959) reported bizarre and exaggerated behavior in schizophrenic children after administration of tryptophan without MAOI. In a nonschizophrenic group these symptoms were less pronounced. We administered 2 g L-tryptophan to 7 schizophrenic patients for 2 days. During that time we observed an accentuation of the psychopathology in 2 patients, while the remaining 5 who were either in partial or full remission showed no change in the clinical symptomatology. These observations, although small in number suggest that in those 2 patients whose psychosis was active the relatively small dose of tryptophan was high enough to add to the already existing disturbance of indole metabolism and thus facilitate the increase in the psychopathology, while in the other 3 patients in full or partial remissions the tryptophan dose was not sufficient to bring about a greater aberration in the metabolism of indoles. This conclusion is in agreement with the findings of Pollin et al. ( I 961) who using higher doses of tryptophan (20 g DL-tryptophan/70 kg/day) in combination with a MAOI observed behavioral alterations including exacerbations of the psychoses in a number of schizophrenic patients. These investigations point to the conclusion that elevation of indoles in the brain by administration of indole amine precursors singly or in combination with MAOX may represent a biochemical factor in the corresponding activations of the psychoses in

91

A M I N E METABOLISM A N D PSYCHOTIC BEHAVIOR

schizophrenic patients. There is, however, no substantial evidence that these substances can induce a psychosis in non-psychotic humans. In contrast, even small amounts of N,N-dimethylated tryptamines evoke psychoses-like states in mentally healthy humans, suggesting that the N , N-dimethyl configuration plays an important role in the psychoactivity of tryptamines, a configuration which was also found to be important for the pharmacological effects of the tranquilizer and the antipsychotic agent, chlorpromazine (Brune et a]., 1963). Thus, the question arose whether or not changes in transmethylation processes would be involved in behavioral alterations. Tn our studies on the clinical effects of methylgroup donors equivalent amounts of methionine and betaine in terms of labile methylgroups were given at two dose levels. Prior to the administration of methylgroup donors in combination with isocarboxazid, the patients received the MA01 alone for 20 days and the behavior observed at the end of the isocarboxazid period was compared with that shown during the combined treatment. In addition, the behavioral effects of the sole administration of methionine were also studied. In 2 of 7 patients receiving DL-methionhe (0.286 g/kg/day) alone TABLE I V EFFECTS

OF

METHYL

DONORS

SINGLY AND

IN

COMBINATION

WITH

A

MONOAMINE

OXIDASE INHIBITOR O N THE BEHAVIOR O F S C H I Z O P H R E N I C PATIENTS

Placebo Drug

Time No. ofpatients studied No. of patients showing behavioral changes

Isocarboxazid (30 mglday) plus

DL-

DL-

DL-

Methionine (0.286 K/kg)

Methionine (0.286 glkg)

Methionine (0.572glkg)

Betaine

Betaine

(0'1'3 gikg)

(o.226 glkg)

5 days

5 days

2 days

10 days

10 days

7

9

9

7

7

2*

7

7

6

-

* Slight behavioral changes. (Taken from Brune and

6 ~~

Himwich, 1963).

for 5 days we observed a shortlked accentuation of the psychopathology (Table IV) There was no change in behavior in the other 5 patients. Incontrast, marked behavioral changes occurred in 7 of 9 schizophrenics when an equal dose of methionine was given in combination with isocarboxazid. The behavioral changes became still more pronounced when the methionine dose was doubled (DL-methionhe, 0.572 g/kg/day for 2 days). The main characteristics of these behavioral changes might be described in terms of two components: the first resembling symptoms commonly observed after alcohol intake as for example euphoria, sleepiness and confusion, while the other component appeared to be an accentuation of the individual psychosis. Euphoria was observed with the lower dose of methionine while sleepiness and confusion occurred when the higher dose was administered. In some of the patients only one of the comReferences p . 95/96

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Ci. C . D R U N E

ponents was predominant, in others, however, each could be distinguished clearly. In the latter case, the first component preceded those behavioral changes which appeared to be an accentuation of individual psychopathology. On the other hand, slight improvement including disappearance of hallucinations was observed in 2 patients. Behavioral effects of the combined treatment with betaine and isocarboxazid were investigated in 7 schizophrenic patients. Each dose level of betaine was administered for 10 days and the behavior during that period was compared with that observed during preceding and following periods when the MAOT was given alone. Administration of betaine evoked behavioral changes similar to those observed with thc lower dose of methionine. In contrast to methionine, however, the changes developed more slowly during betaine administration ; accentuation of hallucinations and delusions as well as the increase in disorganization in thought were more gradual and remained associated with an elevation of mood, a subjective feeling of improvement and reduced anxiety, the latter being accompanied by a decrease in urinary total catecholamines (Pscheidt et nl., unpublished observations). On the whole, the two behavioral components which were described for the lower doses of methionine were also observed with betaine. These data point to dissimilarities between spontaneously occurring recurrences and exacerbations of the psychosis on one hand and accentuations of apparently individual psychopathology during administration of MAOT and methyl group donors, since symptoms such as euphoria, sleepiness and confusion were not characteristics of spontaneous exacerbations. However, it appears possible that the unspecific behavioral component represents side effects due to the relatively high doses of the methyl group donors. Similar behavioral effects as described above were also observed by Pollin et a/. (1961) during combined treatment with a monoamine oxidase inhibitor and methionine. These authors investigated the effects of various amino acids together with a MA01 on the behavior of schizophrenic patients and found that of all amino acids tested only tryptophan and methionine evoked behavioral alterations including activation of individual psychotic patterns. In this connection it is also of interest to recall the report of Summerskill et al. (1956) who found that administration of methionine to patients with hepatic diseases either reproduced or accentuated neuropsychiatric syndromes while at the same time blood ammonia levels remained unaltered. The fact that the essential amino acids, tryptophan and methionine, as well as the methyl group donor betaine but no other amino acids are able to induce behavioral changes in schizophrenic patients points to the possibility that tryptophan and the methyl group donors may be involved in a common biochemical mechanism to produce highly psychoactive methylated tryptaminederivatives. For instance, an elevation of the brain level of serotonin induced by either tryptophan or a MA01 may represent a favorable condition for the formation of N , N dimethyl-5hydroxytryptamine and a rise in the amount of available methyl groups donated by methionine or betaine would further facilitate this process (Fig. 5). The capability of the mammalian organism to produce N , N-dimethylated tryptamines has been proved by Axelrod (1961). Thus, in agreement with our findings on urinary indoles it appears possible that there is a higher level of highly psychoactive amines in schizophrenics with active psychosis than in non-psychotic individuals, either due to

A M l N E METABOLISM A N D P S Y C H O T I C BEHAVIOR TRYPTOPHAN

93

METHIONINE

5-HYDROXYTRYPTOPHAN

N,N-DIMETHYL-5-HYDROXYTRYPTAMINE ( BUFOTENIN )

I I

L

H

I

- - - - - - - - _ _ _ - _ - _ _ - -1 _ - _

Fig. 5 . Hypothetical mechanism of increased formation of methyiated tryptamine derivatives in schizophrenic patients after administration of tryptophan and/or methionine as exemplified by the synthesis of bufotenin.

increased formation or decreased detoxication or both. A few more observations should be mentioned in this regard. In our studies on the behavioral effects of the combined administration of isocarboxazid and methyl donors, we observed in 5 of 9 patients receiving the combination of isocarboxazid and methionine marked behavioral improvement after cessation of the medication. This improvement lasted from 2 to about 5 weeks in individual patients and during that time behavior was markedly better than before the onset of the study. Similar effects, although less pronounced were found after cessation of betaine. There is no ready explanation for this phenomenon. It is, however, tempting to speculate that impaired detoxication mechanisms were activated during the combined treatment to deal with the rapid increase of psychoactive amines and that these detoxication mechanisms continued at a higher speed for some time after the end of the treatment. The fact that the psychosis-like state induced in healthy humans by psychogenic tryptamine derivatives differs in many ways from the psychopathology observed in schizophrenic patients does not necessarily disprove the view that an endogenously determined increase of psychoactive amines may represent a factor in the pathogenesis of the schizophrenic psychosis on account of basic differences in the experimental situation. In the clinical studies with indoleamine precursors, methyl group dono: s and psychotogenic tryptamine congeners, these substances will reach all parts of the body and all regions of the brain including the cortex. In contrast, endogenously occurring changes in indole metabolism will primarily affect those areas of the brain in which indole metabolism is most active, i.e. the hypothalamus, the midbrain, the medulla oblongata, the cingulate gyrus, the hippocampus and the amygdala. Any endogenously occurring altet ations in indole metabolism will therefore affect first the function of those areas of the brain which appear to form the anatomical substrate of emotion (Papez, 1958). Furthermore, in contrast to clinical studies with psychotogenic agents in healthy human beings, a chronic intoxication with all its consequences must be assumed in the case of the schizophrenic patient. Relerenccs p. 95/96

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In conclusion, the results of our investigations are compatible with the view that psychoactive indoleamines are more than casually involved in psychotic behavior of schizophrenic patients. Our observations are also in accordance with the hypothesis that the behavioral effects of antipsychotic drugs such as reserpine and the phenothiazines are mediated by their actions on biogenic amines. These drugs reduce the level of amines in the brain or block their actions. In addition, our findings are in agreement with the generally recognized fact that these drugs have their most beneficial effects on the symptoms of acute psychoses but leaving the mental alterations of the so-called ‘burnt out’ cases unaffected. Tt is of interest, that the optimal therapeutic effects on the psychosis are obtained when simultaneously slight extrapyramidal symptoms are induced. The basis for this relationship is not yet clear. Recent investigations, however, point to the view, that biogenic amines may be also involved in the function of the extrapyramidal system, as well as in the function of the vegetative and the endocrine systems. In this paper I have discussed only certain aspects concerning a possible relationship between indole metabolism and psychotic behavior in schizophrenic patients. I have, therefore, traced only one path of investigation, realizing however, that other biogenic amines as well as other biochemical factors may play a role in psychotic behavior as well as in the actions of psychotropic drugs. SUMMARY

The paper reviews investigations related to the role of biogenic amines in psychotic behavior of schizophrenic patients. Various methodological approaches are presented and discussed, including: (1) determination of urinary indoles and total catecholamines in relation to psychotic behavior and anxiety; (2) evaluation of behavioral responses to reserpine and monoamine oxidase inhibitors, and (3) evaluation of behavioral alterations after administration of indoleamine precursors and methyl group donors. The results indicate that an intensification of psychotic behavior is associated with a rise in urinary tryptamine, total indole-3-acetic acid and 5-hydroxyindoleacetic acid, while urinary total catecholamines were found to correlate to the degree of anxiety and sympathetic activity. Paper-chromatographic studies on urinary excretion of N , Ndimethylated tryptamines disclosed a substance with the paper-chromatographic characteristics of bufotenin in some psychotic patients. Clinical investigations on the actions of psychotropic drugs point to the view that agents which reduce the level of biogenic amines in the brain or block their actions may ameliorate psychotic behavior, while those substances which elevate the amine level in the brain may activate the psychosis in schizophrenic patients. In addition, it was observed that indoleamine precursors as well as methylgroup donors are able to facilitate recurrcnces and exacerbations of psychoses. These findings are in accordance with the view that indoleamine precursors and methylgroup donors may be involved in a common mechanism to form highly psychoactive amines in schizophrenic patients.

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95

Although the presented results are compatible with the view that endogenously formed psychoactive indoleamines are more than casually involved in psychotic behavior and especially in the paranoid-hallucinatory type of psychoses, further studies are necessary to reach really valid conclusions.

REFERENCES AXELROD, J., (1960); The fate of adrenaline and noradrenaline. G . E. W. Wolstenholme and M. O'Connor, Editors. Ciba Found. Symp. Adrenergic Mechanisms. Boston, Little Brown (pp. 28-39). AXELROD, J., (1961); Enzymatic formation of psychotomimetic metabolites from normally occurring compounds. Science, 134, 343. BOGDANSKI, D. F., WEISSBACH, H., A N D UDENFRIEND, S., (1958); Pharmacological studies with the serototiin precursor, 5-hydroxytryptophan. J . Pharmacol. exp. Ther., 122, 182-194. BOSZORM~NYI, Z., AND SZARA,ST., ( I 958); Dimethyltryptamine experiments with psychotics. J . ment. Sci., 194,445-453. BRODIE, B. B., SULSER, F., A N D COSTA, E., (1961); Theories on mechanism of action of psychotherapeutic drugs. J. M. Bordeleau, Editor. Extrapyramidal System and Neuroleptics. Montreal, Editions Psychiatriques (pp. 183-189). BROWN,B. B., (1960); CNS drug actions and interactions in mice. Arch. int. Pharmacodyn. 128,391-414. BRUNE, G. G., AND HIMWICH, H. E., (1961); Biphasic action of reserpine and isocarboxazid on behavior and serotonin metabolism. Science, 133,190-192. BRUNE,G. G., AND HIMWICH,H. E., (1962); Indole metabolites in schizophrenic patients. Arch. gen. Psychiat., 6,324328. BRUNE, G. G., AND HIMWICH, H. E., (1963); Biogenic amines and behavior in schizophrenic patients. J. Wortis, Editor. Recent Advances in Biological Psychiatry. New York, Plenum Press, 5, 144-160. BRUNE, G. G., AND HIMWICH, H. E., Unpublished observations. BRUNE, G. G., KOHL,H. H., AND HIMWICH, H. E., (1964); Uninary excretion of bufotenin-like substance in psychotic patients. J . Neuropsychiat., in the press. BRUNE,G. G., KOHL,H. H., STEINER, W. G., AND HIMWICH, H. E., (1963); Relevance of the N,Ndimethyl configuration to the pharmacological action of chlorpromazine. Biochem. Pharmacol., 12,679-685. BRUNE, G. G., AND PSCHEIDT, G. R., (1961); Correlations between behavior and urinary excretion of indoleamines and catecholamines in schizophrenic patients as affected by drugs. Fed. Proc., 20, 889-893. BRUNE, G. G., PSCHEIDT, G. R., AND HIMWICH,H. E., (1963); Different responses of urinary tryptamine and of total catecholamines during treatment with reserpine and isocarboxazid in schizophrenic patients. Znt. J . Neuropharmacol., 2,17-23. BUMPUS, F. M., AND PAGE,I. H., (1955); Serotonin and its methylated derivatives in human urine. J. biol. Chem., 212, 1 1 1-1 16. COSTA,E., HIMWICH,W. A., GOLDSTEIN, S. G., CANHAM, R. G., AND HIMWICH,H. E., (1959); Behavioral changes following increases of neurohormonal content in selected brain areas. Fed. Proc., 18,379. COSTA,E., PSCHEIDT, G. R., VANMETER,W. G.,AND HIMWICH, H. E., (1960); Brain concentrations of biogenic amines and EEG patterns of rabbits. J . Pharmacol. exp. Ther., 130, 81-88. ERSPAMER, V., (1955); Observations on the fate of indolalkylamines in the organism. J. Physiol., 127, 118-133. FABING, H. D., AND HAWKINS, J. R., (1956); Intravenous bufotenin injection in the human being. Science, 123, 886-887. FISCHER, E., LAGRAVERE, T. A. F., VASQUEZ, A. J., AND DI STEFANO, A. O., (1961); A bufotenin-like substance in the urine of schizophrenics. J. n e w . ment. Dis., 133, 441-444. HAVERBACR, B. J., SJOERDSMA, A., AND TERRY, L. L., (1956); Urinary excretion of the serotonin metabolite 5-hydroxyindole acetic acid, in various clinical conditions. New Engl. J . Med., 255, 270-272. HESS,S. M., CONNAMACHER, R. H., AND UDENFRIEND, S., (1961); Effect of a-methylamino acids on catecholamines and serotonin. Fed. Proc., 20, 344.

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HESS,S. M., REDFIELD, B. G., AND UDENFRIEND, S., (1959); The effect of monoamine oxidase inhibitors and tryptophan on the tryptamine contcnt of animal tissue and urine. J . Pharniacol. exp. Ther., 127, 178-181. HIMWICH, H. E., AND BRUNE,G. G., (1964); Relationship between indoleamine metabolism and schizophrenic behavior, M. Rinkel, Editor. Biological Treatment of Mental Illness. New York, Philosophical Library. HIMWICH, W. A., AND COSTA,E., (1960); Behavioral changes associated with changes in concentrations of brain serotonin. Fed. Proc., 19, S838-S845. IRVINE,G. D., (1961); Apparently non-indolic Ehrlich-positive substances related to mental illnesses. J . Nertropsychiat., 2,292-305. JWSON, J. B., UDENFRIEND, S., AND ZALTZMAN, P., (1959); The enzymic conversion of tryptamine to 6-hydroxytryptamine. Fed. Proc., 18, 254. KLEE,G. D., BERTI (0, J., GOODMAN, A., AND ARONSON, H., (1960); The effects of 5-hydroxytryptophan (a serotonin precursor) in schizophrenic patients. 1. ment. Sci., 106, 309-316. LAUER, J. W.. INS KIP,^. M., BERNSOHN, J., AND ZELLER, E. A,, (1958); Observations on schizophrenic patients after iproniazid and tryptophan. A . M . A . Arch. Neurol. Psychiat., 80, 122-130. MITOMA, C., WEISSBACH, H., A N D UUENFRIFND, S., (1 956); 5-Hydroxytryptophan formation and tryptophan metabolism in ChromobactePium violaceum. Arch. Biochem. Biophy..., 63, 122-1 30. PAPEZ,J. W., (1958); The visceral brain, its components and connections. H. H. Jasper, Editor. Reticular Formation of the Brain. Boston, Little Brown (pp. 591-605). POLLIN, W., CARDON,W. P. V., AND KETY,S. S., (1961); Effects of amino acid feedings in schizophrenic patients treated with iproniazid. Science, 133, 104-105. PSCHEIDT, G . R . , BRUNE, G. G., A N D HIMWICH, H. E., (1960); Effect of therapeutic doses of psychotropic drugs on clinical symptomatology. Physiologist, 3, 126. PSCHEIDT, G. R., BRUNE, G . G., A N D HIMWICH, H . E., (1961); Uniform response of biogenic amines to psychotropic drugs in selected schizophrenic patients. Fed. Proc., 20, 305. PSCHPIDT, G. R., BRUNE, G. G., ANII H I M W I CH. I I ,E., Unpublished observations. SCHUMANN, H. J., (1960); Formation of adrenergic transmitters. G. E. W. Wolstenholme and M. O’Connor, Editors. Ciba Found. Symp. Arfrenergic Mechanisms. Boston, Little Brown (pp. 6-1 6). SHAW,C. R., LUCAS,J., AND RABINOVITCH, R . D., (1959); Metabolic studies in childhood schizophrenia. Arch. gen. Psychiat., 1, 366-371. SJOERDSMA, A., GILLESPIE, L., JR., AND UDENFRIEND, S., (1958); A simple method for the measurement of monoamine oxidase inhibition in man. Lancet, ii, 159-160. SJOFRIISMA, A,, OATES, J. A,, ZALTZMAN, P., AND UDENFRIEND, S., (1959); Identification and assay of urinary tryptamine: Application as an index of monoamine oxidase inhibitor in man. J . Pharmaco/. exp. Ther., 126,217-223. SPECTOR, S . , MALING, H. M., AND SHORE,P. A., (1959); Effect of JB 516, a monoamine oxidase inhibitor, on levels of serotonin and norepinephrine in brain and spinal cord. Fed. Proc., 18, 447. SPECTOR, S., PROCKOP, D., SHORE, P. A., AND BRODIE, B. B., (1958); Effect of iproniazid on brain levels of norepinephrine and serotonin. Science, 127, 704. SUMMERSKILL, W. H. J., DAVIDSON, E. A., SHERLOCK, S., AND STEINER, R. E., (1956); The neuropsychiatric syndrome associated with hepatic cirrhosis and an extensive portal collateral circulation. Quart. J , Med., 49, 245-266. SUTTON, H. E., (1959); Personal communication to Shaw, C. R., Lucas, J., and Rabinovitch, R. D. Arch. gen. P.yychiat., I , 366-371. TEIIFSCIII, D. H., TEDESCHI, R. E., A N D FELLOWS, E. J., (1959); The effect of tryptamine on the central nervous system, including a pharmacological procedure for the evaluation of iproniazid-like drugs. J . Pharmacol. exp. Ther., 126,223-232. UDENFRIEND, S., TITUS,E., AND WEISSBACH, H., ( I 955); The identification of 5-hydroxy-3-indole acetic acid in normal urine and a method for its assay. J . biol. Cheni., 216, 499-505. UDENFRIEND, S., TITUS,E., WEISSBACH, H . , AND PETERSON, R. E., (1956); Biogenesis and metabolism of 5-hydroxyindole compounds. J . biol. Chem., 219, 335-344. VONEULER, U. S., AND FLODING, I., (1956); Diagnosis of pheochromocytoma by fluorimetric estimation of adrenaline and noradrenaline in urine. Scantf. J . d i n . Lab. Invest., 8, 288-295. VONEULER,U . S.. AND LISHAJKO, F., (1959); The estimation of catecholamines in urine. Actaphysiol. scand., 45, 122-132. WEISSBACH, H., K I N D W., , SJOERDSMA, A., AND UDENFRIEND, S.Y(l959); Formation of indole-3acetic acid and tryptamine in animals. J. hid. Chem., 234, 81-86.

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Limited Usefulness of EEG as a Diagnostic Aid in Psychiatric Cases Receiving Tranquilizing Drug Therapy W. G. S T E I N E R ”

AND

S. L. P O L L A C K

Thurlichuni Psychiatric Research Laboratory, Galesburg State Research Hospital, Galesburg, Ill. ( U . S . A . )

In recent years, the EEG has come to be considered an essential diagnostic tool in the psychiatric setting. The EEG evaluation of convulsant phenomena met with early acceptance and the various EEG patterns which are associated with these disorders are widely known. The EEG approach has also proved to be of definite diagnostic value in cases of brain lesion (Bagchi et al., 1961; Bagchi and Kooi, 1961 ; Small et a]., 1961 ; Fischer-Williams et al., 1962; Hasegawa and Aird, 1963) although the interpretation of findings, especially those relating to deep involvements, requires a degree of training and competence outside the scope of many psychiatric units. Concurrent with this development, however, has been the extensive introduction of pharmacological agents in the treatment of psychiatricdisorders. The EEG rhythms are often confounded by medication in psychiatric cases receiving tranquilizing drug therapy in a manner which renders most EEG findings meaningless with respect to the presence of underlying brain pathology. It is the purpose of this report to emphasize some of the consequences of tranquilizing medication in terms of routine clinical EEG practice in the psychiatric setting. The occurrence of EEG slowing is a generally recognized consequence of tranquilizing drugs. Numerous animal studies have reported EEG slowing as a basic action of many chemotherapeutics in psychiatric use and a recent report by our laboratory (Steiner and Himwich, 1963) has indicated that this is a basic action of all phenothiazine compounds studied in addition to a large group of related non-phenothiazine compounds such as imipramine which have an antidepressant action in clinical use. Several of the early reports dealing with human EEG changes (Lehman and Hanrahan, 1954; Turner and Berard, 1954; Shagass, 1955) employed only low dose values of chlorpromazine (25-50 mg i.v. or i.m.) for study and consequently, the full range of EEG effects were not obtained. This dose level sometimes does little more than stabilize the a-range of frequencies (8-12 counts/sec) although Szatmari (1 956) found an increase in temporal and parietal 5-6 counts/sec activity in a psychotic group of

* Present address: Department of Psychology, Yale University, 333 Cedar Street, New Haven, Conn. (U.S.A.). References p . 104/105

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patients at a 50 mg i.v. dosage of chlorpromazine and Fabish (1957) reported the appearance of 3-4 counts/sec &activity in control subjects at 0.3 to 0.5 mg/kg i.v. (approximately 18 to 30 mg) of chlorpromazine. Of greater significance for the psychiatric setting are the reports which utilized dosages in the therapeutically effective range on hospitalized patients. The papers by Steward (1957) and Friedlander (1959) serve as useful reviews on the subject of tranquilizing medication as an activating agent in convulsant disorders. Our concern here is primarily with changes which are not associated with paroxysmal disorders. Jorgensen and Wulff (1 958) utilized orally administered chlorpromazine (300-600 mg) for a period of about 45 days in a group of schizophrenic patients andconcluded that of practical importance was the occurrence of abnormality not present in the pretreatment record. A normal or borderline record was rendered abnormal in 8 of 16 cases. Hollister and Barthel (1959) studied the long term administration of chlorpromazine (400 mg average) to chronic schizophrenic patients and found that I 1 of 40 patients had records which had changed towards an abnormal EEG pattern with focal slowing being thc most frequent change. They concluded that the significance of these changes is not known, but their presence may lead to diagnostic confusion unless considered as a possible drug effect. Towler et al. (1962) embraced this problem directly in the introduction to their paper by stating that the usefulness of EEG as a diagnostic tool may be impaired by the administration of many commonly used drugs prior to electroencephalographic study. It is beyond the scope of this paper to offer a complete review of the literature. A recent symposium on EEG and Human Psychopharmacology (Fink, 1963) held in conjunction with the Third World Congress of Psychiatry, addressed itself specifically to the question of the nature of electrographic patterns following acute and chronic administration of psychopharmacologic agents in man. As expected from data on animals, tranquilizing drugs were found to enhance the resting rhythms of the brain with increased amount of u, increased amplitude of frequencies and slowing being cardinal features of the induced changes. Many paroxysmal patterns were noted by several participants. Slow wave activity which has frequently been associated with brain pathology in the literature was also present in these drug patterns being both rhythmic and dysrhythmic, diffuse and focal in distribution. Activity at both the 0 (4-7 counts/sec) and b (3 and below counts/sec) ranges were observed in conjunction with tranquilizing drugs. Of considerable importance for clinical EEG was the finding by Ulett el al. (Fink, 1963) that these altered patterns persisted in some cases for as long as 10 weeks following a 3-week period of medication. Swain and Litteral(l960) had previously noted that I9 ”/, of their cerebral arteriosclerosis and senile dementia patients had displayed a persistence of EEG changes for a period of 3 months following cessation of drug. The findings in the literature concerning tranquilizing drug action speak for themselves although little attention has been explicitly paid to a discussion of consequences; the comments by Hollister and Barthel (1959) and by Towler et al. (1962) being two notable exceptions. Even in the symposium cited (Fink, 1963), discussion centered primarily on the question of whether EEG change was necessary to behavioral

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change. The remainder of this paper will be devoted to underscoring the difficulties which accompany the use of EEG as a diagnostic tool in psychiatric cases receiving tranquilizing drug therapy. One of the basic difficulties confronting the diagnostic use of EEG in a psychiatric setting is the lack of a pre-treatment EEG record. In contrast to the carefullycontrolled experimental studies of drug action, the ‘uncontaminated’ case is the exception in the non-specialized psychiatric setting such as the state mental hospital or community mental hygiene clinic. Many of the evaluations are conducted on a near ‘blind’ basis with respect to the pre-institutional physical and treatment history of the patient. Needless to say, the majority of evaluations are also conducted without benefit of a pre-drug recording. Under these conditions, the task of evaluation is very difficult at best with EEG findings raising more questions than they answer with respect to the patient’s illness. The result is a diagnostic picture more clouded than clarified in many instances. Two types of drug-induced EEG changes are apt to be particularly troublesome in the evaluation of psychiatric cases who are receiving tranquilizing drug therapy. A very common problem encountered in these cases is the failure to obtain a satisfactory waking record on the patient. In many instances, convulsant EEG patterns are elicited only during the sleep portions of the record and Silverman and Graff (1957) have utilized sleep states for determination of brain tumor depth. Sleep patterns in the absence of a satisfactory waking record, however, only serve to render the evaluation of EEG rhythms more difficult since there is no adequate reference of waking base line activity upon which to make an evaluation. In the tranquilized Id MIN

13th MIN

25th MIN

Fig. 1 . A short, heavy set, 1 I-year-old negro boy admitted in June of 1961. A low intellectual level and an absence of control over impulses prompted the institutionalization. He was found to be moderately mentally deficient on psychological examination. He was receiving chlorpromazine and perphenazine medication at the time of the EEG recording in July of 1961. His impulsivity and immature aggressiveness were largely responsible for the EEG referral. Rpjerenres p. 104/105

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patient, sleep tends to be of a deep stage where all focal abnormalities are less pronounced and the background waking record, when obtainable, is inclined to lack organization of the dominant frequencies. The rhythms have the appearance as if they have a chronic readiness to 'sag' towards the slower frequencies. A5 a way of illustrating the above point, Fig. 1 (case No. I ) presents 3 EEG tracings from a tranquilized adolescent boy. The rhythms of the first minute of recording are clearly slow for an 1 I-year-old boy. Even in drowsiness, frequencies below 5-7 counts/sec are rarely encountered except in very young children. This type of finding is consistent with this boy's mental deficiency and history of immature aggressiveness but it is also a frequently reported finding for tranquilizing drugs. A pre-drug recording could not be obtained in this case because the boy had been in a treatment situation of one kind or another for some time prior to the time when an EEG evaluation was considered desirable. The electroencephalographer is forced to assume, then, that the slowing reflected in Fig. I is drug-induced and that the implied pathology is nothing more than a consequence of the treatment situation. As a result, no infcrcnce can be made concerning the mental deficiency of this boy or of his immature aggressiveness. A drug-free recording was mandatory at the outset in this type of referral. AWAKE AND TALKING

L.F. LEar E a r Zar

W

R.Ear R.P.

i:& !:L b :;::

w

z:

Mpv

kF

MALE AGE 37

l5"WS-Z

Fig. 2. A 37-year-old, 285 pound, married negro male committed to a state hospital in 1955 as mentally ill because of mental confusion, bizarre and violent behavior. The patient reportedly received treatment for syphilis in 1946 and was hospitalized in 1958 for head injury following unconsciousness. On admission in 1955, the blood Kahn was 16 units positive. The patient was transferred to this hospital in 1958. Since that time, repeated spinal fluid examinations have shown no evidence of neurosyphilis and all skull X-rays have been essentially negative. A neurological examination in 1961 (S. L. Pollack) revealed no evidence of any active neurological disease apart from mild drug-induced extrapyramidal symptoms. The patient has been a management problem and 20 attacks on patients and personnel have produced injuries to them requiring medical attention between November of 1961 and July of 1962. These outbursts of aggressive behavior appear to follow auditory hallucinations. At the time of the EEG referral in February of 1962, the patient was receiving reserpine, 3 mg f.f.cf., fluphenazine, 50 mg i.i.d., and chlorpromazine, 100 mg f.i.d.

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A second but related difficulty encountered in the evaluation of psychiatric cases on tranquilizing medication concerns the occurrence of generalized slowing which appears in the EEG record independently of the sleep process. Fig. 2 (case No. 2) presents two tracings of an extreme but not uncommon instance of generalized slowing in a heavily tranquilized male patient. This record was selected for presentation because it contains controiersial aspects which typify many of the evaluations conducted on medicated patients. The left tracing was obtained while the patient was speaking with the technician. No judgement can be made concerning the state of the patient during the right tracing. In either instance, the rhythms are extremely slow for consciousness thus suggesting an active pathological process. One of the conditions which must be given serious consideration in a patient such as this with a history of head injury and markedly aggressive behavior is temporal lobe epilepsy with so-called ‘amygdaloid’ symptoms. The slow activity on the left of Fig. 2 could be regarded as coming from the temporal lobes by way of the ear references. The tracing on the right of Fig. 2, being without an inactive reference (bipolar), renders this view unlikely but TABLE I

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E V A L U A T I O N OF

61

CONSECUTIVE CASES

~~

Number of cases Average age Average background E E G frequency counts/sec Incidence of sleep EEG Incidence of EEG slowing mentioned separately from sleep

I

11*

111

IV**

No drugs reported

Anti-conwlsant drugs only

Anti-convulsant and tranquilizing drug conibination

Tranquilizing drugs On‘.’

11

18%

29

10 36

167;

19 30

31%

9. I

21 24

35%

10 3

27%

8.1 3

30%

5

26%

9.2 13

65%

-

-

4

40%

5

26%

9

43%

* Drugs include: diphenylhydantoin sodium (Dilantin), phenobarbital sodium, methsuximide (Celontin), and primidone (Mysoline). ** Drugs include: chlorpromazine (Thorazine), triflupromazine (Vesprin), prochlorperazine (Compazine), perphenazine (Trilafon), fluphenazine (Prolixin), trifluoperazine (Stelazine), reserpine (Serpasil), thioridazine (Mellaril), chlordiazepoxide (Librium), and the antispasmodics, trihexyphenidyl (Artane), and aminophylline. under a different technical arrangement, the contribution of the reference can be clarified. It is doubtful, however, that a clarification would settle anything in this particular case. The rhythms are also similar in some respects to certain forms of toxic and infectious encephalopathies, cerebral anoxia and with the attendant consciousness, to those presented by Wells et al. (1957) in their paper on the pharmacological agent, 6-azauracil. Several widely differing interpretations are equally in order so long as the patient remains heavily tranquilized and his extremely aggressive behavior renders this possibility almost a certainty. As in case No. 1, a drug-free References p . 104/105

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recording is mandatory to clarify an EEG pattern which is consistent both with the symptoms of the patient and with the known actions of the tranquilizing agent. A brief summary of 61 consecutive cases referred to the authors for EEG evaluation as part of the normal routine of an in-patient psychiatric hospital is presented in Table I. These data were selected for presentation to indicate the extensiveness of the drug problem when attempting to utilize the EEG as a diagnostic procedure in a psychiatric unit, As can be seen from this table, 82 % of all patients were on medication at the time the EEG recordings were obtained. Our chief concern here is with the group IV patients which received tranquilizing drugs only. In most instances, these are patients who have been considered mentally ill or in need of mental treatment but who, for one reason or another, appear ‘organic’ without demonstrable neurological findings. Frequently, there is an accompanying history of vague physical complaints and a progressive deterioration of functional efficiency in conjunction with an altered performance on psychological examination. No conclusions can be reached from these data on the action of tranquilizing drugs specifically since the predrug EEG patterns on these patients are not known but these data do serve the very useful purpose of indicating the scope of thc problem in terms of the kind of material which confronts the person making the EEG evaluation in an impatient psychiatric unit. Rhythms characteristic of sleep are present in 39% of all records evaluated. The incidence of sleep activity was stable across group 1 to 111(26 % to 30%) but group IV contained an unusually high incidence of sleep activity (65%) as gauged by the appearance of the EEG rhythms. Obviously, the sedative or calming action of tranquilizing medication is enhancing the processes associated with the ‘resting’ rhythms of the brain. The groups receiving anticonvulsant medication (I1 and Ill) were no more subject to sleep activity than the non-drug patients even though anticonvulsant medication also has a sedative action. It is a familiar observation, however, that patients on anticonvulsant medication recover from drowsiness after a brief initial period of medication. The difference between these groups may simply be one of length of time on drug although this position seems unlikely in view of the extensive use which is currently being made of tranquilizing medication. Many psychiatric cases have long treatment histories. A second finding of interest concerns the appearance of abnormally slow rhythms (6 and 0) appearing separately from the rhythms associated with sleep. The patients with no drug reported (group 1) did not present this type of EEG activity while these rhythms were present in approximately 30 % of the records of patients receiving anticonvulsant medication (groups I I and 111). The group IV patients were found to have these abnormally slow rhythms in 43 of the cases. A small percentage of these patients might be expected to have underlying organic pathology but in the main, this high incidence points to the marked slowing which accompanies tranquilizing medication. As a consequence, little significance can be attached to these abnormally slow rhythms apart from the fact that the brain processes are reflecting the pharmacological actions of tranquilizing medication. In general, then, tranquilizing drugs have been found to induce changes in the

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human EEG which are similar to patterns which have been reported as being related to the presence of brain pathology. The position might be taken that these ‘tranquilized’ rhythms are also indicative of pathology but this conclusion is unwarranted in view of the extensive clinical experience which has now accumulated in the use of these pharmacological agents. The more tenable position would be to re-examine our concepts of brain function. As an example, Johnson et al. (1960) found that the cognitive functions involved in learning, retention and recall were not necessarily impaired by disruption of the usual patterns of neural activity as measured by the EEG. In keeping with this finding, it has been known for many years that convulsive patients have interseizure abnormalities, yet function effectively. Gastaut (1 954) mentioned several years ago the case of a young girl who had a background rhythm of 3 counts/sec for more than 3 years following post-traumatic coma even though her mental processes remained quite normal. Experiments with chemical agents such as the study by Wikler (1952) with atropine and the study cited earlier by Wells et a/. ( I 957) demonstrate that behavioral alertness can be associated with the presence of EEG slowing; a fact that contradicts much traditional teaching in regard to EEG states. The authors of this last mentioned paper make the following comment, ‘It was a remarkable finding to see a patient quite alert and able to carry on an intelligent conversation while the electroencephalogram showed almost constant S wave activity’. Batsel (19601, working with animals, showed that even in the chronic isolated cerebrum of the ‘cerveau is0lC’ preparation, the capacity for high-frequency, low-voltage patterns reappears without benefit of the ascending reticular system. Each of these reports stand as an exception when taken singly. The question remains as to how many such exceptions are necessary before we question the major premise upon which the exceptions are derived. Considerable research is needed before the exact significance of these ‘tranquilized’ rhythms is understood. It appears advisable in the interim, to attach very little significance to the appearance of EEG slowing in the tranquilized patient until such time as the relationship between medication and pathological brain processes is better understood. From the brief data presented here, EEG slowing frequently appeared to be a result of the treatment situation in these psychiatric cases and no inference could be made concerning the nature of the illness of these patients. The question may be raised as to the efficacy of making an evaluation in the tranquilized patient. The fact remains that tranquilized patients are as subject to brain pathology as any other human. The problem resides in the identification of the particular individual so that he may be brought quickly t o the attention of the more specialized neurological setting. The contribution of the EEG findings in this diagnostic process is not as great as is generally believed with the consequent danger that these findings will produce many false positive reports unless the pharmacological actions of these drugs are properly evaluated. The principal difficulty confronting the diagnostic use of EEG in the psychiatric setting is that the substantial majority of patients are on medication during or immediately preceding the time the EEG recording is obtained. The practice of withdrawing medication 48-72 h prior to a diagnostic EEG recording is of no practical References p 1041105

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W . G. S T E I N E K A N D S. L. P O L L A C K

value in light of the long action of many tranquilizing drugs" and, in most instances, either the severity of the illness does not permit withdrawal for extended periods of time or the neurological findings are not sufficient to warrant such a withdrawal in the severely disturbed psychiatric patient. The net result is that the electroencephalographer is called upon to evaluate EEG patterns for the presence of brain pathology at a time when the patient's medication is producing widespread changes in the EEG similar to those produced by the pathology in question. Consequently, there is little hope at the outset of the EEG contributing anything of value towards the cstablishment of a diagnosis or a prognosis. Fortunately, other procedures are akailable including a careful history and the diagnosis of organic nicntal ilIness, in the last analysis, must be based on the behavior of the patient. SUMMARY

EEG rhythms are often confounded by medication in a manner which renders most EEG findings meaningless with respect to the presence of underlying brain pathology i n psychiatric cases receiving tranquilizing drug therapy. Two types of drug-induced changes are considered: < l ) EEG slowing as a reflection of sleep in the absence of a satisfactory waking record, and (2) EEC slowing which appears in the record independently of the sleep process. Tracings reflecting both types of change are presented and 61 consecutive cases are reviewed a5 to the incidence of each type of slowing. Diagnostic problems are discussed and certain concepts of brain function are questioned. ACKNOWLEDGEMENTS

We wish to thank Mrs. Helen F. Owens and Mrs. Kathryn Bost for technical assistance with the patients. REFERENCES

BAGCHI,B. K., AND KOOI,K. A,, (1961); Electroencephalography and brain tuniois. UNIV.Mrch. men. Bull., 21, 50-60. BAGCHI, B. K., Koo~,K . A., S E L ~ I N13. G ,T., A ~ CALHOUN, D H A Z ~D., L ,(1961); Subtentorial tumors and other lesions: An electroencephalographic study of 121 cases. Efectroenceph. d i n . Nertop h p i o f . , 13, 180-192. -

*

Thc actual duration of effects is dependent upon the pharmacological agent being considered and the responsc system being studied. As an example, Haynes (1960) reported a significant drop in the urinary excretion of chlorpromazine and chlorpromazine sulfoxide during the first 48 h after cessation of medication and a further excretion of small but measurable amounts for several days thereafter. Huang and Kurland (1961) detected the excretion of glucuronides associated with chlorpromazine for a period of 2 months in 1 patient using the techniques of quantitative paper chromatography. Clark ef af. (1 963), working with various psychological modalities, reported that all behavior scale indices regressed significantly by the end of the 12th week after cessation of chlorpromazine medication but psychological test scoreb had not regressed significantly even after 56 weeks following cessation. For our purposes, the study of Ulett et al. (Fink, 1963) which noted EEG changes up to 10 wecks following cessation of medication and the study of Swain and Litteral(1960) which reported EEG changes up to 3 months are the most applicable.

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BATSEL, H. L., (1960); Electroencephalographic synchronization and desynchronization in the chronic ‘cerveau isol6’ of the dog. Electroenceph. elin. Neurophysiol., 12, 421430. CLARK, M. L., RAY,T. S., A N D RAGLAND, R. E., (1963); Chlorpromazine in chronic schizophrenic women: Rate of onset and rate of dissipation of drug effects. fsychosom. Med., 25, 212-217. FABISH, W., (1957); The effect of chlorpromazine on the electroencephalograms of epileptic patients. J . Neurol. Neurosurg. Psychiat., 20, 185-190. FINK,M., (1963); EEG and human psychopharmacology: A symposium. Electroenceph. clin. Neurophysiol., 15, 133-137. FISCHER-WILLIAMS, M., LAST,S. L., LYBERI, G., AND NORTHFIELD, D. W. C., (1962); Clinico-EEG study of 128 gliomas and 50 intracranial metastatic tumors. Brain, 85, 1 4 6 . FRIEDLANDER, W. J., (1959); Chlorpromazine as an E E G activating agent. Electroenceph. clin. Neurophysiol., 11, 799-801. GASTAUT, H., (1954); The brain stem and cerebral electrogenesis in relation to consciousness. Brain Mechanisms and Consciousness. J. F. Delafresnaye, Editor. Springfield, Charles C. Thomas (pp. 249-279). HASEGAWA, K., A N D AIRD,R. B., (1963); An EEG study of deep-seated cerebral and subtentorial lesions in comparison with cortical lesions. Electroenceph. clin. Neurophysiol., 15, 934-946. HAYNES, E. E., (1960); Urinary excretion of chlorpromazine in man. 1.Lab. clfiz. Med., 56, 570-575. HOLLISTER, L. E., A N D BARTHEL, C. A., (1959); Changes in the e!ectroencephalogram during chronic administration of tranquilizing drugs. Electroenceph. clin. Neurophysiol., 11, 792-795. HUANC, C. L., AND KURLAND, A. A., (1961); A quantitativestudy of chlorpromazine and its sulfoxides in the urine of psychotic patients. Amer. J. Psychfat., 118, 428-437. J O H N S ~ N L. , C., ULETT,G . A,, SINES,J. O., A N D STERN,J. A., (1960); Cortical Activity and Cognitive Functioning. School of Aviation Medicine, U.S.A.F. Aerospace Medical Center (ATC), Brooks Air Force Base, Texas. JORGENSEN, R. S., AND WULFF,M. H., (1958); The effect of orally administered chlorpromazine on the electroencephalogram of man. Electroenceph. elin. Neurophysiol., 10, 325-329. LE-IMAN, H. E., A N D HANRAHAN, G. E., (1954); Chlorpromazine. New inhibitory agent for psychomotor excitement and manic states. Arch. Neurol. Psychiat., 71, 227-237. SHAGASS, C., ( I 955); Effect of intravenous chlorpromazine on the electroencephalogram. Efectroenceph. clin. Neurophysiol., 7, 306-308. SILVERMAN, D., AND GRAFF, R. A., (1957); Brain tumor depth determination by electrographic recording during sleep. Arch. Neurol. Psychiat., 78, 15-28. SMALL, JOYCEG., BAGCHI, B. K., AND Koor, K. A., (1961); Electro-clinical profile of 117 deep cerebral tumors. Electroenceph. clin. Neurophysiol., 13, 193-207. STEINER, W. G., AND HIMWICH, H . E., (1963); Effects of antidepressant drugs on limbic structures of rabbit. J . nerv. ment. Dis., 137, 277-284. STEWARD, L. F., (1957); Chlorpromazine: Use to activate electroencephalographic seizure patterns. Electroenceph. din. Neurophysiol., 9, 427-440. SWAIV,J. M., AND LITTERAL, E. B., (1960); Prolonged effect of chlorpromazine: EEG findings in a smile group. J . nerv. ment. Dis., 131, 550-553. SZATMARI, A., (1956); Clinical and electroencephalogram investigation on Largactil in psychosis (preliminary study). Amer. J . Psychiat., 112, 788-794. TOWLER, M. L., BEALL,B. D., AND KING,J. B., (1962); Drug effects on the electroencephalographic pattern, with specific consideration of Diazepam. J . S. tned. Ass., 55, 832-838. TURNER, M., AND BERARD, E., (1954); Etude sur les effets de la chlorpromazine (4560 R. P.) sur les tracks ClectroencCphalographiques et electrodermographiques. Electroenceph. din. Neurophysiol., 6, 538. WELLS,C. E., AJMONE-MARSAN, C., FREI,E., TUOHY,J. H., AND SHNIDER, B. I., (1957); Electroencephalographic and neurological changes induced in man by the administration of 1,2,4Triazine-3,5(2H, 4H)-dione (6Azauracil). E!ectroenceph. clin. Neurophysiol., 9, 325-332. WIKLER, A., (1952); Pharmacologic dissociation of behavior and EEG ‘sleep patterns’ in dogs: Morphine, N-allylmorphine and atropine. Proc. SOC.exp. Biol. ( N . Y . ) , 79, 261-265.

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Behavioral Changes of Dogs Following Injection of Neurotropic Drugs into the Arachnoid Space Overlying the Cerebral Cortex TSUKASA KOBAYASHI* Tl~ulichuiirP.sycliiatiic Research Laboratory, Gale.dwrg State Reseurcl? Hospital, GalesburR, 111. (U.S.A.)

Feldberg and Sherwood (1954) first described the behavioral effects resulting from injection of drugs through chronically implanted cannulas into the lateral ventricle of unanesthetized and unrestrained cats. Their report has opened a new area in brain research, and using their technique about 40 investigators have published approximately 60 papers on drug actions. On the other hand, the author (1962) has reported the behavioral effects produced by injections of drugs into the cerebroarachnoid space of the dog through chronically implanted polyethylene tubes. By this technique, the drug is administered into the cerebrospinal fluid (CSF) overlying the cerebral cortex, without the need of anesthesia or restraints. This technique is named the intraarachnoid injection (I.A.I.) (Fig. 1). In the previous paper (Kobayashi, 1962) it was reported that three drugs, morphine hydrochloride, amobarbital sodium, and imipramine hydrochloride, did not produce any noticeable changes in dog’s behavior following the I.A.I. Nevertheless intraventricular injection with the same dose evoked various eRects in the dog. These results have suggestcd that the I.A.I. can throw light on the mechanism of drug action and brain function, especially by making comparisons with results by other routes. This paper concerns the effects of 8 familiar drugs on the dog’s behavior following the I.A.I. These drirgs were chosen in order to determine whether or not this route of administration produced the usual pharmacological effects, and whether this technique might yield additional information as to the site and mode of action of the drugs. METHODS

The method of implantation of the chronic tube into the cerebroarachnoid space of the dog for the I.A.I. has been described previously (Kobayashi, 1962). The drugs were not administered until 1 week after the tube was implanted. The volume of

* Present address : Department of Psychiatry, University of Tokyo, Bunkyoku, Tokyo, and Neuropsychiatric Research Institute, 91, Shinjuku-Bentencho, Tokyo.

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Fig. 1 . Diagram illustrating method for injection of drugs into the arachnoid space overlying dog’s cerebral cortex. A = dura mater; B = arachnoid (outer layer); C = pia mater (with inner layer of arachnoid); D = arachnoid space (cerebrospinal fluid); E = subdural space; F = cerebral epidural space.

injected drug solution did not exceed 0.2 ml at any one injection except for ether (0.3 ml). Sometimes during the experiment a higher dose was additionally injected. Each dog was used for a single experiment except for 4 animals which were observed for a second time 1 week after the first experiment. To study the distribution of the drug, 0.2 ml of 1 of 4 dyes (1 % methylene blue, 4.7 ”/, methylene blue, 0.2 % bromophenol blue, 0.5 % aniline blue) was administered through the tube after each experiment and the animals were sacrificed at various periods after the dye injection for the necropsy. Results on 38 dogs (6.5-14.5 kg) were selected for this report. The following drugs were used for injection : acetylcholine chloride (Merck) ; chlorpromazine hydrochloride (Smith Kline and French Laboratories) ; D-tubocurarine chloride (Squibb) ; ethyl ether (Merck) ; y-aminobutyric acid (Biochemical Research Laboratory); L-epinephrine bitartrate (Mann Research Laboratory); potassium chloride (J. T. Baker); serotonin creatinine sulfate (Nutritional Biochemical Corp.), and strychnine sulfate (Merck). The amounts injected refer always to the salt. All drugs used were dissolved in physiological saline or distilled water immediately before each injection. The response was the same whether a saline or water was used to dissolve the drugs. Neither does varying the volume of physiological saline or distilled water below 0.5 ml produce any visible change. The pH of each solution of the drugs, ranging from 3.85 to 7.77 was not altered, because even the I.A.I. (0.2 ml) of an HC1 solution with pH 3.5 or NaOH solution with pH 10.5 produced no significant change in behavior. Behavior was noted in a special observation room with air conditioning. The room is octagonal shaped and about 2.4 m in diameter. It has two openings for References p.III9j120

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~~

~

~ _ _ _

Fig. 2. The dog’s posture classified in 10 types.

a movie camera and 8 windows with one-way vision glass. Any specific change in the animal‘s behavior was photographed with 16 mm colored moving pictures. The dog’s postures were classified in 10 types (Fig. 2) for the descriptive record. In order not to interfere with dog’s behavior, the observers did not enter this room, except for necessary examinations. Thus the changes of heart rate were not observed. Chronic electrodes were planted in the skull in 12 dogs and the skull EEG was recorded following the I.A.I. in the EEG room.

RESULTS

The changes of the dog’s behavior are described for each drug. Because there were no great differences in the time courses of the various dogs to any drug, typical examples are used in this report. Potassium chloride The behavioral change observed 10 min after the I.A.I. of 1 mg potassium chloride was not clearly shown. Therefore a 25% solution (50 mg/0.2 ml) of the drug was employed which percentage is usually used in neurophysiological experiments. Three minutes after the I.A.I. of 50 mg KCl, the dog showed slight ataxia and sat down. At 6 rnin he assumed posture VTT but seemed restless. At 10 rnin a cookie was given but he merely looked at it, while usually he ate one readily. Around I 3 rnin he blinked frequently. At 21 rnin he assumed posture X. He closed his eyes but he could not sleep for even with the slightest noise of the camera he would reopen his eyes. At 25 min the size and reflexes of the pupils and of the knee jerks were normal. The gait showed no ataxia. There was little response to sudden noise. At 40 rnin slight ataxia or muscle weakness of extremities was observed. At 45 rnin the eyelids showed ptosis and he seemed to be asleep. At 51 min he came when called and looked considerably recovered from the depression but slept again when left alone. One h and 40 min after the injection, he woke up but remained quiet. Two h after the I.A.I. his behavior was practically normal. Summarizing these effects, KCI depressed the dog’s behavior for about 1 h and 30 min, and theanimal seemed to be drowsy.

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Strychnine suJfate Within 1 rnin after the I.A.I. (5 mg/0.2 ml) the dog’s body bent toward the injected side and exhibited tonic convulsions. The extremities were extended to the maximum and the pupils were widely dilated. Light reflexes were slow but the corneal reflex was normal. At the 4th rnin muscle tone disappeared abruptly and the dog succumbed.

Ether At the injection of 0.25 ml, the dog struggled against the I.A.I. (left hemisphere), perhaps due to local stimulation of the dura mater. Only licking and whining were observed from 2 to 21 rnin after the injection. Then the dog seemed to be restless or apprehensive. At the 30th rnin slight motor ataxia was observed and the dog laid down. He appeared to be drowsy but was unable to sleep perhaps because of some discomfort. Around 45 rnin after injection, drowsiness seemed to be dominant, however, he responded quickly to auditory stimuli. Two h and 10 rnin after the first injection, an additional 0.3 ml of ether was injected. At the second injection the animal cried, defecated, and urinated. After the injection, he could not move at all foi 1 min. He then paced slowly and gradually became depressed. At 9 rnin he laid down and became drowsy with some licking of the snout. At 13 rnin ataxia was first observed. At 17 min he began to pace in a counterclockwise direction and with an ataxic gait. At 20 min profuse salivation was observed. The dog was quite alert and not indifferent. One h after the second injection, he still paced, however sometimes he slept in posture IV. He appeared somewhat catatonic. Two h and 30 rnin after the second I.A.T., the dog paced ceaselessly in a counterclockwise direction only. He seemed to be conscious. After 4 h and 30 rnin the pupil on the injected side was smaller, although both pupils were miotic. The light reflex was not present. Some analgesia was found on the contralateral foreleg at this time. In summary, ether seemed to stimulate the dog rather than to depress him for 4 h and 30 min. Consciousness seemed to be maintained.

D-Tubocurarine chloride Two min after I.A.I. (right side hemisphere, 500 pgIO.2 ml) the dog exhibited licking, barking and restlessness. At the 1 lth min he began panting. At 21 min he sat down and ate a cookie when it was offered. At 22 rnin an onset of twitches was found at first in the left foreleg and at 26 rnin on the left hind leg as well. The dog responded to auditory stimuli. At 34 min he walked without ataxia for 2 min, but assumed posture VII again. At 46 rnin the twitches were more frequent but not so strong. Panting was observed. He barked more often than usual and blinked his eyes. At 40 min, he assumed posture X, and twitches still continued. At 57 rnin an additional I mg/0.2 ml of the drug was injected. Four min later he rubbed his head with his foreleg and licked his snout. Panting was also seen. At 4 min strong twitches of the body, mainly of the upper half, appeared and he then fell down in a generalized convulsion of the recurrent type. Even during convulsions the corneal reflex was maintained. The pupils References p . 119!12#

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were not dilated but the light reflex disappeared during the convulsions. After 24 min the convulsions still continued. The dog's body bent to right side, and convulsive movements were seen mainly on the left half of the body, legs, and face, especially at the left palpebral fissure. At 50 rnin after the second injection, GABA was administered (I.A.T. 100 mg/0.2 ml) to produce an anticonvulsant action. However, at least in this case, GABA did not diminish the convulsions although it seemed to do so in another instance. The convulsions continued for 1 h after the GABA I.A.I. Two h and 30 rnin after the second D-tubocurarine injection the convulsions ceased, and at the third hour his behavior was almost normal except for showing fatigue. Thus the chief effect of the I.A.I. of D-tubocurarine was the production of convulsions, mainly on the contralateral side. L-Epinephrine hitartrate Four rnin after T.A.T. (1 mg/0.2 ml) the dog defecated and licked his snout. Thirty rnin after the injection, he held posture IX and became drowsy. At 45 rnin the maximum effect was attained but 1 h after the injection the dog was still depressed with a fine tremor over the body. One h and 30 min after the injection behavior became almost normal. Acetylcholine chloride Acetylcholine was injected within the range from 10 pg to 30 mg (0.005-15% solution). Tn all cases transient behavioral depression was observed, though the smaller doses evoked weaker effects of shorter duration. However, even the largest dose did not produce posture X, and the animal did not lose consciousness. They displayed no signs of twitches or convulsions. Two rnin after the T.A.T. of ACh (30 mg) a typical dog became somewhat drowsy and the eyelids were almost shut, as in a man unable to resist intense drowsiness. His behavior became less active showing posture VTII followed by IX. At 6 min a slight tremor was observed, while at 8 min he ate some food. By 18 min he showed no response to hand clapping. He walked around in the room at 28 rnin but failed to respond when someone entered the room. At 33 rnin both reflexes and pupils were normal but the animal was still a little depressed. At 41 min the dog seemed rather drowsy and did not come when called. However, it is positive that the dog did not sleep. Though the dog closed his eyes and seemed to want to sleep, he could not do so as if prevented by intense discomfort. Occasionally the dog raised his nose high in the air and slowly shook his head from side to side. At 57 min the animal began walking in a normal manner, and at 63 min appeared to be fully recovered.

Serotonin creatinine sulfate After I.A.I. of 12.5 pg of serotonin blinking was observed and 100 pg produced very slight transient drowsiness. One mg 5-HT evoked definite behavioral depression. Two

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min after the injection the dog assumed postures VTT and VllI in succession and blinked frequently, especially on the non-injected side. The response to call was very depressed. Nine rnin after the I-mg I.A.T., posture X was taken. Respiration rate was 20/min and the dog whined and barked occassionally. At 25 rnin the animal appeared to sleep, and the respiration rate was 16/min. At 55 rnin the dog had apparcntly recovered but continued to lie quietly. Even the high dose of 20 mg 5-HT produced similar behavioral changes.

Chlorpromazine hydrochloride Five rnin after the injection of 2 mg, the dog laid down assuming posture VII. At 7 and 10 min after the injection, the dog ate cookies and paced slowly in the observation room. At 16 min the posture changed to X, the dog was depressed but did not sleep. At 20 min the dog was alert to noise and in posture VITT. At 22 niin attention was not paid to noises and the eyes were half closed but the animal did not sleep. At 26 min the eyes were opened in response to sounds, but the dog did not look in the direction of the sound. At 29 rnin the posture changed to X again, and the animal slept. Thereafter the dog got up occasionally when aroused by auditory stimuli but each time after a few movements laid down again and continued in a drowsy state until 2 h after the injection. Then behavior gradually returned to normal within the following hour.

The dyes The distribution of the dyes was over the upper half of the hemisphere of the injected side but was concentrated in an area about 1 cm2 around the tip of the tube (Fig. 3). The distribution was modified slightly depending upon the direction of the tip of the tube. However, it is striking that even 2 h later the dyes never reached the lower half

Fig. 3. The distribution of the dye injected through the implanted tube (15 rnin later). It is concentrated around the tip of the tube. References p . 119jl20

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of the hemisphere but remained at the upper half. Of course, it did not reach the cisterna magna. Methylene blue or aniline blue injezted before death stained the surface of the brain clearly, however, 0.2 % bromophenol blue stained the area only slightly. There is no difference in penetration into the brain tissue between injections of 1 % and 4.7 % of methylene blue. There also seemed to be no differences between the basic dye (niethylcne blue) and acid dye (bromophenol blue). They both stained the surface of the br‘iin. perhaps only the pia mater or the very surface of the cerebral cortex. The precise depth of penetration was, however, unknown as a microscopic study was not performed. However, certainly. deeper penetration as reported in Feldberg and Fleischhauer’s paper (1960) was not found. The dyes injected after death usually did not stain the surface of the brain but only the dura mater. After the I.A.I. of ether or potassium chloride, dyes injected even after death stained the surface of the brain very well. DISCUSSION

In gcneral, there are great differences in drug action both quantitatively and qualitatively between the systemic administration and the various intra-CSF routes. Drug action following the topical application on the exposed cerebral cortex is little known, except for the neuronographic observations following strychnine (Dusser de Barenne, 1916), De Leiio’s spreading depression caused by potassium chloride, and the cortical epinephrine pressor response (Walaszek, 1960). In the present paper, the results following the I.A.I. were compared with those by other intra-CSF routes. In this way, the site or mechanism of action of the drug following the T.A.I. was studied.

Potassium chloride It has long been known that a potassium-rich solution exerts a complex influence on the nervous system. Fenn (1940) summarizing the literature, states that potassium in small concentrations is excitatory and in larger ones inhibitory. The threshold of electrical stimulation is in general first lowered and then raised by potassium. Large amounts of potassium finally cause inexcitability. A high concentration of potassium salt is known to depolarize tissue cells. However, when a potassium salt is infused systemically until a lethal concentration is attained, the only changes in the EEG of experimental animals are those associated with circulatory failure (Calma and Wright, 1947). Winterstein (1961) reviewed the effects of introducing potassium into the CSF, mainly on the autonomic nervous system. Therefore, the author will not mention them again, but some other results obtained by the I.A.I. Calma and Wright (1947) injected KCl into the subarachnoid space of the cat at the level of the first sacral vertebra, under chloralose anesthesia. Tsotonic potassium chloride solution (1.15 0.1-0.2 ml) regularly produced dilatation of the pupil, a rise of arterial blood pressure, and various changes in the rate and rhythm of the heart, alterations in breathing. and modifica-

x,

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tions of the spinal reflexes. These investigators (1947) reviewed the results of intracisternal and intraventricular injection of potassium salts. When a solution with an abnormally high potassium concentration is injected intracisternally in the dog, initially increased muscular activity (tetany, extensor rigidity of the legs, etc.), stimulation of respiration followed by a rise of blood pressure and cardiac slowing were noted. However, Fazio and Sacchi (1957) observed that KCI injected into the dog’s cisterna magna is inactive, whereas when introduced into the third ventricle it stimulites motor activities and elicits a rapid and apparently tireless trot. The effects of KCI injected into the ventricles of the cat were first reported by Marinesco et a/. (1929). The injection of 1 mg resulted in clonic contractions which lasted for 20 min, then the cat was unable to walk and appeared very excited. About 2 h after the injection, the cat fell asleep for several hours, while respiration was accelerated and the body temperature rose. Feldberg and Sherwood (1957) first described the effects of the KCl injected into the lateral ventricle of conscious cats. The injection of 0.5 mg KC1 increases alertness and accelerates movements for 30 min. Larger doses (2-3 mg) produce tonic seizures of short duration with or without localized clonic spasms followed by a period of increased muscular tone lasting for 20-40 min. John et 01. (1959) injected KCI (1.8 mg) into the cat lateral ventricle, and reported that the drug produced contralateral somatic motor overactivity culminating in epileptiform seizures : In addition, performance of the various conditioned responses deteriorated after the injection. Stern (1945) thought that changes in K/Ca ratio in the CSF raised the tonus of the sympathetic nervous system and caused CNS excitation. The intraventricular injection of a few mg of potassium in man resulted in augmentation of cardiac activity, increased muscle tone and general excitability. Burej (1959) administered 25% KCl solution on the rat cerebral cortex 6 h after trephining under ether. He found that a conditioned avoidance response was blocked for 6 h and called it a pharmacological reversible decortication. However, in the trephine operation the dura mater and even the cerebral cortex are easily damaged in such a small animal. Therefore, it is not clear whether this result is due to the cortical application or jntracerebral administration. In my experiment, the potassium chloride on the cortex of the dog by the T.A.I. did not produce behavior resembling that of a decorticated dog. Several investigators have studied the electrical activity of the cortex following the topical application of the KC1 and it is well-known that this agent produces De Leiio’s spreading depression (Burer, 1959). Whether or not the behavioral depression seen in our dogs results from De LeBo’s spreading depression is unknown. Following the T.A.I., there were never any alterations in muscular tonus, convulsions, muscle spasms, pupillary changes, respiratory difficulty nor excited behavior. This difference of the results between the T.A.I. and other intra-CSF administration implies that the site of action following the I.A.l. is quite different from those of the intracisternal or intraventricular iiijections. D-Tubocurariiie chloride

Feldberg and Fleischhauer (1962) using cats investigated possible sites of origin of R r ~ w i r c e p. s 1191120

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abnormal electrical discharges resembling epileptic seizures which follow the intravenuicular injection of D-tubocurarine. They excluded the subarachnoid space as a possible site, because the intracisternal injection of the drug did not produce such discharges. However, they observed some abnormal discharges even following the subarachnoid injection, though they were not identical to those following the intravcntricular injection of the drug. Perhaps, the drug has two different sites of action, namely the hippocampus-amygdaloid complex and the cerebral cortex. In my cxperiments, the convulsions following the tubocurarine I.A.I. were apparently due to local penetration of the drug into the cortex, since convulsions were confined mainly to the contralateral side. The diffusion of the drug limited only to the ipsilateral kentricles following the I.A.I. does not seem probable. The possibility of the drug penetrating into the hippocampal region from the parietal cortex through the brain tissue within a few minutes also seems unreasonable. Thus the drug more likely penetrated into the cerebral cortex around the site of administration. A relatively delayed onset of the drug effect was observed following the I.A.I. of this drug, in comparison with an intraventricular injection. Feldberg and Fleischhauer (1 962) reported mydriasis or pilo-erection following intracisternal injection, although the I.A.T. of the drug did not produce such autonomic effects. These results indicate that the site of action of the drug following the cisternal injection is different from that following the l.A.I. Strychnine su(fizte Lewandowsky (1900) described the action of subdurally injected strychnine, convulsions, as occurring at smaller doses than after intravenous administration. Many investigators used the local signs of excitation produced by application of minute doses of strychnine (2% solution) to a specialized area of the cortex in the study on sensory localization (Dusser de Barenne, 1916). The I.A.I. of strychnine confirms Lewandowsky’s results. Ethyl ether While the ultimate action of the ether upon neurons is depolarization, it is doubtful whether a depth of anesthesia sufficient to produce conduction failure by depolarization throughout the nervous system is attained. The I.A.I. of the ether did not reveal anesthesia but analgesia, while the injection of the ether into the lateral ventricle of the dog produced behavioral excitation (unpublished data). L-Epinephrine bitartrate Walaszek (1 960) reviewed the literature on the cortical epinephrine pressor response (CEPR) following topical application of epinephrine (about 750 pg) to the exposed cortex of a rabbit anesthetized with urethane. The increase in blood pressure is usually 20-50 mm Hg and lasts for 0.5-2 min. Such an increase persisted after a

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transection of the spinal cord at CS, after hypophysectomy, or after adrenalectomy. But it disappeared after a transection at theJeve1 of the optic nuclei or after sections of the supraoptic hypophyseal tract below the chiasma. These studies led the investigators to the conclusion that the humoral stimulus produced by epinephrine on the cortex was transmitted by a neuronal pathway to a hypothalamic center capable of secreting a substance with hypertensive activity. The study of the CEPR supports the possibility that behavioral changes following the I.A.I. of epinephrine also may be ascribed to effects exerted on the cerebral cortex. Feldberg and Sherwood (1954) reviewed a number of observations showing that intracisternal epinephrine results in analgesia, sleep and anesthesia. These workers also reported that intraventricularly injected epinephrine in the cat produced similar states. Kumagai et al. (1959a) confirmed these observations using Feldberg and Sherwood’s technique (1954) in the dog. In their dogs 500 pg of L-epinephrine hydrochloride produced drowsiness, analgesia, and tremors, 900 pg provoked ataxia, vomiting, hypokinesia, analgesia but no drowsiness. Two mg evoked salivation and static ataxia. Compared with this drowsiness the effect of the I.A.I. of epinephrine was much weaker. Acetylcholine chloride

Suh et a/. (1935) and Von Euler (1938) reported that the intracisternal injection of acetylcholine (ACh) increased blood pressure. Walaszek (1960) noted that the topical application of ACh (750 pg) in the anesthetized rabbit produced a very small depressor response (5-8 mm Hg) on the first application and usually no response thereafter. Dikshit (1935) reported sleep (0.1-0.5 pg) following intraventricular injection in the cat, while Henderson and Wilson (1936) found vomiting, increased intestinal peristalsis and sweating following intraventricular injection of acetylcholine (2.5-7.5 mg) in man. Feldberg and Sherwood (1 954) observed retching, phonation, akinetic seizure and stupor (10-20 pg) and convulsions followed by sleep, stupor and catatonic-like condition following intraventricular injection of 1 mg in the conscious cat. Several workers have reported on the electrical activity of the brain following the topical application of acetylcholine. Chatfield and Dempsey (1941) failed to find any effect after topical application of 1 P: ACh solution, but Brenner and Merritt (1943) observed localized discharges with 2.5-10% ACh. Forster (1945) found a depression of electrical activity followed by high voltage discharges after intracisternal injections of 10-25 mg of ACh in the cat. Forster and McCarter (1945) and Forster (1945)found similar effects on the topical application of 5-250/, ACh. Forster et al. (1946), however, reported that a depression of activity precedes the spike discharges and that the EEG results bear no relationship to systemic effects. Essig et al. (1953) also obtained depression of activity using 2-20 P: ACh solution. Cooke and Sherwood (1954) found increased spindle activity after intraventricular injections of 10-20 pg of ACh in the ‘encephale isolt’ preparation of the cat. In our experiments with acetylcholine the dog never exhibited sleep or catatonia. The animal closed his eyes and was quiet but seemed uncomfortable as he frequently Refmences p. 119ji20

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opened his eyes and shook his head or raised his nose. The dog’s EEG did not demonstrate any depression of clectrical activity but exhibited intermittent rhythmic waves (10-14 cycles/sec) with high voltage for 20 min but only on the injected side. Following the injection of more than 10 mg of acetylcholine convulsions or twitches were observed in several dogs when the drug was given into the cerebral cortex instead of the surface because of the technical failure in the implantation of the tube. Even in these cases, smaller doses, 500 pg or 1 mg did not produce convulsions but twitches, as saline solution injected in a similar position of the cerebral tissue did not evoke seizures. The convulsions were therefore due to drug action rather than merely to the volume effects of injected fluid. However, convulsions werc never following the I.A.I. even in doses as huge as 30 mg of ACh. Serotonin creatiiiine sulfate Walaszek observed no influence on the blood pressure after the topical application of 5-HT (approximately 750 p g ) in anesthetized rabbits. Feldberg and Sherwood ( 1 954), Gaddum and Vogt (1 956), Sturtevant and Drill ( 1 956a), Vogt (1958) all reported behavioral changes following 5-HT iiijection into the cat’s lateral ventricle: 75 to 500 pg of 5-HT produced muscle weakness, tremor, retching, decrease of activity, tachypnea but not sleep, and 1.2-1.8 mg exhibited manic-like hyperkinesiaand a short clonic convulsion. Sacchi el a/. (1956) iiijected 5-HT into the third ventricle of the dog and found catalepsy with a dose of 1.2-4 mg but failed to produce catalepsy after intracisternal injection. Kumagai et al. ( I 959a) reported the behavioral depression of a dog following intraventricular injection: 25 to 100 pg of 5-HT produced hypokinesia and catatonia, 500 ,ug evoked drowsiness and 10 nig induced ataxia, hypokinesia and catatonia. However following the I.A.I. types of behavior different from the above-mentioned were observed and larger doses were required to produce depression. This behavioral change was not like that of catatonia, catalepsy, but rather resembled drowsiness. In another dog, due to a technical failure of the implantation, a small cut was made about 2 mm deep into the cortex. In this dog the injection of 3 mg 5-HT produced behavioral depression soon following the I.A.I., and with 10 mg violent generalized repetitive convulsions continued for I h. At autopsy the dye was found on the surface of the cortex, but minute traccs of dye were also noted at the site of injury in the cortex. Chlorprornazine h.ydrochloride Sturtevant and Drill ( I 956b) injected 2 mg chlorpromazine into the lateral ventricle of a cat, and found a relaxation of nictitating membrane, tachypnea, ataxia, and a marked catatonia, wherein the cat appeared oblivious of visual, auditory, and pain stimuli and maintained an abnormal posture (catalepsy). Weinberg and Haley (1 956) reported that 2.5 mg chlorproinazine injected into the third ventricle of a dog produced a lowered biphasic T-wave in the EKG and tranquilized the animal for 16 min. A higher dosage of chlorpromazine, 10 mg, produced bradycardia followed by tachy-

INTRA-ARACHNOID INJECTIONS OF D R U G S

117

cardia, ventricular extrasystoles, analgesia, tranquilization, shivering, mydriasis, decreased respiratory rate, defecation, occasional barking and wild behavior as dog attempted to bite the observer. But the animal appeared normal 10-12 h after the injection. Kumagai et a/. (1959b) reported that 1 mg of chlorpromazine injected into the lateral ventricle of a dog produced behavioral depression. But after the blockade of aqueduct of Sylvius, the drug injected intraventricularly evoked excitement. Miura et a/. (1957) injected chlorpromazine (0.38-0.5 mg/kg) into the lateral ventricle in man, through the anterior-horn puncture under local anesthesia. They reported that 0.5 mg/kg chlorpromazine produced a hypotensive state, a fall of 20 mm Hg, 5-10 min after the injection and continued for over 2 h. Heart rate was accelerated and respiration rate was decreased for about 1 h, 15 min after the injection the patient fell into a deep sleep for 3 h and did not respond to the pain stimulation for 1-1.5 h. Sometimes nausea, vomiting, and incontinence were also observed. In general, the site of action of this drug is assumed to be the mesodiencephalon. However, according to my results, the drug depressed not only the subcortical areas but also the cerebral cortex. This depression seems lighter than that following intraventricular injection. Possible site of action ojthe drug following Z.A.I. The author confirmed Feldberg and Fleischhauer’s report (1960) that the brain possesses an active transport because dye injected after death usually does not stain the brain. The probable distribution of the drug is mostly confined to the parts of the cerebral cortex nearest the tip of tube and upper half of the hemisphere on the injected side. This limited distribution is due to the very slow circulation of the CSF and the anatomical construction of the cerebroarachnoid space containing many cul-de-sacs. Dobbing (1961) has reviewed the local uneven distribution of the drug following intracisternal injections. The present data indicate that intracisternal or intraventricular injections produce quite different effects from those after the I.A.I. and these differences are regarded as evidence for the localized distribution of the drug. Bakay (1956) showed that any dye could penetrate the brain from the CSF provided that its particle radius was less than 10 A, the dye being absorbed to a distance which was roughly proportionate to the size of the particle. However none of the 3 dyes used in I.A.I., 1 % methylene blue (basic dye), 4.7 % methylene blue (isotonic), 0.2 % bromophenol blue (acid dye), seemed to penetrate into subcortical areas beyond the cortex. In general, however, there is evidence that many substances enter the brain more easily, more quickly, and in greater amounts, when injected into the CSF, than into the blood. There is an extremely rapid exchange of small molecular weight substances between the relatively small volume of CSF, across the large surfaces of the pia, ependyma, and brain tissues. Furthermore, the brain comes into rapid equilibrium with the CSF (Dobbing, 1961). For example, with radioactive inorganic phosphate, a considerable part of the brain attained maximal levels only 10 or 30 min after intracisternal injection (Bakay, 1956). References p . II9jIZO

118

T. K O B A Y A S H I

The dog’s cerebroarachnoid space islonly about 0.9 ml in volume. Substances injected by I.A.I. into such a relatively small volume of CSF tend to be active in relatively small quantities and over a period of some hours. In man, it is known that the CSF may be replaced each 2.5-20 h implying that a substance injected in the CSF is diluted to about 50% during that time (Dobbing, 1961). These data, that is the localized distribution of the dye, the penetration of radioactive substance into the brain and the slow renewal of CSF, support the assumption that the drug administered by the I.A.I. exerts its effects on the brain through localized action rather than absorption into systemic circulation. Furthermore, the evidence shows that the effects following the I.A.T. are quite different from those evoked by the intracisternal or intraventricular injections, as seen for example with potassium chloride, and in the observation of D-tubocurarine chloride producing convulsions mainly on the contralateral side of the body, all suggest a localized attack of the drug following the I.A.I. Another proof for this possibility is Bircher et d . ’ s experiment. They determined the dose-route relationship of pentylenetetrazol and picrotoxin, when given intravenously, intra-arachnoidally on the cortex, and into the lateral ventricle, the third ventricle, and the fourth ventricle in dog brain with chronically implanted cannulas or catheters. The EEG and EKG were recorded from dogs immobilized by succinylcholine chloride. These workers demonstrated that the EEG convulsive discharges induced by pentylenetetrazol applied by intravenous versus cortical route exhibited a dose-effect relationship of approximately 5 : 1; picrotoxin 1000 : I . Thus the intravenous effects were much milder. Noticeable diffcrences jn latent periods of the convulsive discharges following administration of an ED50 dosage were also observed. In the case of pentylenetetrazol, it was 20 sec by the intravenous route, 21 sec after the cortical application, 82 sec via the lateral ventricle injection, 180 sec in the third ventricle, and 65 sec in the fourth ventricle, These results suggest that the effects of the drug applied in the arachnoid space were not due to drug action after absorption into the systemic circulation. Furthermore, the results revealed that the sensitivity of the brain to the drug varied with different sites. SUMMARY

I . The behavioral effects of the 8 neurotropic drugs following the intra-arachnoid injection were observed in 38 dogs. 2. Potassium chloride depressed the dog’s behavior for about 1 h and 30 min and the animal seemed to be drowsy. 3. Strychnine sulfate produced convulsions. 4. Ethyl ether seemed to excite the dog for 4 h while consciousness was maintained. 5. D-Tubocurarine chloride produced convulsions. 6. L-Epinephrine bitartrate exhibited drowsiness. 7. Acetyl choline chloride evoked a behavioral depression but it was neither sleep nor catatonia. 8. Serotonin creatinine sulfate induced sleep.

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9. Chlorpromazine hydrochloride induced strong behavioral depression resembling that of sleep. 10. Dye injections revealed that the distribution was confinedon the upper half of the hemisphere on the injected side. 11. Possible modes of action were discussed and it was concluded that the drugs produced their effects by affecting a localized cortical surface. ACKNOWLEDGEMENTS

The author is indebted to Dr. Harold E. Himwich for his continuous interest and guidance, to Dr. Christopher Bull for his criticism and correction of the manuscript, to Mrs. Catherine Halsey for her technical assistance, to Mr. Lloyd Tenneson for the preparation of figures and recording of motion pictures, and to Squibb Pharmaceutical Company for the special supply of the D-tubocurarine chloride powder. REFERENCES

BAKAY, L., ( I 956); The Blood-Brain Barrier. Springfield, Thomas. BIRCHER, R. P., KANAI,T., AND WANG,s. c.,(1962); Intravenous, cortical and intraventricular doseseffect relationship of pentylenetetrazol, picrotoxin and deslanoside in dogs. Electroenceph. din. Neurophysiol., 14, 256-261. BRENNER, G., AND MERRITT, H. H., (1943); Effect of certain choline derivatives on electrical activity of cortex. Arch. Neurol. Psychiat., 48, 382-395. BURES,J., (1956); Some metabolic aspects of De Leiio’s spreading depression. J . Neurochem., 1, 153-158. BURESJ., (1959); Reversible decort ication and behavior. The Central Nervous System and Behavior. M. A. B. Brazier, Editor. Transactions of the Second Conference. New York, Josiah Macy, Jr. Foundation (p. 207). CALMA, I., AND WRIGHT, S., (1947); Effects of intrathecal injection of KCI and other solutions in cats. Excitatory action of K ions on posterior nerve root fibers. J . Physiol. (Lond.), 106, 211-235. CHATFIELD, P. O., AND DEMPSEY, E. W., (1941); Some effects of prostigmine and acetylcholine on cortical potentials. Arner. J. Physiol., 135, 633-640. COOKE, P. M., AND SHERWOOD, S. L., (1954); The effect of introduction of some drugs into the cerebral ventricles on the electrical activity of the brain of cats. Electroenceph. clin. Neurophysiol., 6, 42543 1. DIKSHIT, B. B., (1935); Action of acetylcholine on the ‘sleep center’. J . Physiof. (Lond.), 83, 42P. DOBBING, J., (1961); The blood-brain barrier. Physiol. Rev., 41, 130-188. DUSSER DE BARENNE, J. G., (1916); Experimental researches on sensory localization in the central cortex. Quart. J. exp. Physiol., 9, 355-390. ESSIG,C. F., ADKINS,F. J., AND BARNARD, G. L., (1953); Observations on electrocorticographic effects of acetylcholine in monkeys and cats. Proc. SOC.exp. Biol. ( N . Y.), 82, 531-551. FAZIO,C., AND SACCHI, U . , (1957); Experimental catalepsy produced by substances introduced into subarachnoid spaces and ventricle. Psychotropic Drugs. S. Garattini and V. Ghetti, Editors. Amsterdam, Elsevier (p. 104). FELDBERG, W., AND FLEISCHHAUER, K., (1 960); Penetration of bromophenol blue from the perfused cerebral ventricles into the brain tissue. J . Physiol. (Lond.), 150, 451462. FELDBERG, W., AND FLEISCHHAUER, K., (1962); The site of origin of the seizure discharge produced by tubocurarine acting from the cerebral ventricle. J . Physiol. (Lond.), 160, 258-283. FELDBERG, W., AND SHERWOOD, S. L., (1954); lnjections of drugs into the lateral ventricle of the cat. J . Physiol. (Lond.), 123, 143-167. FELDBERG, W., AND SHERWOOD, S. L., (1957); Effects of calcium and potassium injected into thecerebra1 ventricles of the cat. J . Physiol. (Lond.), 139, 403416. FENN,W. O., (1940); The role of potassium in physiological process. Physiol. Rev., 20, 377415.

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FORSTER, F. M ,(1945); Actionofacetylcholineonmotorcortex. Arch. Neurol. Psychiat., 54,391-394. FORSTER, F. M., AND MCCARTER, R. H., (1945); Spread of acetylcholine induced electrical discharges of the cerebral cortex. Amer. J. Physiol., 144, 168-173. FORSTER, F. M., BORKOWSKI, W. J., AND MCCARTER, R. H., (1946); Acetylcholine induced depression of cerebral cortical activity. J . Neuropathol. exp. Neurol., 5 , 364373. GADDUM, J. H., AND VOGT,M., (1956); Some central action of 5-hydroxytryptamine and various antagonists. Brit. J . Pharmacol., 11, 175-179. HENDERSON, W. R., AND WILSON, W. C., (1936); Intraventricular injection of acetylcholine and eserine in man. Quart. J . exp. P h y s d . , 26, 83-95. JOHN, E. R., TSCHIRGI, R. D., A N D WENZEL,B. M., (1959); Effects of injections of cations into the cerebral ventricles on conditioned responses in the cat. J . P h y ~ i o l(Lond.), . 146, 550-562. KOBAYASHI, T., (I 962); Drug administration to cerebral cortex of freely moving dogs. Science, 135, 1126-1127. KUMAGAI, H., DOMAE, A., SAKUMA, A., KATO,H., FUKUHARA, T., KOBAYASHI, T., TAMURA, H., OTSUKA, Y., AND SOKABE, H., (1959a); Studies on the central effects of drugs by means of chronic cerebral ventricular fistula. I. Mode of operation and effects of several adrenergic drugs by intraventricular administration in unanesthetized dog. Folia pharmricol. jap., 55, 120P. KUMAGAI, H., KOBAYASHI, T., KATO,H., SOKABE, H., AND YAMAMOTO, s., (1959b); Effects Of intraventricularly injected chlorpromazine on the behavioral change induced by LSD-25 in the aqueduct-blocked dog. In the press. LEWANDOWSKY, M., (1900); Zur Lehre von der Cerebrospinalflussigkeit. Z . klin. Med., 40,480494. MARINESCO, G., SAGER, O., A N D KREINDLER, A., ( I 929); Experimentelle Untersuchungen zum Problem des Schlafmechanismus. Z . ges. Neurol. Psychiat., 119, 277-306. MIURA,Y., KAWASHIMA,Y . ,MURAKAMI, M., AND OHGAWARA, S., (1957); Intraventricular injection of the chlorpromazine and its application to the neurosurgery. Nippon-Rinsho (Jap), 15, 531-540. SACCHI, U., BONAMINI, F., GARELLO, L., A N D DOLCE, G., (1956); Studio dell' azione catalettizzante della 5-HT per introdurione nel sistema ventricolare o negli spazi subarachnoidei del cane. Boll. Soc. ital. Biol. sper., 32, 179-181. STERN,L., (1945); Direct chemical action upon nerve centers in biology and medicine. Nature, 156,7-9. STURTEVANT, F. M.,AND D R I L L , V. A., (1956a); Effects of serotonin injected into the lateral ventricle of the brain of cats. Anat. Rec., 125, 607-608. STURTEVANT, F. M., AND DRILL, V. A., (195613); Effects of mescaline in laboratory animals and influence of ataraxics on mescaline response. Proc. Soc. exp. Biol. ( N . Y . ) , 92, 383-387. SUH,T. H., WANG,C . H., AND LIM,R. K. S . , (1935); Effect of intracisternal injections of acetylcholine. Proc. Soc. exp. Biol. ( N . Y . ) , 32, 1410. VOGT,M., (1958); Drugs interfering with central actions of 5-hydroxytryptamine. 5-Hydroxytryptamine. G. P. Lewis, Editor. London, Pergdmon Press (p. 209). VONEULER,U . S., (1938); Reflektorische und zentrale Wirkung von Kaliumionen auf Blutdruck und Atmung. Scand. Arch. Physiol., 80, 94-123. WALASZ~K, E. J., (1960); Brain neurohormones and cortical epinephrine pressor responses as affected by schizophrenic serum, Inf. Rev. Neurobiol., 2, 138-173. WEINBERG, S. J., AND HALEY, T. J., (1956); Effect of chlorpromazine on cardiac arrythmias induced by intracerebral injection of tryptamine-strophanthidine.Arch. int. Pharmacodyn., 105,209-21 1 . WINTERSTEIN, H., (1961); The actions of substances introduced into the cerebrospinal fluid and the problem of intracranial chemoreceptors. Phurrnucol. Rev., 13, 71-107.

121

Anti parkinson Drugs and Neuroleptics C. MORPURGO Research Laboratories, J. R. Geigy Ltd., Bash (Switzerland}

Soon after the introduction of chlorpromazine and reserpine in psychiatric therapy, several observations of drug-induced Parkinson-like symptoms were reported as occasional side reactions. The wider use of tranquillizing drugs and the introduction of more potent phenothiazine derivatives and of the butyrophenone derivatives gave rise to an ever increasing number of observations and opinions concerning the frequency and the significance of the pharmacologically induced dysfunctions of the extrapyramidal system. The different results reported in the literature about the statistical occurrence of the parkinsonian syndromes induced by neuroleptics, even when the same compounds were used, may be more likely accounted for by different criteria in evaluating the neurological signs than by different dosages or therapeutic schemes employed by the various authors. The different tendency of the individual compounds to induce extrapyramidal reactions and the different susceptibility of the individual patients have been established ; however, the ability to affect the extrapyramidal system can now be regarded as a characteristic property of neuroleptic drugs, of considerable importance both from the practical and the theoretical point of view. An entire symposium has been devoted to the study of these problems (Extrapyramidal System and Neuroleptics, Montreal, 1961). The mechanisms underlying the appearance of the parkinson-like symptoms during neuroleptic treatments are not ascertained. Only a few observations of neurological syndromes persisting after discontinuation of the drugs can be found in the literature (Hall et al., 1956; Uhrbrand and Faurbye, 1960); usually the extrapyramidal reactions are reversible, therefore no related pathological findings have been reported. Our lack of knowledge of the neurotransmitters in the central nervous system prevents any conclusion on functional modifications that bring about the parkinsonian signs. However there are some suggestions for the hypothesis that the drug-induced extrapyramidal reactions result from a central imbalance in favour of cholinergic mechanisms (Toman, 1963). Such a hypothesis is consistent with the postulation that the tremor occurring in the Parkinson’s disease is caused by a denervation hypersensitivity to acetylcholine of the extrapyramidal relays caudal to injuries in the basal ganglia or in the upper brain stem (Magoun, 1954). The curative effect of anticholinergic drugs in regard to parkinsonian signs supports the hypothesis of their central References p. 132-134

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cholinergic origin. Moreover, the phenothiazine derivatives less prone to cause extrapyramidal dysfunctions, such as chlorprothixene and thioridazine, possess considerable anticholinergic activity (Pellmont et a]., 1960; Haley et al., 1960); even chlorpromazine has a stronger cholinolytic activity than perphenazine (Arrigoni-Martelli and Krarner, 1959), and this property may account for its weaker tendency to induce severe neurological syndromes. The therapeutic implication of the drug-induced extrapyramidal reactions is still controversial. Whereas some authors consider the extrapyramidal signs as undesirable side effects, others regard them as a prerequisite for the antipsychotic activity of the drugs. As pointed out by Freyhan (1961), the ability to induce parkinson-like syndromes has a positive correlation with the therapeutic potency of a given drug: however overt neurological syndromes should be considered as excessive reactions and avoided. In an investigation performed at Galesburg State Research Hospital under the directorship of Dr. Himwich (Brune et al., 1962) on the relationship existing between the behavioural alterations and the extrapyramidal disturbances during a neuroleptic treatment, it was found that in general the optimal therapeutic effects were achieved when only very mild parkinson-like symptoms occurred. These findings were consistent with Haase’s statement (1961) that the fine motor extrapyramidal manifestations (that may be detected by changes in the handwriting) are a conditio sine qua nun for the essential efficacy of neuroleptic drugs, while the more severe signs disturb the patients and thus impair the therapeutic action. In view of the fact that pronounced extrapyramidal syndromes are usually considered undesirable, antiparkinson drugs are often given to counteract the neurological signs and sometimes combined treatments are instituted from the beginning. But, if the action of neuroleptic drugs on the extrapyramidal system is essential for their tranquillizing activity, the question may arise whether drugs interfering with that action would also reduce the therapeutic effectiveness of neuroleptics. Clinical investigations do not lend themselves easily to solve the problem of a pharmacological interaction. According to a number of authors the behavioural improvements obtained with tranquillizing drugs are maintained after addition of antiparkinsonian medications or even enhanced when the neurological symptoms were disturbing the patients. However, the dosage factor was usually disregarded in these studies; it is therefore difficult to ascertain whether the addition of anticholinergic agents leads to results equivalent to a reduction of the dosage of the neuroleptic drugs. In fact, in many cases, an individual adjustment of the dose level of neuroleptics makes it possible to attain the therapeutic effect and to control the extrapyramidal signs. On the other hand, after the addition of the antiparkinsonian medication transitory periods of mental confusion have been observed (Brune et al., 1962; Goldman, 1961), and, though in exceptional cases, a return of disturbed behaviour (Freyhan, 1961). The well-known toxic effects of atropine-like drugs (confusion, delirium, hallucinations, delusions) (Doshay and Zier, 1954; Pfeiffer et al., 1959; Bolin, 1960) seem to point to antagonistic effects of antiparkinson drugs and neuroleptics on the psychic level. It is possible that in combined treatments the psychotomimetic effects of anticholinergic drugs are masked by the simultaneous administration of neuroleptics.

ANTIPARKINSON DRUGS A N D NEUROLEPTICS

123

EXPERIMENTS

In the attempt to investigate the interaction between antiparkinson drugs and neuroleptics, we have undertaken a series of experiments on some of the animal tests that proved to be of a predictive value in assessing the clinical effectiveness of psychotropic drugs (Morpurgo, 1960, 1962; Murpurgo and Theobald, 1964). In this paper are reported some results of the effects of anticholinergic compounds on the pharmacological actions of phenothiazine derivatives. Perphenazine and trifluoperazine have been selected because of their pronounced tendency to induce extrapyramidal dysfunctions, scopolamine as a typical cholinolytic agent and the synthetic antiparkinson compound trihexyphenidyl as a drug widely employed in com bined treatments with neuroleptics. ( I ) Cataleptic reaction in rats A characteristic property of neuroleptic drugs is the ability to induce catatonia-like states in experimental animals which may be regarded as the counterpart of the extrapyramidal reactions in human beings (Courvoisier et al., 1957): a positive correlation between the ability to induce parkinsonian reactions in patients and the cataleptic activity in animals can be observed for the different compounds. The main features of the experimental catatonic syndrome are akinesia and passivity, which consents the maintenance of abnormal positions, though the equilibration reflexes are retained. We studied drug-induced catalepsy on male Wistar rats of the average weight of 300 g, by evaluating the failure to correct imposed postures of the body. The intensity of the response was scored for the single animals according to the stages 111 and IV described by Wirth et al. (1958). In stage I11 the rats were placed on the table with one front paw set on a cork 3 cm high, the other remaining on the table. In stage IV a front paw was set on a cork 9 cm high, the other hanging free. The failure to correct the imposed posture within 10 sec was considered as a positive catatonic reaction. Both stages were tested on the right and left sides, and 1/2 point for each side of stage 111 and 1 point for each side of stage IV were arbitraiily assigned for every positive response. The catatonic reactions were evaluated at hourly intervals after the administration of the phenothiazine derivatives. In this test, perphenazine proved to be more effective than chlorpromazine; a delayed onset of activity was observed with fluorinated compounds : trifluoperazine and fluphenazine. We compared the effects of several anticholinergic drugs used as anti parkinson agents on phenothiazine-induced catatonic reactions. Antagonistic effects on catalepsy induced by 5 mg/kg i.p. of perphenazine were obtained with scopolamine, atropine, trihexyphenidyl, benztropine and biperiden. In doses of 2 mg/kg i.p., the inhibition caused by scopolamine was nearly total and persistent, while the other compounds showed a more transient activity; as their actions wore off, a progressive increase in the scoring of the catatonic response appeared (Fig. 1). A remarkable quantitative difference between the antagonistic effects of scopolamine and atropine was observed : scopolamine showed an activity even in a dose of 0.1 mg/kg, while References p . 13.2-134

124

C. MORPURGO 100r Scopolamine

Biperiden Trihexyphenidyl

Benztropine

I

I

I

I

I

I

3

2

1

h

Fig. 1 . Effect of antiparkinson drugs on the perphenazine-induced catalepsy in rats. Scopolamine hydrobromide, atropine sulphate, trihexyphenidyl hydrochloride, benztropine methansulphonate and biperiden hydrochloride were injected in doses of 2 mg/kg i.p. 15 min prior to perphenazine 5 mg/kg i.p. (at 0 time in the curves). Percent inhibition was calculated in comparison to the cataleptic responses of a group of control rats injected with p-rphenazine alone. Each curve represents the average values obtained for 6 rats. ATROPINE

SCOPOLAMINE

'O 80 Oi

0

1

2

3

h

' " OO

1

2

3

h

Fig. 2. Effect of different doses of atropine and scopolamine on the cataleptic reaction induced by 5 mg/kg i.p. perphenazine in rats. Each curve represents the average values of inhibition obtained for a group of 4 rats in comparison t o the controls injected with perphenazine alone.

atropine was about 20 times less active (Fig. 2). This ratio of activity between the two compounds appears of interest since it is in agreement with the findings reported in the literature on oiher experimental tests for the evaluation of antiparkinson drugs (Jenkner and Ward, 1953; Vernier and Unna, 1956). The curative effect of antiparkinson drugs on neuroleptic-induced catalepsy has been found in regard to other compounds (Schaumann and Kurbjuweit, 1961; Taeschler et a].,1962; Buchel et al., 1962; Morpurgo and Theobald, 1964). Moreover, according to our results, it seems to be a rather specific action of anticholinergic compounds, since CNS stimulants were effective only in much highei doses. ( 2 ) Spontaneous motility of mice An obvious sign of neuroleptic activity is the reduction of the spontaneous motility in experimental animals.

I25

ANTIPARKiNSON DRUGS A N D NEUROLEPTICS

We employed two different procedures for studying the locomotor activity of mice: (a) An actographic method was employed for studying the total motor activity of mice. A single animal was placed in a small cage and its movements were transmitted through a rubber floor to an electrographic recolder (Sanborn). The sensitivity of the apparatus was adjusted in such a way that the fine voluntary movements such as rearing or grooming and not the respiratory activity were registered. Diugs were injected into animals already accustomed to the experimental chamber so that the exploratory activity had subsided.

PERPH~NAZINE

SALINE

2 a g / k g i.p

w

-3

PERPH~NAZINE 2 mg/kg

i.p

SCOPO~A MINE

1 MIN

2 w / k 9 i.p

,

Fig. 3. Effect of scopolamine on the perphenazine induced depression of the motor activity in mice

(In the upper tracing a control mouse received an i.p. injection of saline 30 min after perphenazine.)

t

.T

TRIFLUOPERAZINE f m g / k g 1.p

SALINE

t

TRIFLUOPERAZINE I mg/kg

i.p.

1

HIN

t

TRIHEXYFHENIDYL I0 m g / k g i p

Fig. 4. Effect of trihexyphenidyl on the trifluoperazine induced depression of the motor activity in mice. (In the upper tracing a control mouse received an i.p. injection of saline 30 min after trifluoperazine.)

A restoration of the spontaneous motility depressed by neuroleptic drugs was observed after the injection of antiparkinson drugs (Figs. 3 and 4). (b) The photoelectric technique (Dews, 1953) was used to record coordinated running or walking movements. Male albino mice were placed in groups of 5 in rectangular cages (30 x 20 x 10 cm) kept in a sound-proof loom. The number of times the mice broke a beam of light directed to a photoelectric cell through the short axis of References p . 132-134

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TABLE I EFFECT O F P H E N O T H I A Z I N E D E R I V A T I V E S A N D A N T I P A R K I N S O N D R U G S O N T H E S P O N T A N h O U S M O T I L I T Y O F MICE

Number of counts in the initial 15-min period after introduction of the mice in photocell-counting chambers. Perphenazine and trifluoperazine were injected 30 min, scopolamine and trihexyphenidyl 15 min earlier. In parentheses the number of experiments. -1- Scopolamine

0.5 mg/kg i.p.

-1- Tvihexyphenidyl 5 mglkg i.p.

302 (6) 120 (4) 133 (4)

314 (6) 12.5 (4) 1.53 (4)

__

-~ 182 (50) Perphenazine 1 mg1kgi.p. 34 (12) Trifluoperazine 1 mg1kgi.p. 64 (14)

T A B L E 11 I N T E R A C T I O N O F V A R Y I N G D O S E S OF T R I F L U O P E R A Z I N E A N D T R I H E X Y P H E N I D Y L O N T H E S P O N T A N E O U S M O T I L I T Y O F MICE

Number of photocell counts in a 15 min period. Trifluoperazine was injected 30 min and trihexyphenidyl 15 min before placing the mice in the experimental chamber. Each figure represents the average value obtained for 4 groups of mice.

+ Tvihexyphenidyl + Tvihexyphenidyl Trifluoperazine 1 nig/kg i.p. Trifluoperazine 5 mg/kg i.p.

196 78 60

2 mglkg i.p.

10 mglkg i.p.

337 205 55

3 24 198

141

the cage was recorded by means of digital counters kept in an adjoining room. The counts in the initial 15-min period after the mice had been placed in the cages gave a measurement of the exploratory behaviour. Since the animals were tested in a group, the effect of the mutual stimulation is also to be considered with this procedure. Antithetic effects between antiparkinson drugs and neuroleptics were observed in this test. Scopolamine 0.5 mg/kg i.p. and trihexyphenidyl 5 mg/kg i.p. showed a stimulant effect and antagonized to a certain extent the depression caused by 1 mg/kg i.p. perphenazine and trifluoperazine (Table 1). However, the dosage factor should be taken into consideration in evaluating the pharmacological interaction; as reported in Table 11, the reduction in the motility induced by 1 mg/kg of trifluoperazine was counteracted by either 2 or 10 mg/kg of trihexyphenidyl, whereas an antagonistic effect in regard to 5 mg/kg of the neuroleptic could be obtained only with the largest dose of the antiparkinsonian compound.

( 3 ) Avoidance behaviour in rats Techniques for testing conditioned avoidance behaviour are currently used for an experimental analysis of tranquillizing agents, since different results have been ob-

ANTIPARKINSON DRUGS AND NEUROLEPTICS

127

tained with the different classes of compounds. Neuroleptic drugs produce a specific block of the learned avoidance behaviour in doses that do not impair the ability to escape from a noxious stimulus, whereas barbiturates and meprobamate produce a non-specific block of the avoidance I esponses in neurotoxic doses, affecting the escape response concurrently, owing to an incapacitation of the motor function (Cook and Weidley, 1957). According to Tedeschi et a]. (1961) the rank order of potency of the compounds in blocking a conditioned avoidance behaviour has a positive correlation with the therapeutic potency in man. As a test of conditioned behaviour we employed the lever-pressing avoidance technique developed by Sidman (1953), also called non-discriminated or continuous avoidance, which differs from the classical tests of conditioned activity by the lack of an exteroceptive warning signal. It permits an appreciation of either a depression or a stimulation of the avoidance behaviour. Experiments were carried out in a Skinner-type box with a grid-floor for delivering shocks and a lever mounted on a wall. Shocks were delivered at regulai intervals unless the lever was depressed. Each lever depression postponed the occurrence of the shock for a given period of time or, in the case that the shock was not avoided, could terminate it. Shock-shock and response-shock intervals were both 20 sec. The events were programmed and recorded automatically. Male Long-Evans rats learned to depress the lever at a steady rate in order to avoid the shocks; trained animals were tested during a 30-min session, 5 days a week. Drugs were injected i.p. on Tuesday and Friday. The average number of lever-pressing responses in the pre-drug days (ranging from 6 t o 10 responses/min for the single animals) was used as reference value. T A B L E 111 EFFECT OF PHENOTHIAZINE DERIVATIVES A N D A N T I P A R K I N S O N DRUGS O N THE L E V E R - P R E S S I N G

AVOIDANCE BEHAVIOUR IN RATS

The results are expressed as a percent of the rate of lever pressing responses in the non-drug sessions. In parentheses the number of rats. Perphenazine and trifluoperazine were injected 30 min, scopolamine and trihexyphenidyl 15 min before the introduction of the rat in the Skinner-box.

+ Scopolamine + Trihexyphenidyl 0.2 rnglkg i.p.

Perphenazine 0.2 mg/kg i.p. -62 % (7) Trifluoperazine 0.4mg/kg i.p. -52 % (7)

+

36 % (4) -30 % (4) -38 % (4)

2 mgtkg i . p . +19% (6) -35 % (3) - 2 % (3)

Neuroleptic drugs produced a decrease in avoidance lever-pressing rates, thus increasing the number of shocks delivered. In contrast, a stimulant action was observed with scopolamine and trihexyphenidyl, which antagonized to a certain extent the depression caused by phenothiazine derivatives (Table I1 I). References p . 132-134

I28

C. M O K P U R G O

(4) Amphetamine toxicity in aggregated mice

The protective effect against the toxicity of amphetamine in aggregated mice has been suggested as a test for tranquillizing drugs (Burn and Hobbs, 1958). In fact the enhancement of the amphetamine toxicity in mice kept in a crowded environment in comparison to the toxicity in isolated mice has been attributed to the exhaustion following the furious excitement induced by the mutual stimulation. This group toxicity is selectively antagonized by neuroleptic drugs (Lasagna and McCann, 1957). We studied the effects of phenothiazine derivatives and antiparkinson drugs on amphetamine toxicity in male mice, 20-25 g body weight, grouped 5 to a cage with a floor area of approx. 50 cm2, at a room temperature of 24". Amphetamine was injected in the standard dose of 75 mg/kg i.p. and the mortality was recorded after 6 h. Scopolamine and trihexyphenidyl increased the group toxicity of amphetamine and antagonized the protective effect of perphenazine and trifluoperazine (Table IV). TABLE IV AMPHETAMINE TOXICITY I N A G G R E G A T E D MICE

Percent of deaths recorded 6 h after the injection of 75 mg1kgi.p. DL-amphetamine sulphate in mice grouped 5 to a cage. Phenothiazine derivatives were injected 30 min, scopolamine and trihexyphenidyl 15 min prior to amphetamine. Each treatment was tested on 10 mice (number of controls = 30 mice).

Perphenazine 1 mg/kg S.C. Trifluoperazine 1 mg/kg S.C.

70 "/o 20% 20%

i Scopolamine 0.5 mglkg i.p.

+ Trihexyphenidyl

100% 100%

100% 90 X 100%

90%

5 mg/kg i . p .

Death occurred much earlier in mice injected with the anticholinergic compounds (even in combination with neuroleptics) than in controls. No death was recorded within 6 h in single mice injected either with amphetamine alone or with scopolamine and amphetamine in the same dosages as employed for aggregated mice, but scopolamine appeared to enhance the excitatory action of amphetamine. This observation is consistent with the augmentation of the behavioural effects of amphetamine by scopolamine reported by Carlton (1961a). ( 5 ) Apomorphine-induced 'chewing' reaction in rats

Antagonistic or synei gistic effects of apomorphine-induced reactions are currently employed in the laboratory evaluation of psychotropic agents. While in several animal species the chief effect of apomorphine is vomiting, the apomorphine syndrome in rodents consists in signs of central stimulation (restlessness, increased excitability, exophthalmos) and a compulsory masticatory activity; the masticatory movements, like the persisting pecking induced by apomorphine in pigeons, have been interpreted as 'feeding hallucinations' (Koster, 1957). Neuroleptic drugs have been found to antagonize apomorphine effects (Rosenkilde and Govier, 1957; Janssen et al., 1960; Burkman. 1961 ; and others); in contrast, a potentiation of the apomorphine-induced com-

129

ANTIPARKINSON DRUGS AND NEUROLEPTICS

pulsory gnawing by anticholinergic compounds has been observed in mice (Ther and Schramm, 1962). We studied the apomorphine antagonism in rats according to the procedure described by Janssen et al. (1960). Male Wistar rats of the average weight of 80 g were injected intravenously with 1.25 mg/kg apomorphine hydrochloride, and the presence TABLE V INHIBITION OF T H E A P O M O R P H I N E - I N D U C E D C H E W I N G REACTION I N RATS

Perphenazine and trifluoperazine were injected 60 min, scopolamine and trihexyphenidyl 30 min prior to apomorphine hydrochloride, 1.25 mg/kg i.v. In parentheses the number of rats.

Perphenazine 0.2mg/kgs.c. Trifluoperazine 0.5 mg/kgs.c.

100% (27)

95% (33)

+ Scopo-

+ Scopo- + Trihexy- + Trihexy-

lamine 0.5 nig/kg

phenidyl 5 mglkg i.p.

I0 mg/kg

i.p.

lamine I mg/kg i.p.

59% (4) 84% (8)

5 5 % (10)

80% (10)

65% (13) 87% (13)

75% (10) 65% (5)

phenidyl i.p.

of typical chewing movements was recorded at definite intervals of time. Phenothiazine derivatives were injected subcutaneously 1 h earlier. Perphenazine and trifluoperazine seemed to antagonize in a specific manner the apomorphine-induced chewing movements even in doses that did not affect the general symptomatology. Scopolamine and trihexyphenidyl antagonized to a certain extent the inhibitory action of perphenazine and trifluoperazine (Table V).

( 6 ) ‘Test de traction’ in mice The ‘test de traction’ was suggested by Courvoisier (1956) as a procedure that permits thc evaluation of the sedative action of a product. It consists in suspending the mice by their front paws from a wire; any animal that does not succeed in bringing at least one hind paw up to the wire within 5 sec is considered to be under sedation. The ability 10 effect the traction is dependent on equilibration, muscle strength and tonus. A large dissociation of the effects on this and other tests has been observed for the TABLE V I ‘TEST D E T R A C T I O N ’

I N MICE

Percent of mice (in groups of 25) unable to effect the traction. Scopolamine and trihexyphenidyl were injected 30 min after phenothiazine derivatives. The mice were tested 60 and 120 min after the administration of either perphenazine or trifluoperazine.

+ Scopolamine + Trihexyphenidyl Perphenazine 10 mg/kgs.c. Trifluoperazine 75 mg/kg S.C. References p. 132-134

54% 40 %

0.5 mglkg i.p.

5 mglkg i.p.

80 %

80% 72 %

96 %

130

C . MORPURGO

different phenothiazine derivatives, the most potent compounds as neuroplegics being less effective in impairing the ability of mice to perform the traction (Courvoisier et al., 1957). We studied the interaction between phenothiazine derivatives and antiparkinson drugs on male mice, 20 to 22 g body weight. The percentage of animals unable to perform the traction was calculated on the averages of the results obtained 1 and 2 h after the subcutaneous administration of the phenothiazines. Scopolamine and trihexyphenidyl did not show an antagonism but rather a potentiation of the effects of perphenazine and trifluoperazine (Table VI). (7) Body temperature in rats Since the discovery of the hypothermic effect induced by chlorpromazine in several animal species (Courvoisier et al., 1953) the measurement of rectal temperature has been largely employed in the screening for tranquillizing agents. It has even been suggested that the sedative effects of these drugs might be dependent on the induced fall in body temperature (Lessin and Parkes, 1957). Rectal temperatures in mice have been reported to vary parallel to the changes in motor activity induced by a number of CNS stimulant or depressant drugs (Bastian, 1961). However, the parallelism between hypothermic effect and depression of motor activity does not apply to the group of phenothiazine derivatives; on the contrary, a dissociation between the hypokinetic effect and the hypothermic activity can be observed for many compounds. The drug-induced fall in body temperature may be responsible for the potentiation of barbiturate narcosis, but this test also lacks specificity for the characterization of CNS depressants (Riley and Spinks, 1958). We studied the changes in rectal temperature in rats by means of an electric ther*c r 40

"c

-

-

40

-

39 -

39-

39-

38-

-

Perphenazine

-.-. Trihexyphenidyl --- - Scopolamine

2 0 rnglkg I P

10 rng/kg ip

t

1 mglkg ip. +

perphenazinr 2 0 m g / k g ip. perphenazine 2 0 rng/kg i p

Fig. 5. Rectal temperature of rats. Trihexyphenidyl 10 mg/kg and scopolamine I mg/kg were injected i.p. 15 min prior t o perphenazine 20 mg/kg i.p. Each curve represents the average values obtained for 5 rats.

ANTIPARKINSON D R U G S AND NEUROLEPTICS

131

r

0

1-

I

I

I

2

3

4

-Amphetamine _--__ Scopolamine ......... Scopolamine

5

I

I

6

h

I

5 mg/kg i.p. 1 mglkg ip.

1 mglkg ip.

+

amphetamine 5 m g l k g i.p.

Fig. 6. Rectal temperature of rats. Effects of scopolamine 1 mg/kg i.p. and DL-amphetamine 5 mg/kg i.p. singly or in combination. Each curve represents the average of 10 rats.

mometer (Ellab). As reported elsewhere (Morpurgo and Theobald, 1964) we observed a more pronounced fall of body temperature with chlorpromazine than with other phenothiazine derivatives; a significant hypothermic effect by trifluoperazine was obtained only at high dose levels. As shown in Fig. 5, the hypothermia induced by 20 mg/kg i.p. perphenazine was not affected by pretreatment with trihexyphenidyl 10 mg/kg or scopolamine 1 mg/kg i.p. This dose of scopolamine did not induce any significant change in body temperature, but it appeared to enhance the hyperthermic effect of amphetamine (Fig. 6). CONCLUSIONS AND S U M M A R Y

In the experiments presented in this paper an antagonistic interaction between antiparkinson drugs and neuroleptics was displayed in different degrees according to the tests employed. Antiparkinson drugs inhibited the cataleptic reactions induced by relatively high doses of phenothiazine derivatives ;antagonistic effects between the two groups of drugs were observed also in regard to the spontaneous motility of mice, the avoidance behaviour of rats, the group toxicity of amphetamine in mice and the apomorphine effects in rodents, though the antagonistic interaction on these tests appeared to be dose-dependent. It seems possible to conclude that in the presence of anticholinergic agents increased doses of phenothiazine derivatives are required to achieve a neuroleptic effect. In contrast, some non-specific actions of tranquillizing agents, such as drug-induced hypothermia and the effects on the ‘test de traction’, were not reduced by the antiparkinson drugs tested. The evidence of antagonistic effects in various tests of central activity between some phenothiazine derivatives, which may be regarded as adrenergic blocking agents, and anticholinergic drugs supports the theoretical postulation of a central antagonism between adrenergic and cholinergic mediators. The hypothesis can also be advanced Rfferences p . 132-134

132

C. M O R P U R G O

that drug effects on behaviour are related to central autonomic changes. Experimental studies concerning behavioural alterations induced by central cholinolytic drugs refer mostly to scopolamine and atropine, but it is likely that the synthetic compounds in clinical use as antiparkinson agents differ only quantitatively. From the results obtained in various behavioural situations (Hearst, 1959; Carlton, 1961b) it could be assumed that anticholinergic compounds cause a disruption of normal behaviour by blocking inhibitory processes, Amnesic properties of scopolamine and atropine have also been reported (Domer and Schueler, 1960). It is obvious that animal experiments do not have a direct correlation with human psychological states; however, from the results exposed it seems plausible that antiparkinson drugs antagonize neuroleptic agents in regaid to psychomotor inhibition ; this probably plays a role in the tranquillizing effect of disturbed patients. In our opinion therefore - at least from a theoretical standpoint -the routineadministtation of central cholinolytic agents to psychotic patients should not be recommended. Their use should be limited to cases of severe drug-induced extrapyramidal disturbances requiring a prompt resolution, whereas an individual adjustment of the level of the neuroleptic medication should be usually attempted, in order to reach the tranquillizing effect without incapacitating the patients. REFERENCES ARRIGONI-MARTELLI, E., A N D KRAMER,M., (1959); Studio farmacologico di un nuovo derivato fenotiazinico: la perfenazina. Arch. int. Pharmacodyn., 119, 31 1-333. BASTIAN, J. W., (1961); Classification of CNS drugs by a mouse screening battery. Arch. int. Pharmacodyn., 133,341-364. BOLIN, R.R., (1960); Psychiatric manifestations of Artane toxicity. J . nerv. rnent. Dis., 131, 256-259. BIWNE, G. G., MORPURGO, C., BIELKUS, A., KOBAYASHI, T., TOURLENTES, T. T., AND HIMWICH, H. E., (1962); Relevance of drug-induced extrapyramidal reactions to behavioral changes during neuroleptic treatment. I. Treatment with trifluoperazine singly and in combination with trihexyphenidyl. Conzprehens. Psychiat., 3, 227-234. BUCHEL, L., Levy, J., AND TISSIER, M., (1962); Contribution B l'etude phamacologique du Triperidol (R.2498). The'rapie, 17, 1053-1 094. BURKMAN, A. M., (1961); Relative potencies of some phenothiazines as pecking syndrome inhibitors. J . pharrn. Sci., 50, 111-173. BURN,J. H., AND HOBBS, R., (1958); A test for tranquillizing drugs. Arch. int. Pharrnacadyn., 113, 290-295. CARLTON. P. L., (1961a); Augmentation of the behavioral effects of amphetamine by scopolamine. PJychopharrnacologia, 2,311-380. CARLTON, P. L., (1961b); Some effects of scopolamine, atropine and amphetamine in three behavioral situations. Pharmacologist, 3,60. COOKL., A N D WEIDLEY, E., (1957); Behavioral effects of some psychopharmacological agents. Ann. N . Y. Acad. Sci., 66,740-752. COURVOISIER, S., (1956); Pharmacodynamic basis for the use of chlorpromazine in psychiatry. J. din. exp. Psychopathol. quart. Rev. Psychiat. Neurol., 17, 25-31. COURVOISIER, S., DUCROT, R.,ET JULOU, L., (1957); Nouveaux aspects expkrimentaux de l'activite centrale des derives de la phenothiazine. Psychotropic Drugs. S . Garattini and V. Ghetti, Editors. Amsterdam, Elsevier (pp. 373-391). COURVOISIER, S., FOURNEL, J., DUCROT, R., KOLSKY,M., AND KOETSCHET, P., (1953); ProprietCs pharmacodynamiques du chlorhydrate de chloro-3 (dimethylamino-3' propy1)-I0 phenothiazine (4560 R.P.). Arch. int. Pharmacodyn., 92, 305-361. DEWS,P. B., (1953); The measurement of the influence of drugs on voluntary activity in mice. Brit. J. Pharmacol., 8.46-48.

ANTIPARKINSON D R U G S A N D NEUROLEPTICS

133

DOMER, F. R., AND SCHUELER, F. W., (1960);Investigations of the amnesic properties of scopolamine and related compounds. Arch. int. Pharmacodyn., 127, 449458. DOSHAY, L.J., AND ZIER,A., (1954);Drug therapy. Parkinsonism and ifs Treatment. L. J. Doshay, Editor. Philadelphia, London, Montreal, Lippincott (pp. 77-102). FREYHAN. F. A.,(I 961);The relationship of drug-induced neurological phenomena on therapeutic outcome. Extrapyramidal System and Neuroleptics. J . M. Bordeleau, Editor. Montreal, Editions Psychiatriques (pp. 483491). GOLDMAN, D., (1961); Parkinsonism and related phenomena from administration of drugs: their production and control under clinical conditions and possible relation to therapeutic effect. Extrapyramidal Svstem and Neuroleptics. J. M. Bordeleau, Editor. Montreal, Editions Psychiatriques (pp. 453464). HAASE, H. J., (1961);Extrapyramidal modification of fine movements, a ‘conditio sine qua non’ of the fundamental therapeutic action of neuroleptic drugs. Extrapyramidal System and Neuroleptics. J. M. Bordeleau, Editor. Montreal, Editions Psychiatriques (pp. 329-353). HALEY, T. J., FLESHER, A. M., AND RAYMOND, K., (1960);Pharmacological comparison of chlorpromazine and Mellaril, 3-methyl-mercapto-IO-[2-(N-Methyl-2-piperdyl)-ethyl]-phenothiazine hydrochloride. Arch. int. Pharmacodyn., 121, 455-460. HALL,R. A., JACKSON, R. B., A N D SWAIN, J. M., (1956);Neurotoxic reactions resulting from chlorpromazine administration. J . Amer. med. Ass., 161,214218. HEARST, E.,(1959);Effects of scopolamine on discriminated responding in the rat. J. Pharmacol. exp.Ther., 126,349-358. JANSSEN, P. A.J., NIEMEGEERS, C. J. C., AND JAGENEAU, A.H.M., (1960);Apomorphine-antagonism in rats. Arzneimitiel-Forsch., 10, 1003-1005. JENKNER, F. L.,AND WARD,A., (1953);Bulbar reticular formation and tremor. A.M.A. Arch. Neurol. Psychiat., 70,489-502. KOSTER, R., (1957);Comparative studies of emesis in pigeons and dogs. J. Pharmacol. exp. Ther., 119,406417. LASAGNA, L., AND MCCANN,W. P., (1957);Effect of ‘tranquilizing’ drugs on amphetamine toxicity in aggregated mice. Science, 125, 1241-1242. LESSIN, A. W., AND PARKES, M. W., (1957);The relation between sedation and body temperature in the mouse. Brit. J. Pharmacol., 12, 245-250. MAGOUN, H.W.,(1954);Anatomy. Parkinsonism and its Treatment. L. J. Doshay, Editor. Philadelphia, London, Montreal, Lippincott (pp. 5-1 5). MORPURGO, C., (1960); Contributo sperimentale allo studio dell’ interazione fra psico-farmaci e farmaci antiparkinson, con considerazioni sul significato clinico della sindrome extrapiramidale indotta dai farmaci tranquillizzanti. Tesi di Neuropsichiatria. Universita di Modena (Italy), unpublished. MORPURGO, C., (1962);Effects of antiparkinson drugs on a phenothiazine-induced catatonic reaction. Arch. int. Pharmacodyn., 137,8490. MORPUGO, C., AND THEOBALD, W., (1964); Influence effects of antiparkinson drugs and amphetamine on some pharmacological effects of phenothiazine derivatives used as neuroleptics. Psychopharmacologia, 6, 178-19I. PELLMONT, B., STEINER, F. A., BESENDORF, H., BACHTOLD, H. P., AND LAUPPI,E.,(1960);Zur Pharmakologie des ‘Taractan’, eines Neurolepticums mit besonderem Wirkungscharakter. Helv. physiol. pharmacol. Acta, 18, 241-258. PFEIFFER, C. C., MURPHREE, H. B., JENNEY, E. H., ROBERTSON M. G., RANDALL, A. H., AND BRYAN, L., (1959);Hallucinatory effect in man of acetylcholine inhibitors. Neurology, 9,249-250. RILEY,H., AND SPINKS,A.,(1958); Biological assessment of tranquillizers. J. Pharm. Pharmacol., 10, 657-671. ROSENKILDE, H., AND GOVIER, W. M., (1957);A comparison of some phenothiazine derivatives in inhibiting apomorphine-induced emesis. J. Pharmacol. exp. Ther., 120, 375-378. SCHAUMANN, W., AND KURBJUWEIT, H. G., (1 961); Beeinflussung verschiedener Wirkungen von Thiopropazat durch ein zentrales Stimulans. Arzneimittel-Forsch., 11, 343-350. SIDMAN, M.,(1953);Avoidance conditioning with brief shock and no exteroceptive warning signal. Science, 118, 157-158. TAESCHLER, M., WEIDMAN, H., AND CERLETTI, A., (1962); Zur Pharmakologie von Ponalid, einem neuen zentralen Anticholinergicum. Schweiz. med. Wschr., 90, 1542-1545. TEDESCHI, D. H., TEDESCHI, R. E., AND FELLOWS, E. J., (1961);Interaction of neuroleptics with sero-

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tonin in the central nervous system. Extrapyramidal System and Neuroleptics. J. M. Bordeleau, Editor. Montreal, Editions Psychiatriqucs (pp. 113-1 18). T I i E R , L., AND QCHRAMM, H., (1962); Apomorphin-Synergismus (Zwangsnagen bei Mausen) als Test zur Differenzierung psychotroper Substanzen. Arch. int. Pharmacodyn., 138, 302-310. TOMAN, J. E. P., (1963); Some aspects of central nervous pharmacology. Ann. Rev. Pharmacol., 3, 153- I 84. UHRBRAND, L., A N D FAURBYE, A., (1960); Reversible and irreversible dyskinesia after treatment with perphenazine, chlorpromazine, reserpine and electroconvulsive therapy. P~ychopharmacologiu,1, 408418. VERNIER,V. G., A N D UNNA,K. R., (1956); The experimental evaluation of antiparkinsonian compounds. Ann. N . Y . Acad. Sci.,64, 690-104. WIRTH,W., GOSSWALD, R., HORLEIN, U., RISE, KL. H., AND KREISKOTT, H., (1958); Zur Pharmakologie acylierter Phenothiazin-Derivate. Arch. inr. Pharmacodyn., 115, 1-31.

135

Free Amino Acids and Related Compounds in Brain and Other Tissues : Effects of Convulsant Drugs J E A N K. TEWS A N D WILLIAM E. STONE The Departtnent of Physiology and the Epilepsy Research Center, University of Wisconsin Medical School, Madison, Wisc. ( U.S.A.)

I n recent years it has become evident that the free amino acids and proteins in the brain are subject to a continual rapid turnover, and that an imbalance in the metabolic processes involving these constituents may occur in association with convulsive activity (Tower, 1960). With the development of ion-exchange chromatography, precise methods for the separation and quantitative measurement of individual amino acids became available (Moore and Stein, 1954) and could be applied in the analysis of brain tissue frozen in situ. Such studies have been under way in our laboratory. This paper reviews previously published observations on post-mortem changes and on the effects of anoxia, infusion of ammonium chloride, treatment with AOAA", and injection of several convulsants in dogs. New data are given on the effects of MFA and MFB on the free amino acid pattern in the brain of the dog and in that of the mouse, and in the liver, kidney, heart and skeletal muscle of the mouse. Species differences in cerebral amino acid patterns also are discussed briefly. The final section presents a revision of a previously proposed neurochemical classification of seizures. I. G E N E R A L M E T H O D S

In those experiments requiring dogs, adult males were used. Food was withheld for 18-20 h preceding the experiment. The animal was given morphine sulfate (5 mg/kg s.c.) and transient anesthesia was induced 30-40 min later with thiopental sodium (about 12 mg/kg i.v., with subsequent small doses if needed). The cranium was exposed and opened, and most of the calvarium was removed, the dura mater remaining intact. Preparation was made for recording the electrocorticogram from three cortical areas and for freezing the brain in situ with liquid air. Polyethylene cannulas were inserted into a femoral artery for blood pressure recording and into a femoral vein for injections. An endotracheal tube was inserted for the administration of artificial respiration when necessary. After completion of these procedures, a period

* Abbreviations used are: AOAA, amino-oxyacetic acid; y-ABA, y-aminobutyric acid; MFA, methylfluoroacetate; MFB, methyl-y-fluorobutyrate. References p. 160.-163

136

J. K . T E W S A N D W. E. S T O N E

TABLE I C E R E B K A L C O N S T I T U E N T S I N T H h DOG. C O N T R O L L E V E L S A N D L E V E L S A F T E R A M M O N I U M CHLORIDE INFUSION .

-

__ 45 ruin NHaCl infirsion __ ~ _ _

Control

Con.rtitueni *

~

No. of exprs.

Acetylaspartic acid Alanine ./-ABA Ammonia Arginine Aspartic acid Citric acid Glutamic acid Glutamine Glutathione Glycerophosphoethanolamine'* Glycine Glycogen Histidine Lactic acid Leucine Lysine Methionine -1- cystathionhe** Phenylalanine Phosphoethanolamine Serine Taurine Threonine Tyrosine Urea Valine

8 9 9 13 9 19 9 19 13 9

8 9 3 9 17 9 9 9 9 8 9 8 9 9

9 7

Mean f S.D.

Expi. 3

9.74 f 0.79 0.14 & 0.04 0.829 & 0.081 0.26 1 0.07 0.069 t 0.012 2.45 0.22 0.24 t 0.02 7.81 t 0.65 5.6 +C 1.7 1.45 SC 0.29 0.58 f 0.08 0.55 f 0.10 8.33 i 1.20 0.068 i 0.01 I 1.04 t 0.26 0.089 t 0.011 0.15 1 0.03 0.31 & 0.08 0.058 f 0.013 1.28 & 0.20 0.39 f 0.05 1.25 & 0.17 0.29 f 0.15 0.029 & 0.006 3.4 i 1.0 0.13 1 0.03

+

11.0

0.32 0.851 0.89 0.061 I .46 0.24 7.49 9.3 1.63 0.52 0.50 0.107 1.94 0.111 0.18 0.19 0.049 1.67 0.53 1.22 0.56

7.1 0.09

Expf. 4 10.1 0.36 0.906 1.96 0.070 I .54 7.42 10.1

0.89 0.57 0.54 0.087 2.68 0.085 0.13 0.18 0.064 I .40 0.40 1.53 0.62 0.053 3.5 0.08

.~

* Values are in pmoleslg. Glycogen values represent glucose produced * * Color yield per mole assumed equal to that of leucine.

by hydrolysis.

of 40 min or more was allowed for disappearance of the thiopental effects. The rationale for selection of these conditions, and more complete details, have been described elsewhere (Tews et at., 1963). The experimental drugs were dissolved in physiological saline for injection (MFB in 6 % ethanol in saline). Doses of AOAA are stated in mg of the hemihydrochloride. The brain was frozen at the chosen time and the exposed parts of the cortex were removed to a depth of about 1 cm. The tissue was analyzed for amino acids and related compounds by ion-exchange chromatography, and for ammonia, glutamine, lactate, citrate and glycogen by other methods. The analytical details have been described previously (Tews et al., 1963; Carter and Stone, 1961). 11. E F F E C T S O F E X P E R I M E N T A L V A R I A B L E S I N T H E D O G

As an aid in the interpretation of any changes which might be observed during seizures

_

FREE A M I N O A C I D S A N D R E L A T E D C O M P O U N D S

137

T A B L E I1 CEREBRAL CONSTITUENTS I N T H E DOG. POST-MORTEM A N D A N O X I C LEVELS

Values are in pmolesig.

Constituent

Alanine y-ABA Ammonia Aspartic acid Glutamic acid Lactic acid Leucine Methionine cystathionine** Tyrosine

+

Post-inortern* Mean f S.D.

Anoxia* Mean f S.D.

0.62 & 0.06 (4) 1.25 f 0.04 (4) (1) 0.74 2.28 0.32 (4) 7.70 1.22 (4) 21.4 (1) 0.104 & 0.025 (4) 0.24 f 0.03 (4) 0.032 0.006 (4)

0.38 i O . 1 1 (4) 1.06 f 0.09 (4) 0.29 f 0.07 (9) 1.78 0.32 (4) 8.97 1.13 (4) 7.88 j,1.75 (4) 0.117 f 0.015 (4) 0.19 0.05 (4) 0.037 j ,0.006 (4) __ -

* Number of animals in parentheses. * * Color yield per mole assumed equal to that of leucine. or other conditions, it was considered essential to study post-mortem changes and the effects of anoxia and of ammonium chloride infusion. Post-mortem and anoxic changes

Post-mortem tissue was obtained from animals killed by probing the medulla after the brain had been exposed in the usual way. Extraction procedures were applied to the unfrozen sample 20-23 min post-mortem. Anoxia was induced by allowing the dog to breathe a mixture of 4.5 % oxygen with 95.5% nitrogen for 12-13 min before freezing; respiration of the same mixture continued during freezing. Data obtained in control experiments are given in Table I. Changes occurring both post-mortem and during anoxia (Table 11) include significant increases in alanine, y-ABA and lactate (p < 0.01). Ammonia was greatly increased post-mortem but was not significantly elevated by anoxia, Changes observed during anoxia but not postmortem include increases in glutamate and leucine (p < 0.01) and tyrosine (p < 0.05), and decreases in aspartate (p < 0.01) and in the methionine-cystathionine peak (p < 0.02). Discussion. Oxygen deprivation appears to have similar effects on alanine and lactate levels. Conversion of pyruvate to alanine by transamination may represent an anaerobic pathway for pyruvate metabolism alternative to the synthesis of lactate. Increases in y-ABA (Factor I) have been found in rat brain during anoxia (Elliott and Van Gelder, 1960), and within 2 min post-mortem (Elliott and Lovell, 1962). A more extensive discussion of post-mortem changes in brain I/-ABA has appeared recently (Lovell and Elliott, 1963). Among other possibilities, it has been suggested that the accumulation of y-ABA results from anaerobic activity of glutamic acid decarboxylase, the enzyme responsible for the biosynthesis of y-ABA from glutamate. References p 160-163

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Concurrent anaerobic interference with tricarboxylic acid cycle activity would lessen the supply of a-ketoglutarate. Hence the subsequent removal of y-ABA, catalyzed by y-ABA-a-ketoglutarate transaminase, would be inhibited (Roberts, 1960; Elliott and Van Gelder, 1960; Love11 and Elliott, 1963). The surprising, but apparently significant, differences between the anoxic and post-mortem states may be related partly to the fact that, during the 12-13-min anoxic period, function and certain metabolic interrelationships were maintained, while in the post-mortem experiments oxygen deprivation was sudden and complete. Further discussion and comment on observations by other workers are given elsewhere (Tews et al., 1963). It has long been recognized that liquid air fixation is required if values approaching levels in vivo are to be obtained for ammonia, lactate and certain other constituents. It is now evident that this is true also for alanine and y-ABA. Ammonium chloride infusion

Ammonium chloride solutions (0.225-0.47 M ) , buffered with sodium bicarbonate, were infused into the femoral vein at rates sufficient to induce a gradual rise in brain ammonia. Infusion rates were 0.1 I to 0.28 mmoles/kg/min. The infusion periods were for 10 or 45 min, after which the freezing was done. Representative data from 2 animals infused at rates of 0.1 1 and 0.13 mmoles/kg/min for 45 min are presented (Table I). The heart rate became slow and irregular and cortical electrical activity was depressed slightly ; arterial blood pressure and oxygenation were adequate. Results from other animals, including those obtained for 10-min infusion periods, have been tabulated earlier (Tews et al,, 1963). Occasional twitching was seen, but seizures (clonic) appeared in only one animal (near the end of a 10-min infusion period). The cerebral ammonia levels were elevated greatly after the 10- or 45-min intervals. Glutamine was barely affected after 10 min, but very high values were found after 45 min. Alanine was increased significantly in all instances. Histidine also showed a small but significant increase, and urea was elevated by variable amounts. Definite decreases in aspartic acid were noted, and valine levels were slightly below the control values. Glutamic acid levels were slightly decreased, but the change was not statistically significant. Lactate was invariably increased to a level above the control range. Discussion. Several investigators have noted that treatment of the animal with ammonium salts induces a rise in brain glutamine. The formation of glutamine is thought to be a means of detoxifying ammonia. This view has been substantiated by experiments of Berl et al. (1962a), who used 15N-ammonia in cats and found greater incorporation of the label into both the amide and a-amino groups of brain glutamine than into glutamate; this was interpreted as indicating compartmentalization of the glutamate. It has been suggested that ammonia may interfere with cerebral oxidative processes by diverting part of the a-ketoglutarate from the tricarboxylic acid cycle to glutamate and glutamine (see Tews et a]., 1963 for documentation and discussion). Although there may be a small decrease in brain glutamate in the presence of exogenous am-

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monia, the glutamine increase is much greater, and Berl et al. (1962a, b) noted that extra glutamate formation is associated with the synthesis of glutamine. Furthermore, by using 14C-bicarbonate, they demonstrated in vivo synthesis of oxaloacetate by COa fixation, and pathways from the tricarboxylic acid cycle to glutamine and aspartate. Such oxaloacetate formation would tend to counteract any depletion of the cycle intermediates resulting from infusion of ammonium salts. However, when measuring the incorporation in vivo of label from pyruvate-2-14C injected intracisternally in rats, McMillan and Mortensen (1963) did not note formation of glutamate appropriately labelled to indicate increased COa fixation into oxaloacetate after intraperitoneal injection of ammonium carbonate. Theoretically, it would seem that determination of label distribution in glutamine might be helpful. If ammonia stimulates COZ fixation by labelled pyruvate to form oxaloacetate, thereby altering the labelling pattern in glutamate, this effect should be reflected more obviously in glutamine (which is believed to be formed from a small pool of active glutamate) than in the total glutamate which includes a large pool apparently not involved in glutamine formation. Studies in vitro on cat brain cortex have shown inhibition of oxygen uptake and increased aerobic glycolysis in the presence of ammonium ions (Tower et al., 1961). Oxidation in vitro of a-ketoglutarate or pyruvate also was inhibited by addition of ammonium chloride to the system. In other experiments the decarboxylation of pyruvate was shown to be inhibited. Comparable effects of ammonia in vivo could induce an increase in pyruvate with related increases in alanine and lactate. The decrease in brain aspartic acid is consistent with the finding of Berl et al. (1962b) that infused ammonium salts shift the pathway of 14C-labelled intermediates away from aspartic acid and toward glutamine. It may also be suggested that a decrease in oxaloacetate resulting from depletion of its precursors in the tricarboxylic acid cycle might be countered in part by a transamination converting aspartate to oxaloacetate, with a resulting decrease in aspartate. Berl et al. (1962b) demonstrated that interchanges among aspartic, oxaloacetic, malic and fumaric acids take place in the brain. 111. EFFECTS OF D R U G S I N T H E D O G

Amino-oxyacetic acid Wallach (1961a) reported that administration of AOAA causes large increases of I/-ABA in the brains of several species by inhibiting the activity of y-ABA-a-ketoglutarate transaminase. D a Vanzo et a!. (1961) quoted further work of Wallach indicating that AOAA increases the level of brain glutamine, and presented experiments showing that AOAA is an effective anticonvulsant. Therefore, we have made a further investigation of the effects of AOAA on brain constituents in the dog as a preliminary step before studying its anticonvulsant action against thiosemicarbazide, pentylenetetrazol, picrotoxin, MFA and MFB. Physiological and neurochemical efsects. Injection of AOAA (5, 10 or 20 mg/kg) into the otherwise untreated animal induced a profound depression within 15-25 min. References p . 160-163

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M F A Seizure

MFA +AOAA D

W

-

-

I

MFB+ AOAA

Fig. 1. Electrocorticographic effects of amino-oxyacetic acid, rnethylfluoroacetate and methylfluorobutyrate in the dog. A - Control; B = 120 min after injection of amino-oxyacetic acid; C : Beginning of tonic-clonic seizure 73 rnin after injection of MFA. D - 120 min after AOAA, 60 rnin after MFA; E = 110 min after AOAA, 30 rnin after MFB; F = 102 min after AOAA, 22 min after MFB; G = 125 min after AOAA, 44 rnin after MFB.

Recovery occurred after several hours. These effects have been described in detail (Wallach, 1961a; Roa et al., 1964). I n the surgically prepared animal, AOAA (20 mg/kg) induced a gradually developing depression in the electrocorticogram manifested by diminishing amplitude of the normal fast-frequency waves and developing slow wave activity (Fig. 1, A and B). The changes were usually minimal or undiscernible 1 h after injection, becoming quite apparent by 2 h and showing further development at 3 h. Although Wallach (1961a, b) observed a convulsant action of large doses of AOAA (amounts not stated), we have seldom seen this effect in our experiments. Administration of the drug at 20 mg/kg never caused a seizure. In one case, 30 mg/kg induced a convulsion with typical epileptiforni cortical activity at 115 min. However, doses of 30-40 mg/kg in 5 instances induced cortical depression at 2 h. I n one of these (40 mg/kg) a tonic seizure lasting 1 rnin occurred at 171 min while the cortex remained profoundly depressed. AOAA (20 mg/kg) induced changes in 8 of the cerebral constituents measured. A

141

F R E E AMINO A C I D S AND R E L A T E D COMPOUNDS

T A B L E 111 EFFECTS O F A M I N O - O X Y A C E T I C ACID ON LEVELS OF C E R E B R A L CONSTITUEN

rs

IN THE DOG

Values are in pmoles/g Min after AOAA (20 mgJkg)

Constituent

Alanine y-ABA Ammonia Aspartic acid Citric acid Glutamic acid Glutamine Lactic acid Leucine Lysine Serine Threonine Tyrosine

63

93

120

145

181

0.19 1.49 0.69

0.17 2.25 0.72 1.68

0.15 2.07 1.23 1.75 0.18 7.04 8.8 1.37 0.090 0.19 0.37 0.19 0.143

0.28 3.41 I .03 I .69

0.09 3.52 0.85 3.48

8.25 14.0 2.22 0.095 0.21 0.40 0.46 0.256

7.50 9.3 1.09 0.099 0.30 0.32 0.20 0.130

1.85 8.16 9.0 1.60 0.118

0.14 0.3 I 0.54 0.186

7.54 8.3 2.50 0.102 0.17 0.29 0.15 0.171

210

233

0.17 2.60 0.70 2.39 0.24 I .23 13.1 I .23 0.100 0.22 0.33 0.33 0.148

0.14 4.10 0.32 2.42 8.32 4.5 1.09 0.105 0.16 0.4 1 0.33 0.039

rough indication of the progression of these effects was obtained from a series of 7 dogs in which the brains were frozen 1 to 4 h after injection of the drug (Table 111). y-ABA, ammonia, glutamine and tyrosine showed marked increases; except for y-ABA, these returned toward control levels at varying times up to 4 h. Lysine showed a smaller increase which also was no longer apparent at 4 h. Alanine and lactate tended to be high during the first 3 h, but these changes were not entirely consistent. It has been noted previously that alanine and lactate tend to change in parallel fashion, probably because each is closely related to pyruvate. Aspartate was the only constituent to show a decrease; a return to normal took place within 4 h. It was found that blood ammonia increased from a mean control value of 0.12 pmoies/ml to as high as 1.69 pmoles/ml 75 min after administration of AOAA (20 m g / W Discussion. The changes in alanine, aspartate and lactate, as well as in glutamine, may be secondary to the rise in ammonia since all of these alterations occur on infusion of ammonium chloride. The rise in brain ammonia may be due in whole or in part to the increase of this constituent in the blood, and hence presumably to a metabolic effect of AOAA on the liver. The biochemical effects of AOAA appear to resemble those of ~-2,4-diaminobutyricacid, a convulsant which increases brain y-ABA and glutamine and shows hepatic toxicity (Tower, 1963). Although AOAA has been found to induce an accumulation of p-alanine in liver, kidney and spleen, the available reports (Baxter and Roberts, 1962; Roberts, 1963) are not clear as to whether or not such a change was observed in brain. Unfortunately, the method of Moore and Stein (1954) used in this laboratory does not separate /?-aIanine from glucosamine, but the peak representing these two constituents can be measured. The mean and S.D. were 0.038 & 0.010 pmoles/g (calculated as p-alanine) References p . 160-163

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in 9 control animals, and 0.054 0.022 in 10 animals 1-4 h after injection of AOAA (20-40 mg/kg). The apparent increase is not statistically significant. It is possible that AOAA inhibits other pyridoxal phosphate-dependent enzymes in addition to y-ABA-a-ketoglutarate transaminase. Hopper and Segal(l962) observed that AOAA inhibits the glutamic-pyruvic transaminase of rat liver. The increase in tyrosine in the brain could be the result of inhibition of tyrosine-a-ketoglutarate transaminase, which has been found in brain (Albers et al., 1962; Haavaldsen, 1962). However, the tyrosine increment could come from protein in the brain, or from tyrosine production elsewhere in the body since it is known that this amino acid rapidly penetrates the blood-brain barrier in the rat (Chirigos et al., 1960). The elevation in tyrosine suggests the possibility of an alteration in catecholamine metabolism. Methionine sulfoximine

The neurotoxic effects of methionine sulfoximine in many species have been reviewed by Proler and Kellaway (1962). Several reports have implicated this drug as a methionine antagonist and as an inhibitor of glutamine synthesis and of other reactions involving glutamic acid (Gershoff, 1956; Pace and McDermott, 1952; Peters and Tower, 1959; for other references see Tews and Stone, 1964). Our experiments indicate that the influence of methionine sulfoximine on nitrogenous metabolism in the brain is more wide-spread than has been realized previously. TABLE IV EFFECTS OF M E T H I O N I N E S U L F O X I M I N E O N LEVELS OF

C E R E B R A L C O N S T I T U E N T S I N THE D O G

Values are in pmoleslg.

Alanine 7-ABA Ammonia Aspartic acid Glutamic acid Glutamine Lactic acid Leucine Lysine Methionine I cystathionine Phosphoethanolamine Serine Valine _

_

_

-

Mean & S.D.*

Constituent

0.58 f 0.19** 0.744 f 0.054 0.77 + 0.13** 1.30 & 0.26+* 6.56 i0.62** 1.6 0.6** 1.53 &0.37** 0.041 & 0.006** 0.28 & 0.04** 0.14 & 0.04** 1.83 +0.18** 0.86 f 0.14** 0.09 f 0.028

+

~

* 5 animals with symptoms of toxicity;

4 of these had seizures. **Significant change from control level in Table T (p << 0.Ol). 5 Significant change from control level in Table I (p < 0.05).

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Physiological and neurochemical efects. In dogs, intraperitoneal administration of methionine sulfoximine at 10 mg/kg caused severe generalized seizures after 16 to 18 h. At 6 mg/kg, the drug did not always induce convulsions. In one instance marked toxicity was apparent but seizures were not observed; in two other cases the animals showed only minor symptoms and were not studied further. When seizures had been observed, or at 21.5 h in the animal not convulsing, morphine was given and the usual surgical procedures were carried out. The seizures were not suppressed by morphine, but were eliminated by thiopental and did not recur thereafter. Analyses of the frozen brain samples support earlier reports of decreases in glutamate and glutamine (Table IV). The mean decrease in y-ABA is not statistically significant, but if the value from the animal not convulsing(0.838 ,umoles/g) is omitted, the mean and S.D. become 0.720 0.015 pmoles/g and the decrease is significant (p < 0.05). The fact that the value for methionine and cystathionine is decreased supports other indications of a disturbance in methionine metabolism. The great increase in brain ammonia is not due to accumulation in the blood, since the blood levels did not exceed control values. The increased cerebral levels of alanine and lactate and the decreases in aspartate and valine may be secondary to the elevated ammonia concentrations. Other changes include increases in lysine, phosphoethanolamine and serine, and a decrease in leucine. Anticonvulsant action of AOAA. It has been reported that AOAA protects cats from seizures induced by methionine sulfoximine (DaVanzo et al., 1961). We have not investigated this possibility in the dog. Thiosemicarbazide

Recent efforts to elucidate the biochemical mechanisms underlying the excitatory action of the convulsant hydrazides have been reviewed extensively by Roberts and Eidelberg (1960) and by Williams and Bain (1961). These reviews have included discussions of the relationships of y-ABA to glutamic acid decarboxylase, y-ABAa-ketoglutarate transaminase and vitamin B g . The concentration of y-ABA, a substance having an inhibitory action on neuronal activity, is reduced in vivo byconvulsant hydrazides (since these agents inhibit the activity of glutamic acid decarboxylase). Hydroxylamine raises the level of y-ABA by inhibiting y-ABA-a-ketoglutarate transaminase, and tends to reduce cerebral excitability. The use of these drugs has suggested an inverse relationship between y-ABA levels and excitatory responses to electrical stimulation and to pentylenetetrazol (Roberts and Eidelberg, 1960; Roberts et al., 1960). However, it is evident that hydrazide seizures cannot be attributed solely to the decrease in brain y-ABA. In animals treated with hydroxylamine, thiosemicarbazide induces seizures while y-ABA levels remain above normal (Baxter and Roberts, 1960). The study described here supplements previous reports on hydrazide action in which cerebral constituents other than y-ABA usually have received little attention. AOAA has been reported to have anticonvulsant action against thiosemicarbazide in cats, rats and mice (DaVanzo et a]., 1961). Therefore, electrographic and neurochemical References p . 160-163

1 44

J. K . T E W S A N D W . E . S T O N E

TABLE V E F F E C T S OF

T H I O S E M I C A R B A Z I D E O N L E V E L S OF C E R E B R A L C O N S T I T U E N T S I N T H E DOG

Values are in ,umoles/g. Number of animals in parentheses. -.

Constituent

Alanine y-ABA

Ammonia Glutamic acid Lactic acid Tyrosine

Mean f S . D . 0.19 0.552 0.39 8.42 3.61 0.045

f 0.04 (4)*

k: 0.062 (4)** -1- 0.01 (3)s 0.S7 (4)

f 0.36 (4)** 3~ 0.008 (3)**

*Significant change from control level in Table I (p 0.05). **Significant change from control level in Table T (p ': 0.01). $Significant change from control level in Table I (p :c 0.02).

effects of the hydrazide, alone or in combination with AOAA, have been determined in the dog. Anticonvulsant action of AOA A. The administration of thiosemicarbazide (20 mg/kg) to dogs resulted in severe clonic or tonic-clonic seizures after 43 to 87 min. Prior or simultaneous treatment with AOAA (20 or 30 mg/kg) greatly reduced or prevented the excitatory response seen with thiosemicarbazide alone. However, increasing the dose of thiosemicarbazide to 60 mg/kg induced typical generalized seizures despite the previous administration of AOAA. Neurochemicd effects. Analyses of brains frozen while thiosemicarbazide-induced seizures were in progress showed statistically significant changes in 5 cerebral constituents (Table V). y-ABA was decreased while its precursor, glutamic acid, showed a trend toward a higher value which was not statistically significant. Alanine, ammonia, lactate and tyrosine were increased. Levels of the same constituents were altered similarly in one brain frozen during a seizure 57 min after injection of thiosemicarbazide at 60 mg/kg (not included in Table V). Tn another case the brain was frozen 53 min after the hydrazide (20 mg/kg) but before a seizure occurred. The y-ABA had decreased to a level equal to the highest value observed during a seizure (0.63 pmoleslg). Tyrosine was 0.036 pmoles/g, a value higher than the mean control level and equal to the lowest value observed during a seizure, although exceeded in one control experiment. The other constituents did not differ from the control values. The chemical findings obtained with combinations of AOAA and thiosemicarbazide were quite similar to those with AOAA alone. y-ABA levels were always high, even during convulsions. Ammonia, lactate and alanine were sometimes higher than with AOAA alone. Discussion. The original finding of Killam and Bain (1957) that the brain y-ABA

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145

levels were decreased in rats given semicarbazide has been confirmed for other convulsant hydrazides and in other species. De Ropp and Snedeker (1961) also noted that semicarbazide induced an increase in rat brain alanine. I n two cats given thiosemicarbazide, Tower (1963) found low initial levels of glutamate and glutamine in cortical slices. Hydrazides inhibit the activity of several pyridoxal-dependent enzymes in vitro, and that of glutamic acid decarboxylase both in vitvo and in vivo (Killam and Bain, 1957). The observed increase in tyrosine suggests interference with an enzyme such as tyrosine-a-ketoglutarate transaminase; other possible explanations for a rise in tyrosine are given in the section on AOAA. There are numerous indications that the y-ABA level in the brain is one of the factors regulating neuronal excitability. Therefore, it is not improbable that the inhibitory effect of AOAA on seizures is due, at least in part, to the associated increase in y-ABA. This inhibition is rather weak in the dog, since it is easily overcome by increasing the dose of thiosemicarbazide. Da Vanzo et al. (1961) found that in rats the protective action against the hydrazides was maximal during the first 3 h after administration of AOAA, whereas the y-ABA levels continued to rise for about 8 h. This suggests that the anticonvulsant action may be unrelated to the y-ABA level, but an alternative possibility is that an accommodation process occurs, allowing recovery of excitability in the presence of excess y-ABA. Large doses of AOAA sometimes induce seizures. It is possible that the excitatory effects of AOAA and of the hydrazides involve the same basic mechanism, since all of these agents are carbonyl-trapping agents. A decrease in y-ABA might potentiate excitation by hydrazides, and an increase could oppose excitation by AOAA. Other drugs influencing y-ABA levels may also exhibit more than one pharmacologic property; on this basis the lack of correlation between y-ABA levels and neuronal excitability in response to these agents is not surprising. Various examples supporting this suggestion may be found in the literature. Hydroxylamine, which causes a rise in y-ABA and a decrease in neuronal excitability, may also induce seizures during the brief period preceding the rise in y-ABA (Roberts et al., 1960; Baxter and Roberts, 1959). L-Glutamic acid-y-hydrazide increases y-ABA and has depressing effects at low doses, but induces seizures at higher doses (Massieu et al., 1962). Hydrazine (Maynert and Kaji, 1962) and ~-2,4-diaminobutyricacid (Tower, 1963) have been reported to raise the y-ABA level and to induce seizures. L-Anthranilic hydroxamic acid has complex effects, inhibiting brain glutamic acid decarboxylase and decreasing y-ABA, but causing seizures only after daily doses for 3 days (Utley, 1963). A single large dose induces a depressed state resembling that seen in unanesthetized animals given AOAA, but associated with a slight decrease in y-ABA. No correlation has been found between the convulsant actions of various vitamin Bs antagonists and their inhibitory effects on brain glutamic acid decarboxylase in vivo (Rosen ef al., 1960). Variations in intracellular distribution of y-ABA might also be expected to influence the response to these drugs. Our data are not incompatible with the suggestion that the seizures caused by drugs wich inhibit glutamic acid decarboxylase or y-ABA-a-ketoglutarate transamiReferences p . 160-163

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J . K . T E W S A N D W. E. S T O N E

nase result from a reduced rate of metabolism through the y-ABA pathway (Wallach, 1961b; Balzer et al., 1960; Tower, 1963). Pentylenetetrazol and picrotoxin

In an earlier investigation we failed to find distinctive metabolic changes in brain constituents after treatment of the animal with pentylenetetrazol or picrotoxin (Stone et al., 1960). We have expanded the original study by determining the effects of these convulsants on the concentrations of the cerebral free amino acids in the dog. The effectiveness of AOAA as an anticonvulsant for these agents also has been examined. Anticonvulsant action of AOAA. The convulsive threshold to pentylenetetrazol in surgically prepared control dogs was 10 to 15 mg/kg (determined by giving small intravenous doses at 30-sec intervals until a brief generalized seizure occurred). The effect of AOAA on this threshold was examined by injecting AOAA (20 mg/kg in 5 experiments, 30 mg/kg in one experiment) I to 3 h before testing with pentylenetetrazol. The threshold was raised in every instance, the range being from 15-20 mg of pentylenetetrazol per kg to 85 mg/kg; the mean was 50 mg/kg. The thresholds were not correlated with time after AOAA injection nor with the brain y-ABA levels. Picrotoxin ( 1 mg/kg) caused generalized clonic or tonic-clonic seizures 5-17 min after injection. AOAA (20 mg/kg) showed essentially no anticonvulsant action against picrotoxin, although there may have been a slight delay in the onset of seizures. It should be noted that this dose of picrotoxin may be considerably above the threshold level; the long latent period of picrotoxin precludes testing by the procedure used for pentylenetetrazol. Neurochemical eftcis. These convulsants did not induce striking changes in free amino acid content in brains frozen while seizures were in progress. Picrotoxin (1 mg/kg) caused a slight reduction in aspartate to 2.20 & 0.31 pmoles/g (p < 0.02). With pentylenetetrazol (40 mg/kg) this change was not significant, the mean level being 2.27 & 0.36 pmoles/g. Pentylenetetrazol induced significant changes in the levels of glutamate (reduced to 7.08 0.69 pmoles/g; p < O.Ol), alanine (increased to 0.18 -& 0.015 pmoleslg; p < 0.05) and ammonia (increased to 0.55 4~ 0.24 pmoles/g; p < 0.01). A smaller increase in ammonia with picrotoxin (to 0.33 f 0.09 p moles/g) was not significant. A small decrease in malic acid induced by pentylenetetrazol has been noted previously (Stone et al., 1960). These results are in agreement with other reports indicating that major changes in cerebral free amino acids are not induced by these stimulants, despite the fact that increased protein turnover has been observed in the perfused cat brain during pentylenetetrazol seizures (Geiger et al., 1960a, b). Documentation and discussion of various reported changes, sometimes inconsistent and of uncertain significance, have been given earlier (Tews et al., 1963). Fluoroacetate andjuorobutyrate MFA, MFB or their corresponding acids, when administered to animals of various

F R E E AMINO ACIDS A N D RELATED COMPOUNDS

147

species, cause violent seizures as well as alterations in the content of cerebral constituents such as increased alanine and decreased glutamate (Dawson, 1953), decreased aspartate (Dawson, 1953; Stone et al., 1960) and increased citrate (Buffa and Peters, 1949). Since the changes also include a striking increase in free ammonia levels in dog brain (Benitez et a/., 1954; Stone et a/., 1960), a more extensive investigation was made of the effects of fluoro compounds on the concentrations of free amino acids and other constituents. A preliminary report has been published (Tews and Stone, 1963). Convulsions resulting from treatment with fluoro compounds may be caused by mechanisms other than those significant in the appearance of thiosemicarbazide- or pentylenetetrazol-induced seizures. Hence we sought to determine whether or not AOAA has anticonvulsive effects against the fluoro compounds comparable to those which it exhibits against thiosemicarbazide and pentylenetetrazol. Also included were determinations of the effects of AOAA on the neurochemical changes caused by MFA or MFB. Anticonvulsant action of AOAA. MFA in doses of 1 mg/kg induced extremely violent tonic-clonic seizures within 51-88 min (Fig. IC). MFB (1 mg/kg) caused convulsions which seemed identical with those induced by MFA except that the latent periods were only 25-38 min. The anticonvulsant action of AOAA was tested by giving 20 mg/kg 60 min before MFA or 80 min before MFB, the time intervals being chosen to compensate for the fact that the latent period is shorter for MFB than for MFA. AOAA prevented both the generalized seizures and the epileptiform cortical activity typical of MFA alone (Fig. lD), even when exposure to MFA was for as long as 2% h. Moderate to profound electrocorticographic depression was observed in 6 experiments, while only minimal depression occurred in one instance. Somewhat variable effects were obtained when MFB was administered after AOAA. As with AOAA MFA, considerable depression of electrocortical activity usually was found by the end of the experiment (Fig. 1E). In only one instance did an epileptiform seizure occur, appearing after an unusually short interval of 12 min after MFB injection. Otherwise, although some convulsive manifestations usually were seen before depression appeared, there was incomplete correlation of muscular movement with electrocorticographic epileptiform activity. The pattern shown in Fig. 1F was recorded during a tonic contraction, while the grand mal-type activity in Fig. 1G was associated with nothing more than slight tail motion. Various mild convulsive movements took place without cortical involvement, indicating subcortical convulsive activity. It is of interest that, despite the great cortical depression which occurred with either MFA or MFB after AOAA, administration in a few experiments of a massive dose of pentylenetetrazol (1 00 mg/kg) could still induce a cortical convulsive response. Neurochemical efects. Chemical analyses were done on brains frozen during seizures, the application of liquid air beginning about 20 sec after the onset of the convulsive electrical activity. The data from the MFA and MFB groups were combined, since the results were similar in all cases (Table VI). Significant increases were found for alanine, ammonia, citrate, lactate, leucine and serine. Small increases in threonine

+

References p 160-163

148

J . K . T E W S A N D W. E. S T O N E

T A B L E VI C H E M I C A L C H A N G E S I N DOG C E R t B R A L C O R T E X I N D U C E D H Y M E T H Y L F L U O R O A C L T A T E A N D M t T H Y L F L U O R O E U T YRATE, A L O N E O R I N C O M B I N A T I O N WITH A M I N O - O X Y A C E T I C ACID

Values are in pmoles/g. Glycogen values represent glucose produced by hydrolysis. ______________~

Consiituent

_ _ _ _ _ _ _ _ . ~ _ _ _ _ _ _

M F A or M F B seizure* Mean L S.D.

~ _ _ - _ _ _ _ _ _ _ _ _ _ 0.96 i 0.239 (7) 0.897 +C 0.032 (7) 1.52 kO.470 (9) 1.64 4 0.245 (20) 1.18 0.175 (9) 7.20 t 0.8859 (20) 5.6 t 2.2 (9) 3.95 i 0.305 ( 5 ) 5.27 1.095 (10) 0.127 0.0255 (7) 0.18 &0.04 (7) 0.49 i 0.05s (7) 0.39 i 0.18 (7) 0.040 10.018 (7) _ _ ___ ____ ___ ___

Alanine 7-ABA Ammonia Aspartic acid Citric acid Glutamic acid Glutamine Glycogen Lactic acid Leucine Lysine Serine Threonine Tyrosine

A O A A and MFA** Mean S.D.

+

MFB* * 5 S.D.

Mean

_____

._________

*

0.28 0.055 2.16 i 0.52s 3.21 i 0.505 1.62 0.565 0.29 4 0.08 6.35 0.338 10.9 2.95

+

+

4.33 0.121 0.24 0.38 0.64 0.168

*

* Number of animals in parentheses. * * 5 animals 5 Significant change from control level in

AOAA and

-__

5 1.819: f O.Ol85 & 0.028

i0.04 & 0.105

f 0.050s

0.43 2.33 3.89 1.70 0.76 7.30 7.9

+ 0.08s

i 0.375 & 1.125 & 0.600 & 0.195 i.0.85 & 1.45

8.71 t 1.66s 0.151 -t 0.0355 0.23 L 0.039; 0.44 0.09 0.25 i 0.07 0.196 & 0.0565

+

~-__-___

Table I (p < 0.01).

$5 Significant change from control level in Table 1 (p

; =

0.02).

and lysine were not significant (p > 0.05). Decreases were noted for aspartate, glutamate and glycogen. Figures for other constituents were not significantly different from the control values. When dogs were treated with AOAA before administration of MFA or MFB, the resulting neurochemical pattern (Table VI) was essentially that induced by AOAA with some of the effects of the fluoro compound superimposed. (The brains were frozen 1 h after injection of MFA or 25-30 min after MFB; in one case a grand ma1 seizure appeared and the brain was frozen 12 min after MFB.) Reference to the data obtained for AOAA alone (Table 111) indicates that this drug was responsible for the increases in y-ABA, glutamine, tyrosine, and probably lysine. Additive effects of the combined agents are apparent in the very high ammonia values. Other superimposed effects of the fluoro compounds include great increases in lactate, increases in leucine, and decreases in glutamic acid. The decreases in aspartic acid, seen on administration of AOAA, MFA or MFB individually, were not accentuated by the combinations used. The striking and widely observed increase in citrate which almost invariably results from treatment with these fluoro compounds was antagonized by AOAA, the rise in response to MFA being completely prevented and that caused by MFB being significantly reduced (p < 0.01). Changes in serine concentrations showed the same trend. The increases in alanine in response to fluoro compounds

FREE AMINO ACIDS A N D RELATED COMPOUNDS

149

were antagonized by AOAA, the levels attained being above the mean value with AOAA alone but much lower than those with MFA or MFB alone. Threonine showed a significant increase after AOAA MFA, but none after AOAA + MFB. Low values for glutathione occurred in several instances after AOAA MFA, but this change was not consistent. Discussion. The great increase in citrate levels induced by fluoro-fatty acids is thought to be due to inhibition of aconitase activity. This interference results from a synthesis in vivo of fluorocitrate, the actual inhibitor (Peters, 1957). Although MFA and MFB cause essentially identical neurochemical changes in the dog, their conversion in vivo to the active fluorocitrate probably occurs by dissimilar routes. This is suggested by the fact that the latent period for MFB-induced convulsions is shorter than that for MFA. Data on the comparative inhibitory effects of fluoroacetate, fluorobutyrate and fluorohexanoate in vitro (Dominguez et al., 1954) support this view, as do the dissimilarities between the effects of MFA and those of MFB in dogs given AOAA. Furthermore, it has been noted that acetate-containing molecules, such as monoacetin and acetamide, frequently can protect against the effects in vivo of fluoroacetate (Chenoweth et al., 1951 ; Gitter, 1956). Similarly, monobutyrin (but not monoacetin) shows some antagonism to fluorobutyrate (Kandel and Chenoweth, 1952a). Therefore, a conceivable explanation of the ability of AOAA to prevent MFA-induced seizures in the dog is that AOAA is acting as a source of acetate. Consistent with this hypothesis is the observation that AOAA is much less effective in preventing the seizures caused by MFB. However, AOAA by itself causes no accumulation of citrate (Table 111). Gitter (1956) noted that monoacetin and acetamide bring about definite increases in rat brain citrate, even though they tend to diminish the increases induced by fluoroacetate. Another factor to be considered in relation to the anticonvulsant action of AOAA is the increase in brain y-ABA. If the y-ABA level played a major role, one might expect AOAA to be equally effective against MFA and MFB, but this could be tested only if pharmacological data were available permitting the selection of precisely equivalent doses of MFA and MFB. Although AOAA antagonized MFA more effectively than it did MFB at the dose levels used, as regards both convulsive activity and citrate accumulation, a causal relation of high citrate to seizures cannot be inferred. Kandel and Chenoweth (1952a) and Hendershot and Chenoweth (I 955) have presented evidence that citrate accumulation per se is not the cause of fluoroacetate convulsions. The inhibition of citrate formation in animals given AOAA prior to MFA might result from a block in the formation of fluorocitrate, but there is no experimental support for such an explanation. Pertinent to this suggestion is a statement by Potter (1951): ‘It should be emphasized that the failure to form citrate after some pretreatment that is followed by fluoroacetate does not imply that the pretreatment. . . necessarily inhibited some enzyme in the metabolic sequence immediately prior to citrate’. Inhibition by AOAA of transaminase activities might indirectly prevent an increase in citrate levels as a result of decreased production or altered balance of its keto-acid precursors. Studies by Gal et al. (1961) have indicated alternate routes of 14C-

+

References p . 160-163

+

150

J. K . T E W S A N D W . E. S T O N E

fluoroacetate metabolism in the intact rat. It is possible that AOAA might have diverse effects on these metabolic pathways as well as on those of fluorobutyrate. The amino acid data obtained from dogs treated with MFA or MFB confirm and extend those of Dawson (1953) from brains of fluoroacetate-treated rats. Minor differences in the degree of change are apparent; for instance, aspartate appears to be affected more and glutamate less in the dog than in the rat. Dawson also observed that no changes occur in the levels of y-ABA or glutamine. The source of the brain ammonia which appears after treatment with the fluoro compounds has not been identified, but possible precursors have been pointed out (Benitez et a/., 1954). Observations of many workers indicating that protein-bound glutamine is a significant source of endogenous ammonia have been reviewed by Tower et al. (1961). Probably all of the ammonia is of cerebral origin since the blood ammonia levels, although increased, are much lower than those of the brain. It is generally accepted that glutamine formation is the primary means of ammonia detoxication in the brain, as indicated in Section 11. After injection of a fluoro-fatty acid, however, glutamine does not increase in the dog brain although free ammonia is elevated by about 6-fold. The explanation may lie in a second metabolic effect of the fluoro compounds, unrelated to the inhibition of citrate oxidation. Lahiri and Quastel ( 1 963), in experiments on rat brain slices respiring in a glucose-containing medium, found that fluoroacetate at concentrations too low to inhibit the respiration had an effect on the metabolism of glutamic acid. The synthesis of glutamine from ammonia and glutamate was suppressed, and both substrates were shown to accumulate. The labelling of aspartate from 14C-glutamate also was inhibited. In our experiments in vivo the inhibition of glutamine formation and the block in the tricarboxylic acid cycle apparently were superimposed, since the level of glutamate was slightly lowered rather than raised. Inhibition of citrate oxidation would lead to a decreased production of u-ketoglutarate, and hence of glutamic acid. It is unlikely that the block in glutamine formation accounts entirely for the increase in ammonia. Such an explanation would imply that there is normally a rapid turnover of ammonia, with release followed by conversion to glutamine, and utilization of glutamine in transamination or other reactions. However, the glutamine level is not reduced by fluoro compounds as would be expected on the basis of this hypothesis, and in the mouse brain it is measurably increased (as shown in Section IV). Fluoro-fatty acids apparently cause an actual increase in ammonia formation in the brain. The production of ammonia in incubated brain slices is increased by absence of glucose from the medium (Weil-Malherbe and Green, 1955), and an inhibition of glucose oxidation via the tricarboxylic acid cycle might be expected to have the same effect. The decrease in aspartic acid induced by fluoro compounds is probably due to the high concentration of ammonia (see Section I1 for discussion), although the blocking of a pathway by which carbon atoms pass from glutamate to aspartate might also be a factor (Lahiri and Quastel, 1963). The very large increase in alanine probably is due to transamination reactions, but could reflect a secondary route of ammonia detoxication by means of direct amination

FREE A M I N O A C I D S A N D R E L A T E D C O M P O U N D S

151

of pyruvic acid. Fisher and McGregor (1961) have shown that crystalline glutamic dehydrogenase from beef liver is not specific for glutamate or for a-ketoglutarate; alanine and pyruvate are effective substrates. However, the fact that the increase in alanine induced by the fluoro compounds was partially prevented by AOAA is probably related to the inhibitory effects of AOAA on transaminase activities. Cerebral lactic acid levels are elevated greatly during violent seizures, but the increase noted in dogs treated with AOAA MFA cannot be explained on this basis. In these experiments the high lactate may be in part a consequence of the extremely high ammonia levels. In the animals which received AOAA MFB the brain lactate levels were even higher, indicating that the occurrence of usually mild seizure activity also contributed to the elevation of this constituent. Marked decreases in brain glycogen were observed during seizures induced by fluoro compounds. This result is in contrast to earlier findings with pentylenetetrazol; in a small series of dogs this drug did not perceptibly decrease the glycogen level (Gurdjian et al., 1947). However, Klein and Olsen (1947) found that, in the cat, seizures induced by pentylenetetrazol, picrotoxin or other convulsants were accompanied by decreases in brain glycogen, and in mice low glycogen levels were found during seizures induced by pentylenetetrazol, picrotoxin or insulin (Carter and Stone, 1961). Lifson (1960) has noted that fluoroacetate enhanced both the disappearance of glycogen and lactate accumulation in the isolated, perfused gastrocnemius muscle of the dog. Reports emphasizing the biochemical effects of fluoro compounds have been reviewed by Peters (l957), and an extensive survey, including chemical and pharmacological studies, is available in a monograph by Pattison (1959).

+

+

I V . EFFECTS O F F L U O R O - F A T T Y A C I D ESTERS I N THE MOUSE

A few observations were made in the mouse in an effort to note species and tissue differences in response to MFA. The protective effects of AOAA in this species also were examined. Neurochemicaf effects

Female, albino, 8-10-week-old mice (Rolfsmeyer) were given 200 mg MFA per kg i.p. (this level is approximately 10 times the LDloo for mice). After 1 h the animals showed profound depression (seizures did not occur). They were killed by dropping into liquid air. Control animals were frozen in the same manner. The brains (excluding the cerebella) were removed, care being taken to prevent any thawing of the samples. Analyses for amino acids were done on samples pooled as indicated in Table VII. Most of the alterations induced by MFA in the amino acid content of mouse brain are similar qualitatively to those noted in dog cerebral cortex. These include increases in alanine, ammonia, leucine and serine and decreases in aspartate and glutamate (Table Vll). Lysine, which shows an insignificant increase in the dog, is elevated more noticeably in the mouse. Changes apparent in mice but not in dogs include increases Rejerences p 160-163

152

J . K . T E W S A N D W. E. S T O N E

T A B L E V1I C H E M I C A L C H A N G E S I N M O U S E B R A I N I N D U C E D BY M E T H Y L FLUOROACETATE

Values are in pmoles/g. The analyses were done on pooled samples: 3 or 4 brains for ammonia and glutamine; 9-12 brains for chromatographic fractionation. Control*

Constituent

___-

Acetylaspartic acid Alanine y-ABA Ammonia Arginine Aspartic acid Glutamic acid Glutamine Glutathione Glycerophosphoethanolamine* * GI ycine Histidine Leucine Lysine Methionine cystathionine** Phenylalanine Phosphoet hanolamine Serine Taurine T hreonine Tyrosine Urea Valine

8.25 0.45 2.26 0.43 0.133 3.17 11.35 6.76 1.77 0.35

j ,0.61 (3)

& 0.05 (4) i0.29 (4) 0.18 (6) f 0.002 (4) zI=O.II (4) 0.23 (4) I 1.2 (6) i0.05 (4) -1- 0.06 (4) 0.93 A 0.05 (4) 0.054 iO.010(4) 0.064 0.009 (4) 0.21 & 0.04 (4) 0.14 & 0.05 (4) 0.013 (4) 0.048 1.76 i 0.15 (4) 0.95 i 0.04 (4) 0.45 (4) 10.17 0.30 i 0.05 (4) 0.066 0.019 (4) 5.8 i 1.1 (4) 0.091; 0.12

+

-+

+

MFA* -

.__

8.40 1.57 2.65 1.05 0.14 1.40 8.17 8.00 1.60 0.26 1.00 0.068 0.15 0.31 0.16 0.061 1.50 1.12 9.93 0.34 0.082 5.7 0.13

8.87 1.87 3.19 1.66 0.14 1.49 8.93 8.86 0.32 1.02 0.078 0.18 0.34 0.070 1.70 1.14 10.20 0.38 0,097 7.1 0.13

* Number of control analyses indicated in parentheses; individual analyses given for MFA. * * Color yield per mole assumed equal to that of leucine. in brain y-ABA, glutamine and glycine. The high y-ABA values may be caused by a non-specific effect of the drug. If, at the time of freezing, the animals were anoxic as a result of the severe depression, this amino acid probably would be increased.

Effects in other tissues Livers, kidneys, hearts, and muscles from the hind legs were removed from the frozen animals. Analyses were done on the samples pooled as shown in Table V111. Since few quantitative data on the free amino acid patterns of mouse tissues have appeared i n the literature, values are presented for all measured components, whether or not they were affected by MFA. The observations are limited, but do show the general effects of the drug in these tissues. In most jnstances the results were similar to those seen in brain. When compared with the control values aspartic acid was decreased in all cases, the greatest effect being noted in the heart and the least in the liver. Glutamate levels also were decreased for each tissue. No effect of MFA on alanine in

153

FREE A M I N O A C I D S A N D R E L A T E D C O M P O U N D S

TABLE VlII T H E EFFECT OF M E T H Y L F L U O R O A C E T A T E O N A C I D - S O L U B L E N I T R O G E N O U S C O N S T I T U E N T S OF MOUSE T I S S U E S

Values are in ,umoles/g. The analyses were done on pooled samples from the control or MFA-treated animals, respectively: heart, 9 or 1 I ; kidney, 12 or 13; liver, 5 each; skeletal muscle, 9 each.

Constituent

Alanine Aspartic acid Glutamic acid Glutathione, oxidized Glycerophosphoethanolamine* Glycine lsoleucine Leucine Methionine cystathionine* Phosphoethanolamine Serine Taurine Threonine Urea Valine

+

Heart -___ Control MFA

0.93 1.04 2.21 0.85

0.64 0.086 0.1 I 0.07 $

0.32 16.3$ 0.21 6. I 0.12

Kidney

Liver _ _ Control MFA

_

Control

MFA

I .85 0.24 0.78

0.83 1.34 3.07 0.64

1.22 0.55 0.94 1.00

0.97 0.87 1 .92 2.11

0.08 0.90 0.21 0.35

0.88 1.32 0.13 0.12

0.57 1.40 0.19 0.38

0.13 0.32 0.30 15.8 0.30 11.2

0.21 2.42 0.27 9.72 0.21 30.8 0.12

0.16 1.89 0.31 7.57 0.36 16.7 0.18

Skeletal muscle

~

Control

MFA

4.26 0.44 0.74 1.57

1.35 0.37 1.26 0.23

1.22 0.17 0.51 0.72

0.46 1.63 0.11 0.19

0.27 2.02 0.28 0.53

0.04 2.21 0.12 0.11

0.04 2.45 0.22 0.31

0.19 0.45 0.50 14.5

0.27 0.56 0.51 14.1

10.0 0.22

13.2 0.46

0.10 0.13 0.46 34.1 0.46 5.7

0.12 0.19 0.32 29.7 0.32 10.3

**

**

**

**

* Color yield per mole assumed equal to that of leucine. ** Two peaks. 0 Phosphoethanolamine included with taurine.

skeletal muscle was noted, but this constituent inrceased in the other organs. There was approximately a 3-fold elevation of leucine in the samples from animals treated with MFA; isoleucine also showed definite increases. The reported interference in measurement of isoleucine values of brain (Tews et al., 1963) was never found in the other tissues examined. Glycine, especially in heart and liver, showed a tendency toward higher concentrations after treatment of the animal with MFA. In contrast with the findings in brain, serine levels were not raised. MFA treatment resulted in a marked decrease of urea in the kidney, concomitant increases occurring in the other three tissues examined. Some alterations occasionally appeared in other constituents, and unidentified components not found in the brain were present at times. Further experimentation would be required to determine the significance of some of these observations. Our studies of the effects of MFA on free amino acids in mouse tissues are in partial agreement with those of Awapara (1952), who studied the same tissues (except brain) in male rats. Three hours after administration of non-lethal doses of fluoroacetate, the levels of aspartate, glutamate and glutamine were found to be decreased in heart and liver, but not in kidney. Serine was elevated in heart. Further determinations in liver References p . 160-163

I54

J . K . T E W S A N D W . E. S T O N E

of 8 other amino acids, including alanine, glycine, isoleucine and leucine, showed no changes after fluoroacetate treatment (with the exception of a possible decrease in alanine). The fact that we used a much higher dose of the drug may explain some of the differences between Awapara’s results and ours.

Anti-convulsant action of A O A A In the pharmacological studies on mice, it was found that both the LD.50 and LDioo (i.p.) for MFA were between 10 and 20 mg/kg, in agreement with values tabulated by Chenoweth (1949). Studies with MFB indicated the L D m to be below 5 mg/kg. (Data of Kandel and Chenoweth, 1952b, suggest that for the rat also, fluorobutyrate is more toxic than fluoroacetate.) Unfortunately, the behavior of the mice was different from that usually reported (Chenoweth, 1949) in that seizures rarely were observed with MFA in doses from 5 mg/kg (completely non-lethal) to 400 mg/kg. In contrast, mice injected with 5 mg MFB per kg almost always manifested severe, repetitive seizures. Convulsions seldom were seen with doses of 10 or 20 mg/kg, although an occasional extensor spasm occurred. AOAA, at levels of 40 or 100 mg/kg i.p. was not lethal although depression occurred. Administration of either dose of AOAA 1 h before MFA (20 mg/kg) was ineffective in preventing death due to the latter drug. Seizures were less common in mice injected with MFB (5 mg/kg) 1 h after AOAA than with MFB alone, but mortality was unchanged. Thus AOAA was unable to prevent the lethal effects of these drugs in the mouse, even though the doses of the fluoro compounds were far less overwhelming than those used in the dog. Experiments with intact animals of other species might be more informative; Chenoweth et al., (1951) noted that mice showed variable responses to fluoroacetate. Recently Winnick et al. (1963) have reported that AOAA can reduce the toxicity of a-fluoro-p-alanine in mice. V. S P E C I E S D I F F E R E N C E S I N B R A I N A M I N O A C I D L E V E L S

The values which we have found for free amino acid concentrations in the brains of dogs and mice may be compared with results from other laboratories as tabulated by Waelsch (1957) and Tallan ( I 962). The most striking species difference in brain amino acid concentrations is in the high level of taurine in the mouse, where the concentration of this amino acid is about 8 times that in the dog. Although values for taurine are higher in rats than in dogs, they are considerably lower in rats than in mice. Otherwise, many of our data do not differ greatly from those for other species. This is especially true for amino acids which are present in relatively low concentrations. Levels of aspartate, glutamate, glycine and serine are slightly lower in dog cerebral tissue than in whole mouse brain (without cerebellum). Glycine and serine levels also are lower in dog cortex than in the brains of other mammals listed by Tallan (1962). Alanine and y-ABA are much lower in the dog than in the mouse; the available data from other species are not always comparable since these two constituents increase post-mortem. It is unlikely that incomplete extraction is a factor in our analyses, at least in the case of y-ABA, since our use of picric acid for the extraction gives results

FREE AMINO ACIDS AND RELATED COMPOUNDS

155

on frozen mouse brain which are almost identical with those of Lovell and Elliott (1963) based on extraction with 90 "/d ethanol. Work by De Ropp and Snedeker (1960) indicates that picric acid is slightly superior to 80 "/d methanol as an extracting agent for amino acids. Since our frozen specimens of dog cortex contained small amounts of white matter, the effects of this admixture must be considered. Okumura et al. (1959) have reported amino acid analyses of cortical gray matter, cerebral white matter and other parts of the dog brain (data from two females killed by decapitation). For the most part our data are in conformity with their results on cortical gray matter if allowance is made for post-mortem increases in alanine and y-ABA. It appears that the inclusion of white matter could have contributed to the higher value which we found for lysine and to the slightly lower values for glutamate and phosphoethanolamine, but more data would be required for a statistically valid evaluation. Under post-mortem conditions the y-ABA level also is much lower in cerebral white matter than in cortical gray matter; assuming a similar difference in the frozen brain, our values for this constituent are a little lower than the true levels in gray matter of the cortex. The values for acetylaspartic acid given by Okumura and his coworkers may be too low because of insufficient time allowed for hydrolysis of this constituent (Jacobson, 1959). It should be pointed out that our values for mouse brain glutamine may be too high. Nathan and Warren (1959) feel that the method of Richter and Dawson (1948), which we have used, is unsatisfactory for the determination of glutamine in mouse brain. Using a glutaminase procedure, they found the glutamine concentration in this tissue to be 4.14 pmoleslg. VI. N E U R O C H E M I C A L C L A S S I F I C A T I O N O F S E I Z U R E S

It seems altogether probable that certain chemical changes occur in the brain as concomitants of convulsive activity, appearing during the seizure but not preceding it, representing manifestations or results of this activity rather than causal mechanisms, and bearing no relation to the type of stimulating agent involved. The available data suggest that the following changes belong in this category : increased utilization of oxygen and of carbohydrate and other substrates, an increase in the lactic acid level*, a measurable breakdown of phosphocreatine, and a slight or moderate increase of ammonia. There are other chemical changes, however, which may occur as a result of enzyme inhibition or other metabolic interference caused by a convulsing agent. These changes may be observable before the beginning of the seizure, and may persist or increase during its course. They may be excitatory or may provide clues to excitatory mechanisms, or they may represent side effects bearing no relation to the convulsive process. Since different convulsants may have very different metabolic effects, it is possible to distinguish different types of seizures on the basis of specific chemical patterns even

* Hypoglycemic convulsions represent an exception in which lactic acid formation and probably some other metabolic processes are prevented by the lack of glucose as a substrate (Stone, 1938). References p . 160-163

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J . K . T E W S A N D W . E. S T O N E

though the electrographic manifestations are all very much alike. A tentative neurochemical classification of seizures induced by pharmacologic agents was attempted in an earlier paper (Stone et al., 1960). In the light of subsequent investigations, a modified version is now presented. Cholinergic seizures. The cerebral store of acetylcholine decreases in most seizures, probably as a result of an increased rate of release and destruction of this transmitter substance, but a notable exception occurs in the seizures induced by antichohnesterases (Stone, 1957). Here acetylcholine accumulates and appears to be the excitatory agent initiating the convulsive activity. The initial electrographic effect is an intense ‘arousal reaction’, a response not commonly occurring with other types of convulsant drugs. In some instances this is the only effect to be seen, but often a seizure develops characterized by epileptiform cortical activity with associated convulsive movements. The cortical electrographic changes are blocked completely by large doses of atropine or hyoscine, a fact which confirms the unique nature of cholinergic seizures inasmuch as atropine and hyoscine do not block other types of convulsive activity. Longo (1962) has reviewed a number of studies in this area. Acetylcholine itself, and several agents which mimic its peripheral actions, induce spikes and other convulsive wave-forms when applied directly to the cerebral cortex. Intravenou? injection of atropine or hyoscine blocks the action of topical acetylcholine (Longo, 1962) or nicotine (Rarnold, 1962). When cholinergic drugs are given intravenously their peripheral effects tend to mask any central actions, and some of these agents do not pass the blood-brain barrier, but acetylcholine given to rabbits by intracarotid injection induces an arousal reaction which can be blocked by atropine (Rinaldi and Himwich, 1955a, b; Longo, 1955). Nicotine given intravenously to rabbits induces both the arousal reaction and epileptiform cortical activity (Longo et al., 1954). These responses are blocked by tertiary amines such as carmiphen and chlorpromazine, compounds which exhibit atropine-like and weak ganglionic-blocking properties and which presumably penetrate the blood-brain barrier more rapidly than do quaternary ammonium compounds. Carmiphen also blocks the central electrographic effects of diisopropylfluorophosphate, an anticholinesterase (Essig et a/., 1950). In rabbits the cortical responses to intravenous nicotine were not blocked by atropine or hyoscine in the doses tried (Longo et al., 1954), but in dogs Ramold (1962) found that nicotine induces an arousal reaction which can be prevented by a massive dose of hyoscine. We have not examined the cerebral amino acids in cholinergic seizures. Seizures induced by ammonium salts. If the ammonium ion content of the blood is raised rapidly to a high level by injection of an ammonium salt, a seizure usually occurs. With slow intravenous infusion, however, depression of the cortical activity is more common. Whether or not a seizure occurs, the chemical changes in the brain may include increases in ammonia, glutamine, alanine, histidine and lactate, and decreases in aspartic acid and valine. The mechanism by which the convulsive activity is induced is unknown. These observations suggest a possible role of endogenous ammonia as a stimulating or depressing factor in conditions induced by agents such as fluoro compounds, methionine sulfoximine, insulin, and AOAA. However, no

FREE AMINO ACIDS A N D RELATED COMPOUNDS

157

firm conclusions can be reached as yet with regard to the role of ammonia. In one instance we observed a very high level i n the brain (2.90 pmoleslg) with only very slight depression of cortical electrical activity i n a dog given AOAA and MFA; the associated increase in y-ABA (to 1.52 pmoleslg) was of moderate degree. Seizures induced by Juoro-fatty acids. These are characterized by inhibition of the enzyme aconitase, with a resulting accumulation of citrate in the brain; by a reduction in the rate of synthesis of brain glutamine; and by a great increase in brain ammonia. The changes in citrate and ammonia occur prior to the appearance of seizures, but as indicated previously, the convulsive response cannot with certainty be attributed to either of them. With small doses of fluoro compounds, Kandel and Chenoweth (1952a) sometimes observed seizures in the absence of increased brain citrate. Other changes observed during seizures induced by fluoro-fatty acids include an increase in leucine (not yet observed in any other type of seizure), increases in alanine and serine, and decreases in glutamate and aspartate. In mice, but not in dogs, there were increases in glutamine, lysine and glycine, and also a rise in y-ABA which may have been secondary to anoxemia. Methionine sulfoximine seizures. These occur intermittently as part o f a toxic syndrome characterized by an extensive disturbance in the cerebral amino acid metabolism. There are increases in brain ammonia, alanine, lactate, lysine, phosphoethanolamine and serine; decreases in glutamine, glutamate, aspartate, leucine, valine, and the sum of methionine and cystathionine; and a slight decrease in y-ABA. These changes have been found in animals showing the toxic syndrome; no observations made during actual seizures are available. The drug is known to be an inhibitor of glutamine synthetase and of a glutamyl transferase in brain, and is a methionine antagonist. The block in glutamine synthesis represents a point of similarity to the effects of fluoro-fatty acids, although the fluoro compounds do not decrease the brain glutamine concentration to subnormal levels. Hypoglycemic convulsions. Dogs in insulin ‘shock’ frequently exhibit convulsive reactions, but we have found it dificult to produce such a response in animals prepared surgically for the recording o f cortical electrical activity and subsequent freezing of the brain. Profound cortical depression more commonly occurs. Others have found that the cortical activity may remain depressed during a seizure, while convulsive potentials are present in deep structures such as the hippocampus and the amygdala (Van Meter et al., 1958; Berkowitz et al., 1960), but movement artifacts introduced an element of uncertainty in these experiments. In our laboratory one record was obtained during a mild insulin convulsion; in this instance the convulsive activity did invade the cortex, the clonic movements being associated with groups of 3 to 5 large spikes (Stone et al., 1962). Hypoglycemia, unlike all of the other conditions we have studied, is a state in which glucose is not available in amounts adequate for the normal metabolic processes of the brain. In profound ‘shock’ the true glucose in the brain is reduced almost to the vanishing point. The related compounds glycogen, citrate and lactate also are decreased, as are alanine, y-ABA, glutamate and glutamine. Ammonia is greatly increased, aspartate and lysine rise significantly, and breakdown of energy-rich phosRefercnrrs p. 160-163

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phorus compounds occurs. Acetylcholine also decreases. Some of these changes have been observed in several laboratories; the pertinent literature is to be reviewed in a paper now in preparation (Tews, Carter and Stone). The critical factor for the precipitation of seizures remains unknown. Seizures induced by carbonyl-trapping agents. The convulsant hydrazides induce seizures which appear to be in some way related to those of acute vitamin B6 deficiency, being antagonized by various forms of this vitamin whereas those induced by other well-known convulsants are not (Williams and Bain, 1961). The hydrazides are carbonyl-trapping agents which combine with the aldehyde group of pyridoxal phosphate. A characteristic observation is that brain y-ABA levels are decreased as a result of in vivo inhibition of glutamic acid decarboxylase activity. However, it has become clear that the low level of y-ABA is not the major causative factor, since the seizures can occur when this decrease is prevented. The hydrazides evidently have some other excitatory action, possibly through reducing the rate of metabolism via the y-ABA pathway (Balzer et al., 1960; Wallach, 1961b; Tower, 1963) or through formation of excitatory complexes with pyridoxal compounds (Dixon and Williams, 1962). Hence the exact relation of hydrazide action to vitamin B6 deficiency is not clear. Carbonyl-trapping agents such as AOAA and hydroxylamine, which preferentially inhibit y-ABA-a-ketoglutarate transaminase in vivo, do not decrease the brain y-ABA level but rather increase it. These agents may exhibit both inhibitory and excitatory effects. They are tentatively classed with the hydrazides since the basic convulsive mechanism may be the same. Wallach (1961b) has noted that seizures induced by AOAA are inhibited by pyridoxal phosphate, although in vitro the vitamin does not counteract the inhibitory effect of AOAA on y-ABA-a-ketoglutarate transaminase. Both thiosemicarbazide and AOAA increase the level of free tyrosine in the brain. It is suggested that the brain y-ABA level plays a subsidiary role, a decrease potentiating excitation by hydrazides and an increase opposing excitation by agents such as AOAA and hydroxylamine. Picrotoxin-type seizures. The convulsant effects of picrotoxin and pentylenetetrazol appear to be very similar, except for the longer latent period of picrotoxin. No specific effects of these drugs on cerebral intermediary metabolism are known, and they have been tentatively classed together as representing a type of convulsant for which unique and characteristic chemical changes are not apparent. It is conceivable that these drugs mimic the action of an unknown chemical transmitter at certain central synapses. Strychnine-type seizures. The effects of strychnine appear to be different from those of most other convulsants, but there are not enough data available to delineate a chemical pattern associated with strychnine convulsions. Tetanus toxin, brucein and thebaine may belong in the strychnine group. Other possibZe types. There are many known convulsants that cannot be classified until more data become available. It is probable that these include types not yet distinguished.

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SUMMARY

This paper summarizes a series of studies on free amino acids and related compounds in the brain, including previously unpublished data on the effects of fluoro-fatty acid esters. Cerebral tissue was analyzed by ion-exchange chromatography and other methods, and the technique of freezing the brain in situ with liquid air was used to avoid post-mortem increases which otherwise occur in alanine, ammonia, y-aminobutyric acid and lactic acid. Dogs were used for most of the experiments, but supplemental studies on effects of fluoro compounds in mice are included. Anoxia induced by administration of 4.5% oxygen in nitrogen for 12-13 min brought about significant increases in alanine, y-aminobutyric acid, glutamic acid, lactic acid, leucine and tyrosine, and decreases in aspartic acid and a fraction consisting of methionine and cystathionine. Ammonia was not significantly increased. Infusion of buffered ammonium chloride for a 10-min or 45-min period generally caused some degree of depression of cortical electrical activity, although in one animal a seizure developed. The cerebral ammonia was greatly increased in every instance. Glutamine showed only a minimal change at 10 min, but was greatly increased at 45 min. Alanine, histidine and lactate were increased, and aspartate and valine were decreased. Urea values were generally high but variable. Amino-oxyacetic acid, which is known to antagonize the convulsant actions of thiosemicarbazide and methionine sulfoximine, was found to induce increases in brain y-aminobutyrate, alanine, ammonia, glutamine, lactate, lysine and tyrosine, and a decrease in aspartate. Previously known as an inhibitor of y-aminobutyrate-a-ketoglutarate transaminase, this drug may have inhibitory effects in vivo on various transamination or other reactions. The changes in some of the cerebral constituents probably are secondary to the high ammonia concentration in the brain, which in turn may be due at least in part to ammonia accumulating in the blood. The chemical changes usually were accompanied by electrographic depression, but occasionally by signs of cerebral excitation and in one instance by a generalized seizure. Methionine sulfoximine was found to have extensive effects on the nitrogenous metabolism of the brain, causing increases in alanine, ammonia, lysine, phosphoethanolamine and serine, and decreases in aspartate, glutamate, glutamine, leucine, and the methionine cystathionine fraction. y-Aminobutyrate and valine also were slightly decreased, and lactate showed a small increase. Some of the changes may be secondary to the increase in brain ammonia. There was no accumulation of ammonia in the blood. Seizures induced by thiosemicarbazide were accompanied by a decrease in y-aminobutyrate, increases in alanine, lactate and tyrosine, and a relatively small increase in ammonia. Amino-oxyacetic acid had an inhibitory effect on thiosemicarbazide seizures which could be overcome by increasing the dose of thiosemicarbazide although the y-aminobutyrate remained above normal. The combined effects of the two drugs on the chemical pattern was essentially the same as the effect of amino-oxyacetic acid alone. The inhibitory effect of amino-oxyacetic acid may be due to the increase in brain y-aminobutyric acid. The excitatory effects of hydrazides and of amino-oxyacetic acid, which are carbonyl-trapping agents, may involve the same (unknown)

+

References p . 160-163

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basic mechanism, with a decrease in y-aminobutyrate potentiating excitation by hydrazides and an increase opposing excitation by amino-oxyacetic acid. The levels of free amino acids in the brain were remarkably stable during seizures induced by picrotoxin or pentylenetetrazol. With picrotoxin the only significant change noted in this group of compounds was a slight decrease in aspartic acid. In pentylenetetrazol seizures there was an increase in alanine and a slight decrease in glutamic acid. Ammonia was increased moderately by pentylenetetrazol, but not significantly by picrotoxin. Amino-oxyacetic acid raised the convulsive threshold to pentylenetetrazol, but showed little antagonism to a convulsant dose of picrotoxin. In dogs, methylfluoroacetate and methyl fluorobutyrate induced violent seizures accompanied by increases in brain alanine, ammonia, citrate, lactate, leucine and serine, and decreases in aspartate, glutamate and glycogen. Despite very high ammonia levels, glutamine was not increased. In mice, methylfluoroacetate induced profound depression rather than seizures. Chemical changes resembling those in the dog were observed, but a measurable increase in glutamine occurred and there were also increases in y-aminobutyrate (possibly secondary to anoxia) and in glycine and lysine. Changes in liver, kidney, heart and skeletal muscle resembled those in brain, but with minor differences. In dogs, the convulsive response to fluoroacetate was blocked and that to fluorobutyrate was greatly diminished by prior injection of amino-oxyacetic acid. In mice, amino-oxyacetic acid showed an inhibitory effect on fluorobutyrate seizures but failed to antagonize the lethal effects of either fluoroacetate or fluorobutyrate. The neurochemical pattern in dogs given amino-oxyacetic acid followed by a fluoro-fatty acid was essentially that induced by amino-oxyacetic acid with some of the effects of the fluoro compound superimposed, but the rises in citrate and alanine usually seen with fluoro compounds were reduced or prevented. Species differences in cerebral amino acid patterns are discussed briefly, and a revision of a previously proposed neurochemical classification of seizures is presented. ACKNOWLEDGEMENTS

Dr. P. Dante Roa and Mr. Samuel H. Carter collaborated with us on parts of this investigation. We thank Mr. Charles E. Charmley for his invaluable technical assistance. Amino-oxyacetic acid hemihydrochloride was supplied by Dr. P. W. O’Connell of the Upjohn Co., Kalamazoo, Mich. This research was supported by grants NB-00818 and B-3360 from the National Institute of Neurological Diseases and Blindness, U.S. Public Health Service. REFERENCES ALHFRS, R . W., KOVAL,G . J . , AND JAKOBY, W. B., (1962); Transamination reactions of rat brain. Exp. Neuvol., 6,85-89. AWAPARA, J., (1952); The influence of fluoroacetate on the concentration of free amino acids in rat organs. J. biol. Chem., 197,695-699. BALZER,H . , HOLTZ,P., AND PALM,D., (1960); Untersuchungen uber die biochemischen Grundlagen der konvulsiven Wirkung yon Hydraziden. Arch. exp. Puf/zo/.Phuvmnkol., 239, 520-552.

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BAXTER, C. F., AND ROBERTS, E., (1959); Elevation of y-aminobutyric acid in rat brain with hydroxylamine. Proc. Soc. exp. B i d . ( N . Y . ) , 101, 81 1-815. BAXTER, C. F., AND ROBERTS, E., (1960) ; Demonstration of thiosemicarbazide-induced convulsions in rats with elevated brain levels of 71-aminobutyric acid. Proc. Soc. exp. B i d . ( N . Y . ), 104,426427. BAXTER, C. F., AND ROBERTS, E., (1962); Effects of 4-methoxymethylpyridoxine and carbonyltrapping agents on amino acid content of mammalian brain and other tissues. Amino Acid Pools. J. T. Holden, Editor. Amsterdam, Elsevier (p. 499). BENITEZ, D., PSCHEIDT, G. R., A N D STONE,W. E., (1954); Formation of ammonium ion in the cerebrum in fluoroacetate poisoning. Amer. J . Physiol., 176, 488-492. BERKOWITZ,E. C., SUNDSTEN, J. W., AND SAWYER, C . H., (1960); Electroencephalographic and behavioral changes in unrestrained rabbits during insulin hypoglycemia. Neurology, 10, 355-364. BERL,S., TAKAGAKI, G., CLARKE, D. D., AND WAELSCH, H., (1962a); Metabolic compartments in vivo. Ammonia and glutamic acid metabolism in brain and liver. J . hiol. Chem., 237, 2562-2569. BERL,S., TAKAGAKI, G., CLARKE, D. D., AND WAELSCH, H., (1962b); Carbon dioxide fixation in brain. J . hiol. Chem., 237, 2570-2573. BUFFA, P.,ANDPETERS, R. A., (1949); The in vivo formation of citrate induced by fluoroacetate and its significance. J . Physiol., 110, 488-500. CARTER, S. H., AND STONE,W. E., (1961); Effect of convulsants on brain glycogen in the mouse. J . Neurochem., 7, 16-1 9. CHENOWETH, M . B., (1949); Monofluoroacetic acid and related compounds. Pharmacol. Rev., 1, 3834 2 4 . CHENOWETH, M. B., KANDEL, A,, JOHNSON, L. B., A N D BENNETT, D. R., (1951); Factors influencing fluoroacetate poisoning. Practical treatment with glycerol monoacetate. J . Pharmacol. exp. Ther., 102,3149. CHIRIGOS, M. A., GREENGARD, P., AND UDENFRIEND, S., (1960); Uptake of tyrosine by rat brain in v i v a J . biol. Chem., 235,2075-2079. DAVANZO,J. P., CREIG, M. E., AND CRONIN,M. A,, (1961); Anticonvulsant properties of aminooxyacetic acid. Amer. J . Physiol., 201, 833-837. DAWSON, R. M. C., (1 953); Cerebral amino acids in fluoroacetate-poisoned, anaesthetised and hypoglycaemic rats. Biochim. Biophys. Acta, 11, 548-552. DEROPP,R . S., AND SNEDEKER, E. H., (1960); Sequential one-dimensional chromatography: analysis of free amino acids in the brain. Anal. biochem., 1, 424432. DERoPP,R.S., AND SNEDEKER, E. H., (1961); Effect of drugs on amino acid levels in brain: excitants and depressants. Puoc. Soc. exp. B i d . ( N . Y.), 106, 696-700. DIXON, R . H., A N D WILLIAMS, H. L., (1962); The toxicity of pyridoxal and pyridoxal phosphate hydrazones in mice. Fed. Proc., 21,338. DOMINGUEZ, A. M., SHIDEMAN, F. E., MAHLER, H. R., A N D HIFT,H., (1954); Effects of monofluoroacetic, 4-fluorobutanoic and 6-fluorohexanoic acids on fatty acid and Krebs cycle oxidations. Fed. Proc., 13,349. ELLIOTT, K. A. C., AND VAN GELDER; N. M., (1960); The state of Factor I in rat brain: the effects of metabolic conditions and drugs. J . Physiol., 153,423432. ELLIOTT, K . A. C., AND LOVELL, R. A., (1962); The GABA and Factor I content of brain. Fed. Proc., 21, 364. Essrc, C. F., HAMPSON,J . L., BALES,P. D., WILLIS, A., AND HIMWICH, H. E., (1950); Effect of Panparnit on brain wave changes induced by diisopropylfluorophosphate (DFP). Science, 111, 38-39. FISHER, H. F., A N D MCGREGOR, L. L., (1961); The mechanism of the glutamic dehydrogenase reaction. 11. Substrate specificity of the enzyme. J . biol. Chem., 236, 791-794. GAL,E. M., DREWES, P. A., AND TAYLOR,N. F., (1961); Metabolism of fluoroacetic acid-2-I4C in the intact rat. Avch. Biochem. Biophys., 93, 1-14. GEIGER, A., HORVATH, N., A N D KAWAKITA,Y., (1960a); The incorporation of 14C derived from glucose into the proteins of the brain cortex at rest and during activity. J . Neurochem., 5, 311-322. GEIGER, A., KAWAKITA,Y . ,AND BARKULIS, S. S., (1960b); Major pathways of glucose utilization in the brain in brain perfusion experiments in vivo and in situ. J . Neurochem., 5, 323-338. GERSHOFF, S. N., (1956); Biological effects of methionine sulfoximine. 11. Comparative sulfur metabolism studies in rats and rabbits. Amer. J . Physiol., 184,4346. GITTER, S . , ( I 956); The influence of acetamide on citrate accumulation after fluoroacetate poisoning. Biochem. J., 63, 182-187. GURDJIAN, E. S., WEBSTER, J. E., AND STONE,W. E., (1947); Cerebral metabolism in metrazol convulsions in the dog. Proc. Ass. Res. new. ment. Dis., 26, 184-204.

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HAAVALDSEN, R., (1962); Transamination of aromatic amino acids in nervous tissue. Nature, 196, 577-578. HENDERSHOT, L. c.,A N D CHENOWETH, M. B., (I 955); Fluoroacetate and fluorobutyrate convulsions in the isolated cerebral cortex of the dog. J . Pharmacol. exp. Ther., 113, 160-168. HOPPER, S., A N D SEGAL, H.L., (1962); Kinetic studies of rat liver glutamic-alanine transaminase. J. biol. Chem., 237,3189-3195. JACOBSON, K. B., (1959); Studies on the role of N-acetylaspartic acid in mammalian brain. J. gerr. Physiol., 43,323-33 3. KANDEL, A., AND CHENOWETH, M. B., (1952a); Metabolic disturbances produced by some fluorofatty acids: relation to the pharmacologic activity of these compounds. J. Pharmacol. exp. Ther., 104,234-247. KANDEL, A., AND CHENOWETH, M. B., (1952b); Tolerance to fluoroacetate and fluorobutyrate in rats. J . Pharmacol. exp. Ther., 104,248-252. KILLAM, K. F., AND BAIN, J. A., (1957); Convulsant hydrazides. I. In vitro and in vivo inhibition of vitamin Bs enzymes by convulsant hydrazides. J. Phaumacol. exp. Ther., 119, 255-262. KLEIN,J. R., AND OLSEN, N. S., (1947); Effect of convulsive activity upon the concentration of brain glucose, glycogen, lactate and phosphates. J . biol. Chem., 167, 747-756. LAHIRI,S., AND QUASTEL, J. H., (1963); Fluoroacetate and the metabolism of ammonia in the brain. Biochem. J., 89, 157-163. LIFSON,N., (1960); Effect of fluoroacctate on the metabolism of isotopic acetate and lactate by the isolated perfused dog gastrocnemius. Amer. J . Physiol., 198, 107 1-1 074. LONGO,V. G., (1955); Acetylcholine, cholinergic drugs, and cortical electrical activity. Experientia, ii,76-78. LONGO, V. G., (1962); Rabbit Brain Rewarch. Vol. TI. Electroencephalographic Atlas for Pharmacological Research. Effects of Drugs on the Electrical Activity of the Rabbit Brain. Amsterdam, Elsevier. LONGO,V. G., VON BERGER, G. P., A N D BOVET,D., (1954); Action of nicotine and of the ‘ganglioplegiques centraux’ on the electrical activity of the brain. J . Pharmncul. exp. Ther., 111, 349-359. LOVELL, R. A., AND ELLIOTT,K. A. C., (1963); The y-aminobutyric acid and Factor I content of brain. J . Neurochem., 10,479488. MASSIEU, G. H., TAPIA,R., AND ORTEGA, B. G., (1962); Free amino acids in brain of mice treated with L-glutamic acid-y-hydrazide. Biochem. Phaumacol., 11, 976-979. MAYNERT, E. W., AND KAJI,H. K., (1962); On the relationship of brain y-aminobutyric acid to convulsions. J. Pharnzacol. exp. Ther., 137, 114-121. MCMILLAN, P. J., A N D MORTENSEN, R. A., (1963); The metabolism of brain pyruvate and acetate in the tricarboxylic acid cycle. J . biol. Chem., 238, 91-93. MOORE, S., AND STEIN, W. H., (1954); Procedures for the chromatographic determination of amino acids on 4 % crosslinked sulfonated polystyrene resins. J . biol. Chem., 211, 893-906. NATHAN, D. G., AND WARREN, K. S., (1959); A colorimetric method for the measurement of the brain ammonia of the mouse: The effect of glutamine on the total measurable ammonia. Arch. Biochem. Biophys., 81, 377-381. OKUMURA, N., OTSUKI,S., AND FUKAI,N., (1959); Amino acid concentration in different parts of the dog brain. Acta Men. Okayama, 13, 27-30. PACE,J., A N D MCDERMOTT, E. E., (1952); Methionine sulphoximine and some enzyme systems involving glutamine. Nature, 169, 415416. PAmsoiw, F. L. M., (1959); Toxic Aliphatic Fluorine Compounds. Amsterdam, Elsevier. P~TERS, E. L., A N D TOWER, D. B., (1959); Glutamic acid and glutamine metabolism in cerebral cortex after seizures induced by methionine sulphoximine. J . Neurochem., 5, 80-90. PETERS, R. A,, (1957); Mechanism of the toxicity of the active constituent of Dichapetalurn cymosum and related compounds. Adv. Enzymol., 18, 113-159. POTTER, V. R., (1951); Sequential blocking of metabolic pathways in vivo. Proc. Suc. exp. Biol. ( N . Y . ) , 76,41-46, PROLER, M., AND KELLAWAY, P., (1962); The methionine sulfoximine syndrome in the cat. Epilepsia, 4th series, 3, 117-130. RAMOLD, M. C., (1962); Effects of cholinergic and other stimulating agents on the electrical activity of the cerebral cortex. Thesis, University of Wisconsin. RICHTER, D., AND DAWSON, R. M. C., (1948); The ammonia and glutamine content of the brain. J. biol. Chem., 176, 1199-1210. RINALDI, F., AND HiMwicii, H. E., (1955a); Alerting responses and actions of atropine and cholinergic drugs. A.M.A. Arch. Neural. Psychiat., 73, 387-395.

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RINALDI, F., AND HIMWICH, H. E., (1955b); Cholinergic mechanism involved in function of mesodiencephalic activating system. A . M . A . Arch. Neurol. Psychiat., 73, 396402. ROA, P. D., TEWS,J. K., AND STONE,W. E., (1964); A neurochemical study of thiosemicarbazide seizures and their inhibition by amino-oxyacetic acid. Biochem. Pharmacol., 13, 477-487. ROBERTS, E., (1960); Free amino acids of nervous tissue: some aspects of metabolism of y-aminobutyric acid. Inhibition in the Nervous System and y-Aminobutyric Acid. E. Roberts, Editor. Oxford, Pergamon Press (p. 144). ROBERTS, E., (I 963) ; y-Aminobutyric acid (y-ABA)-vitamin Bs relationships in the brain. Amer. J . clin. Nutr., 12,291-307. ROBERTS, E., BAXTER, C. F., AND EIDELBERG, E., (1960); Some aspects of cerebral metabolism and physiology of y-aminobutyric acid. Structure and Function of the Cerebral Cortex. D. B. Tower and J . P. Schade, Editors. Amsterdam, Elsevier (p. 392). ROBFRTS, E., A N D EIDELBERG, E., (1960) ; Metabolic and neurophysiological roles of y-aminobutyric acid. Int. Rev. Meurobiol., 2, 279-332. ROSEN,F., MILHOLLAND, R.J., AND NICHOL,C. A., (1960); Studies on the inhibition of L-glutamic acid decarboxylase in vivo. Inhibition in the Nervous System and y-Aminobutyric Acid. E. Roberts, Editor. Oxford, Pergamon Press (p. 338). STONE,W. E., (1938); The effects of anaesthetics and of convulsants on the lactic acid content of the brain. Biochem. J., 32, 1908-1918. STONE,W. E., (1957); The role of acetylcholine in brain metabolism and function. Amer. J. phys. Med., 36,222-255. STONE,W. E., TEWS,J . K., AND CARTER, S. H., (1962); Chemical changes in the brain during insulin hypoglycemia and recovery. Physiologist, 5, 21 8. STONE,W. E., TEWS,J. K., AND MITCHELL,E. N., (1960); Chemical concomitants of convulsive activity in the cerebrum. Neurology, 10, 241-248. TALLAN, H . H., (1962); A survey of the amino acids and related compounds in nervous tissue. Amino Acid Pools. J. T. Holden, Editor. Amsterdam, Elsevier (p. 471). TEWS,J. K., CARTER, S. H., ROA,P. D., A N D STONE,W. E., (1963); Free amino acids and related compounds in dog brain: post-mortem and anoxic changes, effectsof ammonium chloride infusion, and levels during seizures induced by picrotoxin and by pentylenetetrazol. J . Neurochem., 10, 641-653. TEWS,J. K., AND STONE,W. E., (1963); Neurochemical effects of fluoro-fatty acidestersalone and in combination with amino-oxyacetic acid. Fed. Proc., 22, 633. TEWS,J. K., AND STONE,W. E., (1964); Effects of methionine sulfoximine on levels of free amino acids and related substances in brain. Biochem. Pharmacol., 13,543-545. TOWER,D. B., (1960); Cerebral amino acid metabolism and vitamin Be. Ch. IJI. Neurochemistry o Epilepsy. Springfield, Thomas. TOWER, D. B., (1963); Interrelationships of oxidative and nitrogen metabolism with cellular nutrition and function in the central nervous system. Amer. J . clin. Mutr., 12, 308-320. TOWER, D. B., WHERRETT, J. R., A N D MCKHANN, G. M., (1961); Functional implications of metabolic compartmentation in the central nervous system. Regional Newrochemistry. S. S . Kety and J. Elkes, Editors. Oxford, Pergamon Press (p.65). UTLEY,J. D., (1963); The effects of anthranilic hydroxamic acid on rat behavior and rat brain y-aminobutyric acid, norepinephrine and 5-hydroxytryptamine concentrations. J . Neurochem., 10,423432. VAN METER,W. G., OWENS,H. F., AND HIMWICH, H. E., (1958); Cortical and rhinencephalic electrical potentials during hypoglycemia. A . M.A. Arch. Neurol. Psychiat., 80, 314-320. WAELSCH, H., (1957); Metabolism of proteins and amino acids. Metabolism of the Nervous System. D. Richter, Editor. London, Pergamon Press (p. 43 I). WALLACH, D. P., (1961a); Studies on the GABA pathway. I. The inhibition of y-aminobutyric acida-ketoglutaric acid transaminase in vitro and in vivo by U-7524 (amino-oxyacetic acid). Biochem. Pharmacol., 5,323-331. WALLACH, D. P., (1961b); Studies on the GABA pathway. 11. The lack of effect of pyridoxal phosphate on GABA-KGA transaminase inhibition induced by amino-oxyacetic acid. Biochem. Pharmacol., 8,328-33 I . WEIL-MALHERBE, H., AND GREEN,R . H., (1955); Ammonia formation in brain. I. Studies on slices and suspensions. Biochem. J., 61, 210-218. WILLIAMS, H. L., AND BAIN,J. A,, (1961); Convulsive effect of hydrazides: relationship to pyridoxine. Inr. Rev. Neurobiol., 3,319-348. WINNICK,T., WINNICK,R. E., AND BERGMANN, E. D., (1963); Some metabolic and enzymatic experiments with a-fluoro-8-alanine. Biochim. biophys. Acta, 69, 48-58.

164

The Excretion of 5 - Hydroxyindoleacetic Acid in Mental Patients A. J . V A L C O U R T

Veterans Administration Hospital, Wockton, iMass. ( U.S.A.)

Several aspects of the etiology of mental disease are being studied quite intensely. Not the least of these is the biochemical aspect, especially in regard to schizophrenia. One of the chemicals thought to be involved in brain function is 5-hydroxytryptamine or serotonin (Woolley, 1957; Brodie et al., 1955). An especially interesting observation was that the release of serotonin from various body sites was limited to those of the Rauwolfia drugs with tranquilizing action (Brodie et al., 1956). This release of serotonin has been demonstrated not only directly, but also indirectly by following the urinary excretion of 5-hydroxyindoleacetic acid (5-HIAA), a major end product of serotonin metabolism (Shore et al., 1955). The experiments described herein deal with the effect of reserpine on the urinary excretion of 5-HIAA in mental patients. The patients used in this study were carefully selected for long-term studies on aging and mental disease and were also available for short-term experiments. Such an arrangement made possible the collection of 24-h urine specimens and the quantitative study of the effect of reserpine upon the urinary excretion of 5-H IAA in these patients. EXPERIMENTAL

Each experiment was conducted on a group of 4 to 6 male patients in order to ensure maximum supervision in the matter of urine collections. The urine samples were collected in bottles containing toluene to minimize exposure of the urine to air. When collection was complete, the urine volume was determined and an aliquot placed in the refrigerator for subsequent assay. The creatinine content of the urine samples was measured. The values thus obtained served as a check of the completeness of the 24-h collection and were essentially unchanged whether or not reserpine was being administered. 5-HIAA was extracted and assayed by a modification of Udenfriend’s procedure (Udenfriend at al., 1955). It was found advisable to run internal standards with each sample assayed, since the recovery of 5-HIAA added to urine was incomplete. In addition, the recovery varied from sample to sample, and especially from patient to patient. The recovery usually ranged from 5 5 % to 75%. Therefore, all values for

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5-HIAA were calculated so as to compensate for the varied recoveries obtained, and expressed in mg of 5-HIAA per 24 h and mg of 5-HIAA per g of creatinine. The placebo control values were determined before reserpine medication, except for one group of patients, in which these values were measured 10 weeks after cessation of the first course of reserpine. Practically all values were obtained from samples collected on successive days. The patients were placed on a course of reserpine for at least 1 week; then the medication was discontinued. Five to 9 weeks after cessation of medication, the patients received another course of reserpine. In all cases, 2 mg of reserpine was administered twice daily to each patient. When not on medication, the patients were always kept on placebo. Both reserpine and placebo were given by mouth. RESULTS

Expt. No. 1 . One group of experimental subjects consisted of 6 schizophrenic patients (1 simple, 1 hebephrenic, 1 catatonic, 3 paranoid). An increase of 5-HIAA

excretion of almost 25% over placebo control levels was noted on the first day of medication during the first experimental trea ment with reserpine. The excretion of 5-HIAA after the first day showed no significant change from the control values. There was again a marked rise on the first day of medication when reserpine treatment was repeated. This rise amounted to more than 30 %. After the first day the excretion of 5-HIAA diminished, but still remained slightly above the placebo level. Following cessation of the latter course of reserpine the excretion of 5-HIAA remained at the control level. However, on the 8th day a large increase occurred. Expt. No. 2. In this experiment a different group of 5 schizophrenic patients was used (3 simple, 1 hebephrenic, 1 hebephrenic-paranoid). During the first course of reserpine there was a rise of approximately 30% in the excretion of 5-HIAA on the first day of medication. After cessation of medication, the excretion of 5-HIAA fell below the placebo control level. The greatest reduction amounted to 18 % calculated on the basis of 24-h excretion, and 27 % calculated on the basis of creatinine excretion. During the second course of reserpine, there was an even larger increase in the excretion of 5-HIAA on the first day of medication. This rise amounted to 45 % calculated on the basis of 24-h excretion, and to 70% calculated on the basis of creatinine excretion. 5-HIAA excretion again fell to placebo control levels or less after the first day of medication and after cessation of medication. Expt. No. 3. For this experiment 6 mentally defective patients were used. In this group excretion of 5-HIAA on the first day of initial reserpine administration rose 52 % calculated on the basis of 24-h excretion, and 37 % calculated on the basis of creatinine excretion. A fall in 5-HIAA excretion was again noted after cessation of medication, the greatest fall amounting to 25% calculated either way. During the second course of reserpine the percentage rise in the excretion of 5-HIAA on the first day of medication was practically the same as during the first course. After the first day, and after cessation of reserpine administration, the excretion of the serotonin metabolite again reached placebo levels or less. References p. 168

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t

RESERPINE

PLACEBO

zmg

CONTROL

0 I

AFTER CESSATION OF RESERPINE

8 I D.

2

3

DAY

I

2

7-8

Fig. 1 . Average daily urinary excretion of 5-HIAA in schizophrenic and mentally defective patients on reserpine, 2 mg orally b.i.d., and on placebo before and after administration of reserpine. Summary of data from all experiments (see text).

Fig. 1 summarizes the results of these 3 experiments. Tt can be seen that the most pronounced effect of reserpine on the urinary excretion of 5-HIAA in these patients is the marked rise on the very first day of medication. This increase averaged 36% and occurred in almost all patients. Although this first-day response to reserpine varied over a relatively wide range, the increase was 30%-70% in nearly two-thirds of the urine samples. Other experiments. An attempt was made to pinpoint the increase in the excretion of 5-HIAA by collecting the 24-h urine in several fractions. However, the results obtained in a pilot experiment were inconclusive because of the much greater variation in the daily excretion of 5-HIAA occurring within the shorter collection periods. A study of the effect of other tranquilizers, especially the phenothiazine derivatives, on the excretion of 5-HIAA in these patients was also considered. However, some preliminary uses of chlorpromazine brought out an unforeseen difficulty. It was found that when the urine of 1 patient placed on chlorpromazine was extracted and assayed for 5-HIAA, the recovery of authentic 5-HIAA added to the urine was lowered markedly. This was confirmed by placing 4 additional patients on chlorpromazine. The recovery of added 5-HIAA ranged from 6 % to 3 5 % and averaged 17%. This is quite low when compared with the usual recovery of 55 ”/, to 75 %. Thus, low values for urinary 5-HIAA of chlorproniazine-treated subjects will be noted when using Udenfriend’s procedure. In its present form, therefore, this procedure cannot be used to determine whethei or not chlorpromazine has any effect on the urinary excretion of 5-HIA A. DISCUSSION

From the data presented it is evident that the most consistent effect of reserpine administration on the urinary excretion of the serotonin metabolite in these patients

THE EXCRETION OF

5-HYDROXYINDOLEACETIC A C I D

167

was a large rise on the first day of medication. This reflects an effect of reserpine on serotonin itself. From the work of others (Brodie et al., 1957) it is logical to conclude that this effect consists in the displacement of serotonin from its binding sites by reserpine and an impairment of the binding sites, resulting in a greatly diminished capacity to bind serotonin. This excess free serotonin then is susceptible to iapid conversion to 5-HIAA by amine oxidase. After this initial effect, the synthesis and breakdown of serotonin in the body apparently reach equilibrium. After medication is stopped, serotonin is taken up again by its binding sites. However, this retention is too slow to be detected by measuring the urinary excretion of 5-HIAA. I t is impossible to estimate by the experimental procedures used how much, if any, of the excess urinary 5-HIAA reflects brain serotonin. Larger amounts of serotonin are present in the intestine and the blood, and these sites are known to be reduced in serotonin content in animals when sufficient reserpine (Pletscher et a/., 1955; Haverback et a/., 1956) is administered. However, since serotonin from the brain of animals can also be displaced (Haverback et al., 1956, 1957; Pletscher et al., 1956) perhaps some of the excess urinary 5-HIAA does reflect brain serotonin. This seems especially likely since, in animals, brain serotonin is more sensitive to reserpine than the serotonin present in the largest depot, the intestine (Haverback et a/., 1956, 1957; Pletscher et a/., 1956). The rise in the excretion of 5-HIAA on the first day of reserpine medication was greater in the mentally defective patients than in the schizophrenic patients. However, sufficient data are not available to determine whether this interesting observation is significant. It is tempting to relate these results to the clinical effects of reserpine. It has been proposed that the tranquilizing effect of reserpine is mediated through serotonin (Pletscher et a/., 1955). However, reserpine must not exert its behavioral effect in humans through the initial release of serotonin, since this effect of the drug usually does not appear until several days after oral medication is initiated (Kline, 1956). The problem of how serotonin fits into the function of the nervous system is under intensive study in several laboratories. From this work it can be said that the change observed in the excretion of 5-HIAA reflects an effect of reserpine on the metabolism of serotonin in man. SUMMARY

Reserpine, 2 mg twice daily, was administered to 11 schizophrenic patients and to 6 high-grade mentally defective patients. The excretion of 5-HIAA was increased on the first day of medication in both groups of patients. The excretion of the serotonin metabolite after the first day of medication and after its cessation was reduced to premedication levels or less. When these patients were given another course of reserpine at a later date, the excretory pattern of 5-HIAA was essentially the same as during the first course. The most consistent feature of the experiments was the marked rise in the excretion of the serotonin metabolite on the first day of reserpine medication. RefrrcncpJ p . 168

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A . J. V A L C O U R T

REFERENCES BRODIE,B. B., PLETSCHER, A., AND SHORE,P. A., (1955);Evidence that serotonin has a role in brain function. Science, 122,968. BRODIE,B. B., SHORE,P. A., AND PLETSCHER, A., (1956); Serotonin-releasing activity limited to rauwolfia alkaloids with tranquilizing action. Science, 123, 992-993. BRODIE,B. B., TOMICH,E. G., K u N r m A N , R., A N D SHORE,P. A,, (1957);On the mechanism of action of reserpine: effect of reserpine on capacity of tissues to bind serotonin. J . Pharnracol. exp. Ther., 119,461467. HAVERBACK, B. J., DUTCHER, T. F.,SHORE,P. A,, TOMICH,E. G., TERRY, L.L., AND BRODIE,B. B., (1957);Serotonin changes in platelets and brain induced by small daily doses of reserpine: lack of effect of depletion of platelet serotonin on hemostatic mechanisms. New Engl. J . Mcd., 256, 343-345. HAVERHACK, B. J., SHORE,P. A., TOMICH,E. G., AND BRODIE,B. B., (1956); Cumulative effect of small doses of reserpine on serotonin in man. Fed. Proc., 15, 434-435. KLINE,N. S.,(1956); Clinical applications of reserpine. Psychopharmacology. N. S. Kline, Editor. Publication No. 42. Washington, D. C., American Association for the Advancement of Science (pp. 81-109). PLETSCHER, A., SHORE,P. A., A N D BRODIE, B. B., (1955); Serotonin release as a possible mechanism of reserpine action. Science, 122, 374-375. PLETSCHER, A., SHORE,P. A., AND BRODIE,B. B., (1956);Release of brain serotonin by reserpine. J . Pharmacol. exp. Ther., 116, 46. SHORE,P. A., SILVER, S. L., AND BRODIE,B. B., (1955); Interaction of reserpine, serotonin, and lysergic acid diethylamide in brain. Science, 122, 284-285. UDENFRIEND, S.,TITUS,E., AND WEISSBACH, H., (1955); Identification of 5-hydroxy-3-indoleacetic acid in normal urine and a method for its assay. J. biol. Chem., 216, 499-505. WOOLLEY, D.W., (1957);Manipulation of cerebral serotonin and its relationship to mental disorders. Science, 125,752.

169

Some Motor and Electrical Signs of Drug Action R I C H A R D P. W H I T E Department of Pharmacology, University of Tennessee Medical Units, Memphis, Tenn. ( U.S.A.)

D I S S O C I A T I O N O F E V O K E D R E S P O N S E S IN T H E M I D B R A I N R E T I C U L A R FORMATION A N D THE ELECTROENCEPHALOGRAMS BY D R U G S

In this preliminary study, 21 unanesthetized curarized albino rabbits were employed and all surgical procedures were performed under local anesthesia to avoid prior administration of CNS depressants. Bipolar electroencephalograms were obtained from the right motor and left surface areas of the cortex. Evoked potentials in the midbrain reticular formation were produced by single shocks of 0.1 msec duration and near maximal strength applied to the contralateral sciatic nerve. These were recorded by means of a dual beam cathode-ray oscilloscope and attached camera. The position of the electrode was verified anatomically. The single shocks were applied at random intervals before and after drug administration so that about 30 responses were noted before and following each agent studied. Normal body temperature was maintained with a heating pad and the blood pressure carefully assessed for possible effects on the electrographic recordings. The EEG deactivator drugs studied were pentobarbital, chlorpromazine and atropine; the EEG activators were metrazol, d-amphetamine, and physostigmine. The results with the EEG deactivators (Fig. 1 and Table I) generally concur with others in that (1) atropine produces EEG synchrony without affecting or may even increase the single shock potentials (Loeb et al., 1960), (2) chlorpromazine produces EEG deactivation and augments the evoked response (cf. De Maar et al., 1958), and (3) low doses of barbiturates may attenuate (pentobarbital: De Maar et al., 1958) or augment (thiopental : King. 1956; pentobarbital : Longo and Silvestrini, 1958) midbrain evoked potentials while producing EEG synchrony, whereas high doses can abolish the evoked response (cf. Arduini and Arduini, 1954). In contrast to pentobarbital, it may be seen that even enormous doses of atropine fail to reduce the evoked potential (Fig. I). It also became evident that 10 mg/kg metrazol (two animals) caused an EEG activation without appreciably changing the evoked response (see also Arduini and Arduini, 1954), that amphetamine (Fig. 1) usually produced effects like metrazol but that physostigmine alone always abolished the evoked potential and induced EEG activation References p. 181-183

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Fig. I . This figure shows how the electroencephalographic and midbrain evoked responses studied are affected differently by different drugs or different doses of the same drug. Tracing A illustrates (from left to right) the EEG and the midbrain evoked response before drugs (control), after 5 rng/kg of pentobarbital, following 0.1 mg/kg of physostigmine, and subsequent to2mg/kg of atropine. Note that theeffect of physostigmine (3rd excerpt) is diametrically opposite to that of low doses of pentobarbital. It should also beemphasized that atropine promptly reversed these effects of physostigmine and if given in the dose shown prior to 0.1 mg/kg physoqtigmine will prevent the latter drug from rnanifesting these actions. Tracing B shows how the electrographic effects of chlorpromazine (5 mg/kg) are completely antagonized by 0.1 mg/kg of physostigmine and that 20 mg/kg of atropine readily reversed these actions of physostigmine but has no greater effect than 2 mg/kg of atropine (compare 4th excerpts of A and B as well as the respective control records). TracingC illustrates again how sedative doses of pentobarbital ( 5 mg/kg) markedly change the EEG without suppressing the evoked potential and further how amphetamine can antagonize the EEG effects of pentobarbital without significantlychanging the evoked response (3rd excerpt). Moreover, that additional dosage of pentobarbital (15 mg/kg) again inducesEEG synchrony but also flattens the evoked potential is shown in the 4th excerpt. Note that the action of hypnotic doses of pentobarbital is not shared by high doses of atropine (above record). Tracing D reveals how amphetamine ( I mg/kg) can produce EEG activation without suppressing the evoked potential, whereas physostigmine given subsequently (3rd excerpt) produced no further EEG change but flattened the evoked response. Lastly, I mg/kg of atropine (4th excerpt) reversed the combined actions of amphetamine and physostigmine.

(Fig. 1). Hence, these data emphasize a marked dissociation between EEG patterns and the single shock responses. Such a ‘dissociation’ is further revealed by additional reports. Scopolamine (0.025-1 mg/kg) produces EEG synchrony and fails to alter significantly the midbrain evoked response (Longo and Silvestrini, 1958). This agent, therefore, resembles atropine. Arduini and Arduini (1 954) reported that low doses of chloralosane augment single shock responses, whereas high doses depress the same. Parenthetically, these investigators found that strychnine exerted effects similar to metrazol (Table I). It is also

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TABLE I D R U G EFFECTS O N EVOKED RESPONSE I N M I D B R A I N R E T I C U L A R

SUBSTANCE AND ON THE

EEG

I N RABBITS

Zero refers to no change, plus to augmentation, minus to decrease and an x to abolishment of evoked responses in midbrain reticular formation.

Drug

Dose mdkg

EEG

Evoked response

Report of others (see text)

A . EEG deactivators:

Ether anesthesia Chloralosane Thiopental Pentobarbital Chlorpromazine Scopolamine Atropine

0.025-1 .O 0.54 10-20

Synchrony Synchrony Synchrony Synchrony Synchrony Synchrony Synchrony Synchrony Synchrony Synchrony Synchrony Synchrony

0.1 1-2 1-15 10 0.12

Activation Activation Activation Activation ?

15-35 50-60 5 10-20 5-10 15-20 45 5-10

0,

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B. EEG activators: Physostigmine d-Amphetamine Caffeine Metrazol Strychnine

X

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interesting that the onset of drug action as revealed on the EEG and single shock potentials are often widely separated (French et al., 1953; Longo and Silvestrini, 1958). That thalamic electrostimulation thresholds for behavioral arousal are higher than for EEG activation following 1-4 mg/kg chlorpromazine in cats (Killam, 1957) indicates another type of ‘dissociation’ which contrasts with that produced by atropine (Wikler, 1952) where dogs appear behaviorally alert - though not normal - and the brain waves are synchronous. The apparent independent variation in these two electrographic effects of drugs lead Longo and Silvestrini (1958) to express the view that the anesthetic state is not necessarily related to alteration of the reticular response to single sensory stimulation; nor, for that matter, slowing of the EEG activity or blockade of the EEG alerting response. Our results with physostigmine further emphasize that abolition of the single shock response is not the characteristic of the hypnotic or anesthetic state since animals given 0.1 mg/kg of physostigmine intravenously are indeed behaviorally alert. Lastly, electrophysiological studies reveal that cortical evoked responses may be abolished in motor cortex but augmented in somatosensory cortex simultaneously References p. 181-183

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R. P. W H I T E

during EEG arousal (Frommer and Livingston, 1963) indicating another form of ‘dissociation’. The results of our drug study compared with those of others are shown in Table I. Preliminary experiments performed with EEG antagonists (Group A and B, Table I) also emphasize the dissociation of the EEG and the shock response. The EEG synchrony produced by 5 mg/kg of pentobarbital is readily antagonized by 1 mg/kg of amphetamine, but the midbrain evoked responses continue in a manner similar to that seen when either agent is given alone (Fig. 1). Furthermore, physostigmine in doses capable of antagonizing the EEG effects of atropine will also abolish the evoked response. In this regard, physostigmine appears the perfect antagonist to atropine since amphetamine or metrazol are without this characteristic. Conversely, atropine will readily reverse the flatten evoked recordings and EEG activation produced by physostigmine (Fig. 1) and, moreover, in high doses (10-20 mg/kg) will block both effects of even 0.5 mg/kg of physostigmine. Despite the incongruousness of such findings, several gross inferences may be derived from these data and current knowledge of drug action. The similarity between low doses of pentobarbital and atropine may be related to the sedative effects of low doses of atropine demonstrable in some laboratory animals (White et al., 1961 ) and humans (White et a/., 1956; Ostfeld et al., 1960). Also, the electrographic effects produced herein with low doses of pentobarbital and atropine were antagonized by physostigmine. Moreover, in contrast to the other CNS stimulants, physostigmine completely antagonized both electrophysiological signs of CNS depressants induced at low dosage levels (e.g. pentobarbital) or which are apparently inherent in their action (e.g. chlorpromazine) even at high dosage. This finding might help explain why physostigmine (in contrast to amphetamine) is such a marked EEG antagonist to many CNS depressants (White, 1963). Parenthetically, it seems questionable that physostigmine produces such antagonism by reversing brain acetylcholine changes induced by drugs, since pentobarbital increases and atropine decreases acetylcholine levels, but when given in combination they cause an inordinate rise (Giarman and Pepeu, 1962). Also, McLennan and Elliott (1951) reported that pentobarbital will decrease by 50 % brain acetylcholine synthesis in vivo so that further studies are indicated before biochemical correlations with the electrographic phenomena recorded are possible. Another general characteristic of the drugs on Table I which may hold true in future analysis is that only those agents which markedly reduce evoked midbrain reticular responses and produce simultaneously EEG synchrony are true anesthetics or hypnotics (Table I, Fig. 1). Although atropine, for instance, can produce hypnosis or coma in several laboratory animals (Wikler, 1952; White et a]., 1961 ; Vernier and Unna, 1956) and humans (Forrer, 1951) in high doses, this action is delayed and atropine is not considered an anesthetic nor is its primary action even in high doses hypnosis. Hence, high doses of atropine and pentobarbital will initially produce EEG synchrony but only the latter conspicuously affects the midbrain evoked response (Table I, Fig. 1). Even in this regard, however, this classification of hypnotics or anesthetics apparently cannot be absolute since Longo and Silvestrini (1958) demon-

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strated that scopolamine prevents the effects on the midbrain evoked potentials, but not the anesthetic state, produced by high doses of pentobarbital. Among the EEG activators it is apparent that physostigmine differs from amphetamine, metrazol, or caffeine (Jouvet et al., 1957) in reversibly abolishing the evoked response (Table I, Fig. 1). Even whcn supramaximal stimuli are employed no evoked potential is elicited. This may be due to the multifarious CNS sites of action of anticholinesterases (cf. Essig e l al., 1950; White and Himwich, 1956) causing extra-reticular stimuli to produce suppression of the reticular evoked response. A presumably related inhibition of evoked response has been reported by Bach-y-Rita et al. (1960) and especially by Desmedt and La Grutta (1957) to be induced by physostigmine in the cerebral cortex. The disappearance of the sciatic-to-brain stem response caused by physostigmine is so complete and predictable it resembles that obtained by high frequency excitation of afferent nerve. Whether physostigmine and amphetamine manifest fundamental differences on the midbrain evoked response at all dose ranges or following surgical isolation of the prosencephalon and cerebellum from the mesencephalon remains to be seen. Whether the EEG synchrony and hypnosis induced by naturally occurring compounds like fatty acids (White and Samson, 1956; Holmquist and Ingvar, 1957) and other drugs can be grouped according to the above scheme into sedatives, hypnotics, anticholinesterases, etc., also awaits further study. Regardless of the ultimate outcome of such studies, the data presented indicate ( I ) drugs inducing either EEG activation or deactivation may have entirely different effects on single shock responses recorded in the midbrain reticular formation and ( 2 ) studies with pharmacological synergists and antagonists, employing a wide dosage range, on such electrophysiological phenomena may provide a better means of comparing and classifying drugs. COMPARISON OF CENTRAL ATROPINE-LIKE

CHARACTERISTICS OF D R U G S

Rinaldi and Himwich (1955) were the first to demonstrate in the ‘cerveau isolC’ preparation of Bremer (1935) an EEG antagonism between atropine and several cholinergic comnounds. Also demonstrated was the fact that atropine could block the EEG activation caused by electrostimulation of the midbrain reticular formation and that many antiparkinson agents shared this blockade (Rinaldi and Himwich, 1955b). However, the use of the EEG in this manner failed to reveal differences between several closely related compounds such as diethazine (an antiparkinson agent) and chlorpromazine (an ataractic agent) (Rinaldi and Himwich, 1955b; White and Westerbeke, 1961). The author and associates (White and Boyajy, 1960; White and Westerbeke, 1961 ; White and Carlton, 1963) have recently attempted to compare potencies of compounds which may display central atropine-like properties. One approach is to administer intravenously the series of drugs in question in minimal doses to ascertain their effects o n : (1) the spontaneous EEG, (2) the EEG alerting response evoked by several exteroceptive stimuli or by direct stimulation of the midbrain reticular formation, (3) the EEG activation produced by d-amphetamine or other long-acting central adrenerReferences p . 181-183

R. P. W H I T E

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Fig. 2. This figure illustratcs how piperidyl benzilates can induce EEG synchrony and abolish the EEG efl'ects of noise in rabbits but that only JB 329 and JB 318 (tracings 1 and 2 respectively) blocked the EEG activation pattern of physostigmine. As suggested in tracing 1 and 2, JB 329 was more potent in this regard than was JB 318. It should also be emphasized that the EEG effects of amphetamine, in contrast, were blocked by all of these compounds. Note also the low dosage of JB 329 and JB 318 employed compared to the remaining JB compounds. Numbers below records are milligrams per kilogram injected. The blood pressure record below tracing number 4 was obtained simultaneously from the same animal and was selected to illustrate that even JB 340 which exerted the greatest transient depressor action could be administered so as to avoid persistent effects. Leads: right occipital cortex (RO). Blood pressure scale should have read 150 and 50 at extremes. For further details see White and Carlton, 1963.

gic agents and (4) the activation induced by physostigmine or other anticholinesterases. The dose of the drugs in question is then increased progressively, usually until near lethal doses are employed, and their effect noted on the test battery in separate animals. It should also be mentioned that many EEG deactivators (e.g. atropine and pentobarbital) in low doses fail to block the EEG activation of a variety of compounds such as amphetamine or physostigmine, but in slightly higher doses an obvious difference in blockade appears. Hence, it is preferable to use a variety of compounds and dosages in order to ascertain differences among groups of compounds. In addition to this comparison in potency, the duration of drug action is observed and experiments are performed which indicate that the antagonistic drugs studied exert their actions centrally. It is important that the sequence and time interval between drug administration be standardized (White, 1963) with the EEG deactivator given first. For instance, chlorpromazine or atropine (Steiner and Himwich, 1962), methaminoazepoxide or pentobarbital (White and Carlton, unpublished data) will shorten the

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duration of action of physostigmine (i.e. as the effect of physostigmine is waning) but atropine will unequivocally prevent the EEG effects of cholinergic drugs (Bradley and Elkes, 1957; Rinaldi and Himwich, 1955; Steiner and Himwich, 1962; White and Boyajy, 1959) whereas chlorpromazine will not (Bradley and Hance, 1957; Steiner and Himwich, 1962; White and Boyajy, 1959; White and Westerbeke, 1961), indicating a marked difference in potency relative to atropine. Indeed, since chlorpromazine may produce pseudoparkinsonism (which atropine ameliorates), miosis, adrenergic blockade, inhibition of cholinesterase (Johannesson and Lausen, 1961) and other actions, it is apparent that several drugs should be compared under a wide dose range and against different pharmacodynamic antagonists. A paramount effect of atropine and atropine-like compounds on such a battery of tests is their ability to abolish the EEG effects of anticholinesterases like physostigmine. For example, promazine, chlorpromazine, trifluoperazine, proclorperazine, thioridazine, ethopropazine, and atropine in adequate dosage will significantly elevate the dose of d-amphetamine capable of producing EEG activation, but only the latter two drugs - both interestingly antiparkinson agents - will block the EEG effects of 0.1-0.2 mg/kg of physostigmine. Fig. 2 shows one such comparison with piperidyl benzilates and physostigmine. Note that in adequate dosage all of the JB compounds produced similar EEG effects but only two blocked the EEG actions of physostigmine. Another property of atropine (2 mg/kg) which could be added to this series of tests ascertaining atropine-like characteristics is its ability to block the EEG activation of a wide variety of drugs, presumably because of an important cholinergic link in the alerting mechanism (cf. White, 1963). In contrast, many drugs such as phenoxybenzamine and chlorpromazine preferentially inhibit the EEG actions of adrenergic agents (Goldstein and Muiioz, 1961; Muiioz and Goldstein, 1961). The main results obtained to date with this method employing many drugs over a wide dose range which may be related to clinical actions suggest: (1) compounds with a high order of atropine-like characteristics possess proven efficacy in treating Parkinsonism and (2) those anticholinergic piperidyl benzilates with potent central atropinelike properties are reportedly psychotomimetic (White and Carlton, 1963). Hence, differences between anticholinergic psychodysleptics and antiparkinson agents are not discernible by this method, nor clearly by other laboratory methods. The method only suggests whether a compound is atropine-like in potency and not the clinical actions of such compounds. That these two findings may be related, however, is indicated by the report of Pfeiffer (1959) that compounds presently used for the therapy of Parkinsonism in adequate dosage are without exception capable of producing effects which subjects liken to LSD-25. Since the more potent atropine-like compounds may be classified as non-hypnotic EEG deactivators, it is possible that some psychic energizers like imipramine possess slight atropine-like properties which may account for their ability to induce EEG synchrony experimentally without hypnosis, as the work of BeneSovA et al. (1962) indicates. In their experiment 5 mg/kg imipramine given prior to physostigmine (0.1 mg/kg) markedly dampened the EEG activation of the latter and, in this regard, it may be mentioned that this type of EEG finding can be obtained with 0.5 mg/kg or less Referenccs p . 181-183

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of atropine. Although atropine produces numerous neuropharmacological effects indistinguishable from many CNS depressants (cf. White, 1963), its outstanding action in blocking the EEG activation of cholinergic agents seemingly makes this type of drug antagonism a basis for comparing atropine-like potencies of various drugs, provided suitable dose-response studies are employed. Such gross findings suggcst that the EEG alerting mechanism contains cholinoreceptive neurons which are important links in controlling this mechanism. Thus, with perhaps the exception of several hydroxybenzencs (White and Nash, 1963), atropine is able to block the EEG activation produced by cholinergic and non-cholinergic drugs, physiological stimuli, and direct electrostimulation of the midbrain reticular formaWhite, 1963). Conversely, anticholinesterases like physostigmine can induce tion (~f. EEG activation in animals given a wide variety of drugs as pentobarbital and phenoxybenzamine which produce EEG synchronization, but the central site of action of such drugs is still in question ((5White, 1963) and apparently difficult to determine (see Discussion). E X C I T A T I O N O F T H E N U C L E U S C A U D A T U S W IT H P H A R M A C O D Y N A M I C AGENTS

The early findings of Ferrier ( I 886) that electrostimulation of the caudate nucleus produced contraversive body movements was criticized by many subsequent investigators and the predominant view held until recently by most neurophysiologists was that the caudate nucleus was principally inhibitory in function ((5Fulton, 1949). The primary objection being that electrical stimulation spread to adjacent structures (i.e. internal capsule) and destruction of the caudate, which produced effects opposite to electrostimulation, likewise damaged structures concerned with body movements. Recently, evidence was obtained in unanesthetized cats (Hassler, 1956) and rabbits (White and Himwich, 1956) which supported Ferrier’s observation. Forman and Ward (1957) in a careful study showed by physiological technics i n cats that these contraversive movements were independent of corticospinal systems, as did Laursen (1962). White and Himwich (1957b) evoked contraversive circus movements in rabbits by electrostimulation which could not be imitated by such stimulation of cortical gray or white matter. Furthermore, ablation of one caudate usually produced ipsiversive movements (cc White and Himwich, 1957a; Mettler and Mettler, 1942). Such neurophysiological studies, together with many others (cJ Jung and Hassler, 1960), indicated that unilateral electrostimulation of the nucleus caudatus can evoke body movement$ which are diametrically opposite to extirpation. Intracerebral injections of pharmacodynamic agents also indicate that unilateral stimulation of the caudate nucleus can provoke contraversive body movements where the problem of current spread or significant brain damage is avoided. In small volumes diisopropylfluorophosphate (DFP) produced such an effect even after chronic decortication. Furthermore, no contraversive movements occurred with single or multiple injections into cortical gray or white matter, into the internal capsule or following the ablation of the caudate nucleus in rabbits (White and Himwich, 1957a). Additional

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analysis indicated that no significant spread of the DFP resulted under these conditions and that cholinesterase activity is reduced 60 % or more in the injected caudate (White, 1956). However, the fact that mecholyl or metrazol also evoke contraversive movements with unilateral injection revealed that agents other than anticholinesterases may produce such behavior (White and Himwich, 1957b). Similar results obtained in cats with mecholyl (methacholine), carbachol and acetylcholine injections (Stevens and MacLean, 1961) demonstrate that this pharmacodynamic action was not species dependent. These results, together with ablation studies on rabbits manifesting circus movements following unilateral intracarotid injections of DFP (White and Himwich, 1957a), support the inference of Essig ef al. (1950) that unilateral stimulation of the caudate nucleus is of paramount importance in the adversive movements induced by intracarotid injections of DFP i n monkeys. It should be emphasized that caudate stimulation affects learning, inhibits certain body movements, causes EEG activation and other manifold effects (cJJung and Hassler, 1960; Stevens and MacLean, 1961; Laursen, 1962) indicating further that this conspicuous structure plays an important function in many CNS activities beyond that of simple inhibition. DISCUSSION

Many authors have stressed differences in the behavioral and EEG effects produced by drugs and some inferred that only when a certain behavior is associated with a given EEG pattern the relationship is significant. The excellent correlation between the EEG activation and hyperactive behavior produced by amphetamine, in contrast with physostigmine, prompted several investigators to the hypothesis that the reticular formation was the site of action for adrenergic agents (Bradley and Elkes, 1957). This latter view was emphasized by Rothballer ( I 956) who stated, ‘cholinergic potentiating and blocking agents (physostigmine, atropine) produce EEG effects with the conspicuous absence of any behavioral counterpart, i.e. dissociation’. However, many studies suggest that drugs affect behavior differently in degree and kind than in the manner of classical CNS stimulants and depressants. Killam (1957) showed that chlorpromazine elevated thalamic electrostimulation thresholds more for behavioral than for EEG arousal. The experiments of Longo (1956) emphasize that drugs can alter corticipetal activity more than gross centrifugal activity. In this regard, the behavioral effect induced by high doses of atropine or scopolamine resembles decortication in dogs (White et al., 1961). Moreover, mild (e.g. touch) and strong stimuli produce similar EEG, but dissimilar behavioral effects, suggesting that drugs could change the EEG pattern of animals without greatly changing overt behavior. Indeed, physostigmine produces electrophysiological effects on both neocortex and hippocampus which more closely resemble that produced by electrostimulation of the reticular formation than does methamphetamine (Briicke and Stumpf, 1957). In addition to producing similar EEG effects, physostigmine or amphetamine will shorten pentobarbital sleeping time (Barnes and Meyers, 1963), and evoke contraversive body movements when injected into the carotid artery (Jarvik and Rothballer, 1959). Physostigmine alters conditioned avoidance phenomena which is antagonized by anticholinergic References p . 181-183

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compounds (Herz, 1960) and elicits ‘lucid’ moments in schizophrenic patients (Pfeiffer, 1959). Also atropine potentiates (White et af., 1961 ; Ciarman and Pepeu, 1962) the hypnotic actions of pentobarbital and the EEG effects of the latter (Bradley and Hance, 1957). These drugs also produce whining and restlessness followed by intermittent sleep in dogs (Wikler, 1952; White et a/., 1961) and marked behavioral effects in monkeys (Vernier and Unna, 1956; White et al., 1961) as well as in man (Forrer, 1951; White et al., 1956; Ostfeld et al., 1960). Such reports, together with many unmentioned, show that cholinergic and anticholinergic agents do produce conspicuous behavioral effects and suggest that any drug which imposes a particular ‘regulated’ EEG pattern can impose significant behavioral effects. Much data have been advanced to support hypotheses that the EEG alerting mechanism contains cholinoreceptive, adrenoreceptive or, generally, non-cholinoreceptive neurons. Direct recordings from neurons within the mesencephalic tegmentum clearly show that eserine markedly increases unit activity (Desmedt and Schlag, 1957) and that acetylcholine produces variable effects as did epinephrine (Bradley, 1957). That this activity may be related to EEG activation was suggested by previous experiments of Rinaldi and Himwich (19554 who demonstrated that atropine could block the EEG activation caused by several cholinergic drugs in the ‘cerveau isole’ preparation. They concluded that the activation mechanism is fundamentally cholinergic in nature. From similar experiments Hiebel et al. (1954) stressed the importance of adrenergic mechanism in EEG activation. Goldstein and Muiioz (1 961) inferred from experiments using adrenergic blocking agents such as chlorpromazine and phenoxybenzamine that a- and p-receptors are involved in this mechanism. Rothballer (1957) showed in cats that cocaine potentiated the EEG activation caused by adrenergic drugs and showed further (Rothballer, 1956) that as the midbrain was destroyed progressively rostra1 to the ‘cerveau isole’ lesion the activation by adrenergic agents became progressively less, i.e., higher doses were required. Although no cholinergic agents were given in these experiments, he expressed the belief that if cholinoreceptive neurons exist in the alerting mechanism they were probably in the diencephalon or above. Bradley and Elkes (19571, however, reported that amphetamine did not produce activation in ‘cerveau isolC’ cats whereas physostigmine did. Longo and Silvestrini (1957) and White and Daigneault (1959) reported that both amphetamine and physostigmine caused activation in ‘cerveau isole’ rabbits indicating again that both cholinergic and adrenergic sensitive neurons were involved in the alerting mechanism in this preparation. The latter workers demonstrated that atropine blocked the activation of both these agents in this preparation, suggesting that amphetamine also acts through an atropine-sensitive mechanism lying between the lesion and the cortical recording. They further reported that destruction of the midbrain not only abolished the activation induced by amphetamine, as Rothballer ( I 956) found with other adrenergic drugs, but also that of physostigmine (0.1-0.3 mg/kg). The latter result indicated that connections between the midbrain and cortex must be intact for EEG activation by such agents, but Steiner and Himwich (1962) demonstrated in curarized rabbits that usual toxic doses of physostigmine (0.3-0.9 mg/kg) elicited EEG desynchrony in animals sectioned at the pre-collicular level, whereas 3 mg/kg of amphetamine failed to induce EEG acti-

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vation in such animals. The latter observation agrees with that of Desmedt and Franken (1957) in cats who, in addition, showed that a marked behavioral ‘alarm’ reaction occurs in decorticate animals following amphetamine but not after eserine. Killam et al. (1959) similarly found that electrolytic lesions destroying the midbrain reticular formation at the level of the oculomotor nucleus abolished EEG activation due to 1 mg/kg of amphetamine but not that of 0. I to 0.2 mg/kg of physostigmine. However, in variance with Steiner and Himwich (1962), these investigators reported that 0.4 to 0.6 mg/kg of physostigmine caused a marked depression ofcortical activity. Furthermore, in contrast to the last two reports, Hiebel et al. (1954) found that amphetamine continues to elicit EEG activation unless the plane of section was far enough rostra1 to pass dorsally through the posterior third of the thalamus, thereby severing the cephalad portions of the midbrain reticular formation with the cortex (see also discussion by Van Meter and Ayala, 1961). In view of Rothballer’s (1956, 1957) earlier findings, it would be interesting to repeat such experiments using inordinately high doses of adrenergic agents, perhaps in combination with the potentiator cocaine, to see if EEG arousal is possible in ‘high’ decerebrate animals with adrenergic drugs. Interestingly, caffeine (1 5 mg/kg) but not d-amphetamine ( 5 mg/kg) will induce EEG activation in ‘high’ decerebrate animals (Jouvet et al., 1957) so that such activation is not apparently limited to purely cholinergic drugs. Regardless, the present data indicate that both cholinergic and adrenergic drugs produce EEG activation in ‘cerveau isolC’ preparations; that total destruction of the midbrain will either abolish or markedly reduce the ability of such drugs to produce EEG activation; that physostigmine more readily produces EEG activation than amphetamine in ‘decerebrate’ preparations, and that atropine can block activation caused by such agents (cf. White, 1963). The neuropharmacological evidence indicates that drugs can produce EEG activation by means of thalamic mechanisms (or thalamocortical circuits), independent of the midbrain reticular formation, since cholinergic agents evoke EEG activation after total transection at pre-coliicular levels (Desmedt and Franken, 1957; Steiner and Himwich, 1962) but do not cause activation in the ‘isolated hemisphere’ preparation (Rinaldi and Himwich, 1955a). Apparently caffeine can also produce EEG activation in the absence of the midbrain reticular formation (Jouvet et al., 1957). However, the electrophysiological experiments of Schlag et af. (1961) clearly indicate that thalamic stimulation will not produce EEG activation without the participation of the midbrain reticular formation. Such findings emphasize that results obtained with electrophysiological experiments may not imitate neuropharmacological results. For one, the effect of drugs given intravenously is apt to be more diffuse than electrostimulation of, or electrophysiological responses in, particular areas. Apparently drugs can also exert specific actions not matched by electrostimulation. Epinephrine, for instance, when applied to specific cortical areas evokes a rise in blood pressure by neuronal processes which could not be imitated by electrical excitation (Minz and Goldstein, 1955), and chemicals may cause synaptic discharges where electrostimulation fails (Grundfest, 1961). Conversely, drugs can have significant central actions which recordings of evoked responses may fail to reveal. Atropine will block EEG activation induced by Refrrences p (81-183

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numerous drugs (White, 1963) and direct electrostimulation of the midbrain reticular formation (Rinaldi and Himwich, 1955), but fails to block evoked potentials recorded from the cortex (Marrazzi, 1953; Bach-y-Rita et al., 1960) and other telencephalic structures (Bach-y-Rita et al., 1960) or from the midbrain reticular formation (Loeb et al., 1960). Hence, these data from evoked responses alone would suggest that atropine is inert centrally. Further experimentation, however, reveals that it can block or reverse changes produced by cholinergic drugs on evoked potentials recorded from cortex (Marrazzi, 1953; Bach-y-Rita et al., 1960), ventralis anterior (Bach-y-Rita et a]., 1960) and the midbrain reticular formation reported herein (Fig. I), indicating again that pharmacological antagonists may elucidate actions not readily apparent by electrophysiological methods alone.

SUMMARY

It is apparent from the above data and discussion that drugs can cause sensory induced evoked potentials recorded within the midbrain reticular formation to vary independent of electroencephalographic recordings. Thus, in high doses, atropine (10-20 mg/kg) or pentobarbital (15-20 mg/kg) produce EEG synchrony but only the latter drug attenuates or abolishes evoked responses. Conversely, amphetamine, metrazol, physostigmine induce EEG activation but physostigmine abolishes the evoked potential whereas the remaining two may even enhance this phenomenon. It is possible, however, that when the primary action of a drug is sedative, EEG synchrony and enhanced evoked responses prevail ;whereas, anesthetic agents abolish the latter in addition to producing EEG sleep patterns. Hence, chlorpromazine, atropine and pentobarbital in low doses produce sedation, EEG synchrony and augmented evoked potentials, but only pentobarbital in higher doses clearly abolishes the evoked response and is primarily anesthetic. Evidence has been presented indicating that an outstanding characteristic of atropine and atropine-like agents is their ability to block the EEG activation produced by cholinergic drugs. Although this finding was anticipated from many studies, the importance of employing adequate dose-response studies and numerous drugs to establish atropine-like specificity was stressed. Also emphasized was the concept that any drug which imposes a particular ‘regulated’ EEG pattern, whether adrenergic, cholinergic or otherwise, may be expected to alter significantly some important facet of behavior. Lastly, data were presented and discussed emphasizing that electrophysiological findings, though very suggestive, may not predict effects of drug antagonists nor imitate primary drug actions. Conversely, pharmacodynamic agents may often be used advantageously to confirm and further elucidate information obtained by neurophysiological technics. The effects of several drugs injected directly into one caudate nucleus, for example, support the results obtained from electrostimulation and ablation of the nucleus caudatus indicating that this structure can induce contraversive head and body movements.

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ACKNOWLEDGEMENTS

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Aspects of Amino Acid Metabolism in Phenylketonuria and Other Amino Acidopat hies H A N S H. BERLET Tl~irtlicliuniPsychiatric Research Laboratory%Calesburg Stare Research Hospital, Caleshurg, Ill. ( U . S . A . )

INTRODUCTION

Why some amino acidopathies result in cerebral dysfunction while others do not is unknown. The problem is of great importance since in amino acidopathies, in contrast to other types of mental retardation, the afflictions of the brain appear t o develop during the maturation of the central nervous system rendering an individual of potentially normal intelligence mentally disabled. It is for this reason that the search into the secondary effects of amino acids in these disorders is eminently challenging. The answers can help a small but nevertheless significant number of newborns and infants escape their otherwise almost inevitable fate of mental deterioration. Furthermore, the question of what causes mental retardation in amino acidopathies as well as, more generally speaking, what may be the common biochemical principle of dysfunctions of the central nervous system in mental diseases is still open to speculation in spite of the host of data accumulated on this matter. Indeed, it has yet to be decided whether there is a common principle at all or a specific mechanism or agent for each of the wide variety of diseases associated with amino acidopathies and which are specifically or unspeciiically accompanied by disturbances of mental functions. The literature of the past decades on the inherited metabolic disorders of amino acids has dealt predominantly with two aspects of these disorders. One aspect has been to search for metabolic disorders of amino acids and to give a comprehensive description of their clinical and biochemical features. This approach will continue to retain its validity in the future when a number of additional amino acidopathies is predicted to be found (DiGeorge and Auerbach, 1963). The second aspect was made possible by the opportunities offered by affected subjects to elucidate more clearly the normal and abnormal metabolic pathways for the degradation of amino acids in man and thus to lay the basis for our present knowledge of the metabolism of amino acids in man. Only in recent years the specific effects of amino acids upon the central nervous system have been studied. The emphasis for this work has increased since the identification of serotonin (Erspamer, 1948; Rapport et al., 1948) finally led to the rec-

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ognition of serotonin and other biogenic amines as important constituents of the brain (Twarog and Page, 1953; Amin et a/., 1954; Bogdanski et a]., 1956). Consequently the precursors of these compounds, tryptophan and phenylalanine as well as other amino acids were studied for their behavioral effects in both man (Pollin et a/., 1961; Brune and Himwich, 1962; Berlet et a/., 1964) and animals (Costa et a/., 1961; Himwich et al., 1964). Indeed the theoretical concept that an undisturbed brain serotonin level provides the basis for a normal behavior pattern (Woolley and Shaw, 1954) has proven useful in many respects even though variations in serotonin levels do not necessarily account for the impairment of mental functions in animals with a simulated disorder of amino acid metabolism (Yuwiler and Louttit, 1961). To emphasize other factors which might contribute to the mental deterioration in primary amino acidopathies some well known facts about amino acidopathies might be briefly recalled. Of the presently known amino acidopathies the following ones are accompanied either invariably or in the majority of the cases with symptoms of cerebral dysf~~nction such as mental retardation, neurological defects and psychotic traits : argininosuccinic acidemia, citrullinemia, cystathioninemia, glycinemia, histidinemia, hyperammonT A B L E Ia AMINO ACIDOPATHIES ASSOCIATED WITH MENTAL RETARDATION OR C E R E B R A L D Y S F U N C T I O N S

Amino acitiopatlty

Enzyme defect: Argininosuccinic acidemia Citrullinemia Cystathioninemia Glycinemia Histidinemia Hydroxyprolinemia Hyperammonemia Maple syrup disease Methioninemia (homocystinuria) Phenylketonuria Tryptophanuria Tyrosyluria

Transport: Prolinemia

References p. 209-215

Amino acid(s) involved

Plasma compound characterisrically elevated

Argininosuccinic acid

Argininosuccinic acid

Allan et ar., 1958

Citrullin/aspartic acid

Citrullin, histidine, lysine, methionine Cystathionine Phenylalanine, tyrosine Histidine Hydroxyproline Glutamic acid (glutamine) ammonium Leucine, isoleticine, valine Methionine

McMurray et al., 1963 Frimpter et al., 1963 Childs et a[., 1961 Ghadimi et al., 1961 Efron et al., 1962 Russell et al., 1962

Cystathionine Glycine Histidine Hydroxyproline Ornithine (carbainyl phosphate) Branched chain amino acids Methionine

Reference

Dancis et al., 1959 Gerritsen et al., 1962

Phenylalanine Tryptophan Tyrosine

Knox, 1960 Phenylalanine, histidine Tada et a/., 1963 Tryptophan Tyrosine, leucine, isoleucine, Auerbach er a/., 1963 lysine, methionine, phenylalanine, proline, threonine, valine

Proline

Proline (hydroxyproline, glycine)

Schafer et al., 1962

I86

H. H. B E R L E T

emia, maple syrup disease, methioninemia (homocystinuria), phenylketonuria, prolinemia, hydroxyprolinemia and some cases of tyrosyluria (Table Ia). Only recently, the first case of an inborn error of the metabolism of tryptophan has been added to the list of known amino acidopathies. Tada et af. (1963) reported on a child with mental defects whose fasting plasma tryptophan level was slightly but significantly elevated as compared to normal controls. The nomenclature for primary acidopathies is adopted from DiGeorge and Auerbach (1963) who classify amino acidopathies according to the elevation of plasma amino acids thus eliminating amino acid disorders which might be merely due to failures of intestinal uptake or renal reabsorption of amino acids. In contrast the following amino acidopathies are not accompanied by mental symptoms, or if so, as in some cases of Hartnup disease (Baron et ul., 1956; Hersov and Rodnight, 1960) in a reversible manner or without a relationship to the coexisting disorder of amino acid metabolism: albinism, alcaptonuria, a defect in the thyroid hormone synthesis, oxalosis, tyrosinosis and Hartnup disease, the latter being the result of a renal dysfunction and failure of intestinal absorption of tryptophan, rather than a disorder of amino acid metabolism. The Tables Ia and Ib show that T A B L E Ib A M I N O A C I D O P A T H I E S NOT A S S O C I A T E D W I T H M E N T A L R E T A R D A T I O N OR C E R E B R A L D Y S F U N C T I O N S

Amino acidopathy Enzyme defect: Alkaptonuria Albinism Defect of thyroid hormone synthesis Oxalosis

Tyrosinosis Transport : Hartnup disease

Amino acid(s) involved

Plasma compound characteristically elevated

Tyrosine Tyrosine Mono- and diiodoMonoiodotyrosine, tyrosine diiodotyrosine Glycine (glyoxylic acid) Oxalate Tyrosine

p-Phenylpyruvic acid (tyrosine?)

Tryptophan

Indoles

Reference

La Du, 1960 Fitzpatrick, 1960 Stanbury, 1960 Marshall and Horwith, 1959 Medes, 1932

Evered, 1956 Hersov and Rodnight, 1960

amino acidopathies predominantly involve essential amino acids. This distinction does not, however, differentiate the amino acidopathies with mental defects from the amino acidopathies without mental defects since essential amino acids are distributed rather evenly between both groups. It is worth noting, however, that in those amino acidopathies which are accompanied by mental symptoms, the characteristically elevated compound is either a naturally occurring amino acid or another amino acid which is formed in the process of detoxicating or degrading reactions of amino

A M I N O A C I D METABOLISM I N A M I N O A C I D O P A T H I E S

187

acids. In contrast plasma compounds which are elevated in the second group without prominent mental symptoms are for the most part metabolites of the amino acid involved or as in other amino acidopathies, there may be no clear-cut elevation of any abnormal intermediate. It should not be overlooked, however, that the plasmas of the amino acidopathies of the first group do contain a number of deaminated abnormal intermediates in addition to the elevated amino acid per se. Hence the most important biochemical feature of the amino acidopathies with mental retardation appears to be the plasma elevation of amino acids. Whether there is a truly etiological relationship of this feature to the affliction of the central nervous system in amino acidopathies is unclear as yet and the cause effect relation may not exist at all. Nonetheless the phenomenon is common to all of the amino acidopathies of the first group and therefore deserves particular attention as it may offer the means to establish patterns for a common mechanism of certain abnormalities encountered in amino acidopathies. This more general point of view may be all the more useful since, disappointingly, none of the many rare or abnormal metabolites appearing in conditions with a disturbed amino acid metabolism have proven with certainty to be specifically neurotoxic for the developing or mature brain. The following discussion will therefore concentrate on two main points: (1) the interactions of one amino acid with the absorption and transport of other amino acids and (2) a discussion of the specific actions of one amino acid on the enzymatic reactions in the body concerned with the metabolism of other amino acids and the biosynthesis of important humoral constituents such as biogenic amines from amino acids. These aspects of amino acid metabolism in conditions of elevated plasma amino levels will be evaluated on the amino acid phenylalanine, predominantly with relation to tyrosine, dopa* and tryptophan. These amino acids and their mutual interactions are most thoroughly studied of all amino acids and offer therefore a good opportunity to carry out the purpose of this discussion. The role of amino acids in general, but especially that of aromatic acids in mental disorders has long been recognized and has been the subject of numerous evaluations (McGeer et al., 1957; Sprince, 1961). The following discussion of certain aspects of the metabolism of phenylalanine offers therefore an opportunity to include more recent findings on phenolic compounds which arise from alternative reactions of phenolic amino acids and are believed to be specific for mental diseases such as schizophrenias. It is not intended to give a complete review of the literature but rather to refer mainly to selected references which illustrate a pertinent point of the subject as outlined above. For a more detailed discussion of interactions between amino acids and the central nervous system the reader is referred to recent comprehensive reviews on the following aspects : primary amino acidopathies (DiGeorge and Auerbach, 1963); biogenic amines and behavior (Brune and Himwich, 1963); the brain-barrier system (Dobbing, 1961; Lajtha, 1962); development of the blood-brain barrier system (Himwich, 1962); intestinal absorption of amino acids (Wilson, 1962) ; biochemical alterations in mental disease (Sourkes, 1962). lt is hoped that the results of this study,

*

3,4-dihydroxyphenylalanine.

References p . 209-215

188

H . H. B E R L E T

eventually may become generally applicable to interactions of amino acids, provided that one also keeps in mind that certain effects of an individual amino acid are specific for certain physiological reactions and metabolic requirements and that these effects are, therefore, a priori excluded from a generalization. ALTERATIONS O F THE P H E N Y L A L A N I N E METABOLISM I N

PKU

The best known and most thoroughly investigated of all inherited metabolic disorders of amino acids is that of phenylalanine (PA), phenylketonuria (PKU). It is of particular interest because the majority of the cases exhibits mental defects and occasionally psychosis-like symptoms (Bjornson, 1964) and so offers an opportunity to correlate mental retardation with certain biochemical alterations. PKU is due to an almost complete lack of phenylalanine hydroxylase activity in the liver of the affected individuals (Jervis, 1953). This enzyme is found only in the liver and normally converts phenylalanine to tyrosine by means of a p-hydroxylation (4-position). This reaction does not take place or only to a small extent in phenylketonuria and thus leaves the major portion of the dietary phenylalanine unchanged and available for metabolism through different pathways such as transamination, decarboxylation and to some extent o-hydroxylation. This metabolic alteration gives rise to a number of phenylalanine metabolites which are normally not found in the tissues and fluids of the body in more than trace amounts. The main biochemical finding of this disease is therefore a 10-40 fold elevation of phenylalanine in plasma and urine (Folling et al., 1938; Jervis et al., 1940). A proportional increase of phenylalanine levels was also found in cerebrospinal fluid (Borek et al., 1950). A number of other related phenolic compounds arise from the accumulation of phenylalanine. 111plasma, phenylpyruvic acid is elevated as well as phenylalanine (Jervis, 1952) whereas phenylketonuric urine contains increased levels of phenylalanine, phenylpyruvic acid, phenyllactic acid (Zeller, 1943) and phenylacetic acid and phenylacetylglutamine (Woolf, 1951) as major constituents. Urinary o-hydroxyphenylacetic acid is also markedly elevated, namely, to 100-400 mg/g creatinine as compared to a normal excretion of less than 1 mg/g creatinine and appears to be more directly related to blood phenylalanine levels than phenylpyruvic acid (Boscott and Bickel, 1953; Armstrong et al., 1955). INTERACTIONS OF PHENYLALANINE WITH TRYPTOPHAN, TYROSINE, DOPA A N D OTHER AMlNO ACIDS I N EXPERIMENTAL STUDIES

Many of the biochemical interactions between PA and other products of the metabolic pathways of indoles and catecholamines in inherited phenylketonuria which will be described later have been clarified through the experimental use of phenylalanine in animal experiments. Beyond this immediate goal phenylalanine is now chiefly being used to simulate phenylketonuria and the accompanying behavioral symptoms related to phenylketonuria which infer genuine biochemical and structural damages of the central nervous system. Experimental phenylketonuria may finally bring the

AMINO ACID METABOLISM I N A M I N O ACIDOPATHIES

189

answer to the questions: what are the specific lesions of the CNS in phenylketonuria, what causes them and how do these lesions render an individual a mental defective subject? It is not too daring to hope furthermore that this type of ‘model defect’ of the mind may bring us closer not only to the recognition of a common mechanism of mental deficiencies in inborn amino acidopathies but may also lay a new basis for the investigational approach in other types of mental retardations and mental diseases. Two animal species, rats and monkeys, have been used for the simulation of PKU and production of biochemical changes typically associated with inborn phenylketonuria were obtained (Auerbach et al., 1958; Waisman et al., 1959, 1960; Yuwiler and Louttit, 1961). Rats appear to be least susceptible to a high PA diet because their hydroxylase activity is 12-16 times greater than that of man and monkeys (Waisman, 1963). Monkeys are more responsive to a high phenylalanine diet and offer the additional advantage of being easier to evaluate and to rate as to their mental status, especially when infant monkeys are used (Waisman et al., 1960, 1962). The type of diet used also plays an important role in the successful induction of phenylketonuria. In early investigations a diet high in DL-PA, or DL-PA together with DL-tyrosine was used. Tyrosine was hoped to improve the effect of DL-PA through an end product inhibition of the hydroxylation of phenylalanine. Although tyrosine did not fulfill all the expectations it is still used in combination with L-phenylalanine (Waisman et al., 1959). As the result of numerous variations of their regimen Waisman (1963) finally arrived at an optimal diet according to their experience consisting merely of L-phenylalanine. They also showed that L-phenylalanine is superior to a racemic mixture of phenylalanine in elevating the plasma PA levels

-+-

5-HYDROXYINDOLEACETIC

ACID

1

VANILMANDELIC ACID EPINEPHRINE NOREPINEPHRINE

*

5-HYDROXYTRYPTAMINE

5 -HYDROX YTRYPTOPHAN DOPAMINE

\

0-TYROSINE

1

0-TYRAMINE

\ PHENYLPYRUVIC ACID PHENYLLACTIC ACID PHENYLACETIC ACID

\

P-HYDROXYPHENYLPYRUVIC. -LACTIC, -ACETIC ACID

\ \

t

\

P-TYROSINE

\ \

Alternative Pathways Of D O P A 0-METHYLATION, T R A N SA M I NA T I 0N

0-HYDROXYPHENYLPYRUVIC ACID

0- H Y D R O X Y P H E N Y L. ACETIC ACID

MELANIN

$ ..+.

Established enzyme inhibition

P r e s u m p t i v e enzyme i n h i b i t i o n

Fig. 1. Some biochemical interactions of phenylalanine in phenylketonuria. Solid arrows designate metabolic reactions of PA. Broken arrows represent the influence of PA and PA-derivatives upon unrelated enzymic reactions. The brackets are used to implicate hypothetical metabolic pathways. References p . 209-215

190

H. H . 8 E R L E T

because the D-form of phenylalanine interferes with the intestinal absorption of L-phenylalanine resulting in an insufficiently high level of the biologically active L-phenylalanine in the plasma. L-Phenylalanine is also superior to the mixture of L-phenylalanine and L-tyrosine. The administration of /I-Zthienylalanine, an inhibitor of the phenylalanine-hydroxylase, could not induce phenylketonuric features when given together with L-PA to rats (Neil1 and Langford, 1961). W. A. Himwich and her group are presently engaged in a multidisciplined work on the production of phenylketonuria in newborn dogs which are fed L-PA from the first day of life. If this can be done successfully, the use of dogs presents some advantages over rats or monkeys. Biochemical changes due to excess plasmaphenylalanine (Fig. 1): It may be useful to differentiate between primary and secondary biochemical changes in experimental phenylketonuria. The elevation of plasma phenylalanine and the appearance of urinary phenylketones in addition to the inherited enzyme defect in phenylketonuria may be considered primary changes and represent the criteria for the successful experimental reproduction of phenylketonuria. Alterations, which arise from the accumulation of the phenylalanine in tissue, plasma and urine might be considered secondary since they are produced by the primary action of PA. In phenylketonuria plasma serotonin and 5-HIAA* and the urinary excretion of the latter compound are depressed (Pare et a/., 1957), while urinary indolepyruvic, indolelactic and indoleacetic acid as well as indican are elevated (Armstrong and Robinson, 1954). The metabolism of tyrosine, DOPA and catecholaniines is also affected in these individuals and low levels were found for plasma norepinephrine and epinephrine (Weil-Marherbe, 1955; Nadler and Hsia, 1961) as well as for the urinary excretion of dopamine, norepinephrine and epinephrine. As expected the urinary excretion of vanilmandelic acid is also low (Armstrong, 1963). The alterations of catecholamines are reversible even in phenylketonuric subjects as soon as the plasma levels of phenylalanine are restored to normal levels (Nadler and Hsia, 1961) as are also the alterations of the indole metabolism (Pare et al., 1958; Baldridge et al., 1959). Increased excretions of urinary p-hydroxyphenylpyruvic, -lactic and -acetic acid reflect the secondary effect of PA or, more specifically, of phenylpyruvic acid in phenylketonurics on the metabolism of tyrosine (Boscott and Bickel, 1953). In seeking an explanation for the secondary biochemical effects of phenylalanine the enzymatic reactions involving the indole-metabolism of tryptophan are first considered. Tryptophan metabolism: The clinically observed indole patterns in phenylketonuria can be reproduced by in vivo experiments. When rats or monkeys were given high doses of phenylalanine tissue levels of serotonin and 5-HIAA and the urinary excretion of 5-HTAA were promptly depressed (Huang et al., 1961 ;Yuwiler and Louttit, 1961; Boggs et al., 1963a) while the excretion of other indoles (e.g.indoleacetic acid) was increased. These findings indicate that phenylalanine interferes with the normal metabolism of tryptophan through the indole pathway.

*

5-Hydroxyindole-3-aceticacid.

191

A M I N O A C I D METABOLISM I N A M I N O A C I D O P A T H I E S

The depression of brain serotonin levels may be caused by an effect of phenylalanine upon the hydroxylation of tryptophan, for in contrast to previous studies (Cooper and Melcer, 1961), the hydroxylation of tryptophan was recently demonstrated in vivo for mammalian and avian brain (Gal et a/., 1963, 1964). Tn the light of this finding consideration should be also given now to plasma tryptophan levels and uptake of tryptophan by the brain in the presence of high plasma levels of phenylalanine, because low plasma levels and reduced uptake of tryptophan by the brain could account partly for alterations of brain serotonin provided that the intracerebral hydroxylation of tryptophan would be unaffected by phenylalanine. Thus there are four different ways in which PA may interfere metabolically with the endogenous distribution and breakdown of tryptophan: inhibition of 5-hydroxylation of tryptophan, an inhibition of decarboxylation of 5-HTP, a reduced intestinal absorption of tryptophan and an impairment of the uptake of tryptophan and 5-HTP by brain. Subsequently the implications of each of these possibilities will be discussed in detail (Fig. 1 and Fig. 2). CEREBROSPINAL

FLUID

Phenylalanine

4

4 I

II

SPACE

t

4 .

I

+ I

'.,

.

+---

BRAIN

9

P h e n y l a1 a n i n e Phenylethylaminet

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And \

Other

/*I

\

c-

I INDOLE

t l

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I

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COMPOUNDS

~

~

~

~

Acids

+ I

t INTESTINAL

I I I

ABSORPTION

?

4 Bacterial

Amino

ALTERED PLASMA AMINO ACID PATTERN

4

t

~

. I

RENAL OVERFLOW (AMINO ACIDS. PHENOLIC METABOLITES O F PHENYLALANINE, INDOLES)

I

Dopamine

~

TRYPTOPHAN

?

(And Other Amino Acids)

Decomposition

f+

Free exchange

.$ 4k

Inhibition of t r a n s p o r t mechanisms Increase o r decrease

Fig. 2. Some unspecificeffects of phenylalanine upon transport and absorption of other amino acids. Broken arrows represent the interaction of phenylalanine with transport and uptake mechanisms.

Hydroxylation of tryptophan to 5-hydroxytryptophan: Clinical evidence for a defective 5-hydroxylation in PKU was reported by Baldridge et al. (1959) and by Perry et a/. (1964). When the urinary excretions of serotonin and 5-HIAA following an oral tryptophan and an oral 5-HTP load respectively were compared an increase of urinary serotonin and 5-HIAA similar to that of controls was found only with the 5-HTP load. For the biosynthesis of serotonin tryptophan is normally hydroxylated in a 5position to form 5-HTP. Two different enzyme systems are at present known to catalyze this reaction. Cooper and Melcer (1961) were first able to demonstrate an enzyme References p. 209-215

~

p

h

r

i

n

e

192

H. H. B E K L E T

activity for the conversion of tryptophan to 5-HTP in cells of the mucosa of the small intestine and in kidney tissue of the guinea pig. Later the brain of pigeons and rats was found to be capable of catalyzing this reaction (Gal et al., 1963, 1964). A second system was found in liver tissue from rats and sheep (Freedland et al., 1961 ; Renson et nl., 1962). The latter one, however, is considered to be primarily a phenylalanine hydroxylase system with the ability to hydroxylate both phenylalanine and tryptophan. This system was found to be strictly confined to liver tissue (Renson et al., 1962). The hydroxylase system of liver tissue appears to be of little physiological significance for the hydroxylation of tryptophan, although L-phenylalanine exerted a competitive inhibition upon the tryptophan hydroxylation, eventually offering a simple explanation for the reduced biosynthesis of serotonin in PKU. However, the affinity of this system is so much lower for tryptophan than for phenylalanine, based on Km values of 3 x 10-3 M for tryptophan and 1 x 10-5 M for phenylalanine, that a hydroxylation of tryptophan to an appreciable extent by means of the liver system appears unlikely even under normal conditions, not to speak of PKU. The relationship between these two hydroxylations becomes even more doubtful when the restoration of normal serotonin and 5-HIAA patterns following the institution of a low phenylalanine diet in PKU is considered. The residual activity of the phenylalanine hydroxylase of 5 to 10 of normal in PKU can hardly account for normal hydroxylation of tryptophan even at normal plasma levels of phenylalanine. There remains one other possibility if one considers a defective hydroxylation of tryptophan in PKU to be the main reason for a reduced formation of serotonin and 5-HIAA, namely an interference of phenylalanine with the specific tryptophan hydroxylase activity of the gut, kidney and brain. If this system is the main physiological source of hydroxylated tryptophan the demonstration of its inhibition by phenylalanine or one of its metabolites would furnish experimental proof that hydroxylation is the cause or at least one of the causes of low serotonin levels in conditions with an elevated plasma phenylalanine level. Another question remains also unanswered, namely why there is a reduction of brain serotonin levels in spite of the recently proven presence of a tryptophan hydroxylation in the brain. 1s there also an inhibiton of this reaction in the brain by phenylalanine? Aspects of an impaired amino acid uptake by the brain will be discussed later in so far as it may explain a reduction of brain serotonin levels in the presence of a plasma elevation of phenylalanine. Tryptophan-decarboxylase (Fig. 1) : The inhibition of the hydroxylase activity cannot be the sole mechanism for the reduction of hydroxyindoles. Renson et al. (1962) and McKean et al. (1962) found that in comparison to the untreated controls, 5-hydroxytryptophan failed to yield an increase of brain serotonin when given to animals which were pretreated with phenylalanine. This finding points to an interference of phenylalanine with either the uptake of 5-HTP by the brain or the 5-HTP decarboxylase activity. The latter possibility was previously suggested in 1958 by Pare et al. who found a lower 5-HT and 5-HIAA excretion upon i.v. administration of 5-HTP in untreated phenylketonuric children as compared with matched controls. This view is supported by the in vitro finding of a strong competitive inhibition of the

193

A M I N O ACID METABOLISM I N AMINO ACIDOPATHIES

5-hydroxytryptophan decarboxylase activity by phenylalanine, phenylpyruvic, phenyllactic and phenylacetic acid (Davison and Sandler, 1958; Huang and Hsia, 1963). Furthermore, 5-hydroxytryptophan, 0- and m-tyrosine and tryptophan, are all substrates of dopa-decarboxylase (Sakami and Harrington, 1963). In fact 5-HTP and dopa-decarboxylases are probably identical enzymes (Westermann et al., 1958; Yuwiler et al., 1959; Udenfriend et al., 1960). Dopa-decarboxylase, however, is also strongly inhibited not by phenylalanine itself, but by its related keto acids in vitro (Hartman et al., 1955; Fellman, 1956). Thus an impaired decarboxylation of tryptophan as an additional mechanism for the low serotonin and 5-HIAA levels in the presence of excess phenylalanine appears well founded. Brain uptake: Data on the uptake of amino acids by brain in vitro and in vivo were obtained chiefly for L-tyrosine and 5-hydroxytryptophan (Table 11). The studies of the latter compound are of special interest due to its failure to raise the serotonin content of the brain in the presence of excess phenylalanine. It was observed in animal experiments that L-phenylalanine reduces brain 5-HTP levels 45 % (in vibro) and 58 % T A B L E I1 EFFECT OF AROMATIC A N D OTHER A M I N O ACIDS ON T H E U P T A K E O F A M I N O A C I D S BY B R A I N ~

Amino acids tested

L-Tyrosine

Amino acids tested for interference

L-Phenylalanine

Inhibition of uptake in % of control

In vitro 49 %

p-Fluoro-oL-phenylalanine 44 X I L-Tryptophan 35 % 68 D-Tryptophan

<;

L-Histidine L-Cysteine L-Leucine L-Isoleucine nL-Norleucine L-Valine p-Hydroxyphenylacetic acid 5-Hydroxytryptophan L-Phenylalanine D-Phenylalanine L-Tyrosine L-Phenylalanine -tL-Tyrosine L-Dopa DL-Dopa L-Tryptophan D-Tryptophan 5-Benzyloxytryptophan L-Leucine L-Tsoleucine L-Proline ~

References p. 209-215

References

In vivo 25-55 % Chirigos et al., 1960 (10%) 33% Curoff er al., 1961 33 % Guroff et al., 1962 53-59 % Guroff and Udenfriend, 1964 58 76 51 %

33 % 33 % 38 % 43 96 %

<:

55 p:

42%

56 % 100%

50 7; 97 %

-

85%

59 % 57 %

McKean et al., 1962 Schanberg, 1963

194

H. H. B E R L E T

(in vivo) respectively. Other aromatic amino acids like L-dopa, L-tryptophan, Ltyrosine are also effective in lowering the uptake of 5-HTP by brain both in vitro and in vivo. The D-forms of amino acids have less activity or are ineffective (McKean et al., 1962; Schanberg, 1963). A comparison with tyrosine uptake shows a very similar pattern in vitro and in vivo (Chirigos et al., 1960; Guroff et al., 1961; Guroff and Udenfriend, 1962, 1964). The latter studies reveal that aliphatic amino acids such as valine, leucine, isoleucine and cysteine also inhibit the uptakeof L-tyrosine. p-Hydroxyphenylacetic acid, however, was not effective. In turn, phenylalanine was seen to interfere with the uptake by the brain of arginine and leucine in vitro as well as in vivo (Linneweh et al., 1963). These data indicate clearly that both aromatic and aliphatic amino acids are competing with each other for transport sites across the blood-brain barrier. Bebides the interaction of one amino acid with othcr amino acids the question is also relevant whether an elevation of a plasma amino acid such as phenylalanine will be reflected in an elevation of the brain levels of this amino acid. This problem is of particular interest wheii we consider the possibility of an abnormal intracerebral amino acid metabolism in the amino acidopathies which might result in the formation of neurotoxic intermediates. In experiments with phenylalanine it was shown that an increase of a plasma amino acid is followed by an increase of brain levels of this amino acid, However, quantitatively, the increase of brain levels was less arid represented roughly only 1/4-1/5 of the increase of plasma levels representing a brain plasma ratio of 0.20-0.25 (Lajtha and Toth, 1961; Guroff and Udenfriend, 1964). A comparable ratio in the distribution between brain and plasma levels appears to exist for the elevation of other amino acids too. Cerebrospinaljuid: I n 1950, Borek et al. found blood levels of 19-38 mg”/, for phenylalanine in phenylketonuric subjects as compared to a concentration of phenylalanine in the cerebrospinal fluid of 6.1 to 8.2 m g x . These values correspond to a CSF/plasma ratio of 0.2-0.3, which matches closely the brain/plasma ratio for phenylalanine obtained by Lajtha and Toth (1961). Although there is accordingly a concentration gradient between plasma and CSF, the same ratio may not apply universally to all amino acids and may indeed vary from 0.9 to 0.07 (or 1.1-13.4 for the plasma CSF/ratio) as it was shown recently in a survey of free amino acid concentrations in plasma and cerebrospinal fluid at normal plasma amino acid levels (Knauff et al., 1961). From what has been said on the competitive transport of amino acids across the blood-brain barrier the question now arises whether there exists a similar phenomenon of competitive transport inhibition for the barrier between blood and cerebrospinal fluid. This does not seem to be the case. On the contrary the increase of one plasma amino acid seems to enhance the transport of other amino acids into the cerebrospinal fluid indicating that different rules apply to the uptake of amino acids by the CSF (Wiechert, 1963). In conclusion to the considerations of amino acid uptake by brain and CSF it can be assumed that there normally exists a concentrationgradient between plasma on one hand and brain and cerebrospinal fluid space on the other hand. This concentration

AMINO A C I D METABOLISM I N A M I N O A C I D O P A T H I E S

195

gradient is also maintained when there is an elevation of plasma amino acids as in phenylketonuria, even though an elevation of one amino acid in the plasma will be likewise reflected by a proportional increase of this amino acid in brain and cerebrospinal fluid. Conversely the competitive inhibition of the active transport of one amino acid by another one affects only the blood-brain exchange of amino acids. Intestinal absorption: Boggs et a/. (1963b) point to the fact that a high dietary supplement of PA per se may prevent a sufficient absorption of tryptophan from the small intestines in experimental PKU. This interference with absorption occurs with other amino acids since even an excess of dietary valine was found to reduce significantly the brain and liver content of serotonin. In addition, the elevation of phenylalanine plasma levels could be abolished or prevented when 5 tryptophan was added to a high phenylalanine-tyrosine diet in rats (McKean et at., 1962). These data provide strong, although indirect, evidence that amino acids compete among themselves for the catalytic sites of intestinal amino acid transport (Fig. 2). It is interesting to note that the competition of metabolically unrelated amino acids for intestinal absorption corresponds to the interference of aliphatic amino acids like leucine and valine with the transport of 5-HTP and of tyrosine across the blood-brain barrier, as it was mentioned earlier. The question remains whether a high plasma level of phenylalanine or of any other amino acid without the presence of excess dietary phenylalanine as it occurs in PKU interferes with the intestinal absorption of other amino acids especially that of tryptophan (Boggs et at.,1963a). A failure of absorption could furnish an explanation for the indole defect in PKU as well as for the imbalance of plasma amino acids. The amino acid absorption across the wall of the small intestine is believed to be an active transport, the initial step being an accumulation of amino acids within the cells of the intestinal wall (Agar et al., 1954; Samiy and Spencer, 1961). An ‘uphill’ exchange of plasma amino acids with the intestinal lumen was demonstrated for l-aminocyclopentane carboxylic acid (Christensen et al., 1963). High plasma levels of this amino acid tended to decrease until a steady state ratio was reached between plasma and intestinal lumen indicating an active backward exchange of amino acids from plasma to the intestines. The net uptake of amino acids from the intestines was also seen to be depressed by high tissue levels of phenylalanine, and, vice versa, intestinal L-phenylalanine transport was depressed by elevated tissue levels of L-tryptophan and Lmethionine in vitro (Spencer and Samiy, 1961). It thus becomes likely that high tissue levels of PA retard the absorption of tryptophan and other amino acids from the intestines. In fact, when labeled arginine or leucine were fed to phenylketonuric children, the intestinal absorption of these amino acids was markedly depressed as compared to the absorption by normal children or by phenylketonuric children maintained on a low phenylalanine diet (Linneweh et al., 1963). The poor absorption of tryptophan would result in a retention of this amino acid in the intestines where it becomes exposed to bacterial decomposition. Tryptophan could be converted to tryptamine and indoles such as indoleacetic acid and indican, which then are readily absorbed into the blood and excreted in increased amounts through the kidneys. A similar type of mechanism for the indole defect in Hartnup References p . 209-215

196

H . 11. B E R L E T

disease was demonstrated by Asatoor et al. (1963). In that condition the content of free tryptophan is increased in fecal material due to a poor intestinal absorption of tryptophan. Incubation of feces from patients with Hartnup disease resulted in an increased formation of indole compounds : indole, indoleacetic acid and tryptamine. Thus the plasma and urinary pattern of indoles in PKU is a result of two factors. one being the poor intestinal absorption of tryptophan, the second being the enhanced intestinal absorption of indole products. arising from the bacterial decomposition of intestinal tryptophan. If this assumption of a competitive transport inhibition between the intestinal lumen and the blood stream applies to tryptophan, plasma levels of tryptophan should be low. Low serotonin and 5-HIAA levels could be explained under those circumstances by a partial lack of substrate for the hydroxylation. Further evidence for this possibility could be obtained by measuring plasma tryptophan levels in phenylketonuria. In considering the possible ways in which phenylalanine may interfere with the physiological metabolism of other amino acids especially tryptophan, it is not possible to point exclusively to one particular interaction. It appears that phenylalanine, in a complex way, acts simultaneously on enzymatic reactions like hydroxylation and decarboxylation as well as on catalytic transport sites across the intestinal wall and on the blood-brain barrier to bring about the indole defect in PKU (see Fig. 2). Which one of the factors is the most important or which one may even have to be finally disregarded as irrelevant for the reduction of hydroxylated indoles will remain the subject of future investigation. Currently the view has become prevalent that the interference of phenylalanine with active transport mechanisms is the most important feature (Boggs et al., 1963a; Hsia et al., 1963; Linneweh et a/., 1963). Irrespective of that the inhibitory action of an elevated amino acid upon the enzymatic metabolism of unrelated amino acids will remain an important aspect and this mechanism may also be found in amino acidopathies other than phenylketonuria. L-Leucine (10 g/day) for example, administered for 5 days to normal individuals, reduced markedly the urinary excretion of tryptophan, indican, 5-HIAA, free 3-1AA* and while there was an increase of urinary that of l-methyl-2-pyrridone-5-carboxamide quinolinic acid. In the presence of a nicotinamide load the effect of L-leucine on the excretion of the oxidized 1-methylnicotinamide was much less. The effect of leucine is therefore presumed to be due to an enzyme blockage of one of the several possible pathways for the biosynthesis of nicotinic acid (Belavady et ul., 1963). Interference of absorption such as described above by an elevated plasma level of an amino acid apparently affects not only the intestinal absorption and brain uptake of tryptophan, but also that of other amino acids (Linneweh et al., 1963; Guroff and Udenfriend, 1964) and the inhibitory action of amino acids is not limited to PA. In particular it would be expected that the effect of phenylalanine upon intestinal transport of amino acids would have more general implications as to the plasma amino acid pattern. However, not very many data are available at the present time

*

Indole-3-acetic acid.

197

A M I N O A C I D METABOLISM I N A M I N O A C I D O P A T H I E S

on plasma amino acid patterns in experimental and inherited metabolic disorders of amino acids. Knox (1960) reported an evaluation of plasma amino acids in a phenylketonuric subject. It was observed that, in addition to the expected elevation of phenylalanine, all other amino acids were present in amounts smaller than normal with the exception of glycine, which was in normal range, and histidine, which was increased. The total a-amino nitrogen content of the plasma, however, was normal. Similar observations were made by Linneweh and Ehriich (1960) and Carver eta/. (1962). In a case of a delayed maturation of the tyrosine enzyme system which converts p-hydroxyphenylpyruvic acid to homogentisic acid high plasma levels of tyrosine and phenylalanine were found. Other amino acids were present in abnormal amounts: plasma levels of proline, valine, leucine, isoleucine, methionine were elevated, those of aspartic acid, glutamic aLid and arginine were depressed (Auerbach et al., 1963). Table 111 gives a comparison of the plasma amino acid pattern of a case of phenylketonuria, maple syrup disease (Dancis et al., 1959), citrullinuria (McMurray et al., 1963) and a case of a delayed maturation of the tyrosine metabolism. Although one T A B L E 111 PLASMA AMINO ACID PATTERNS I N PRIMARY AMINO ACIDOPATHIES

No absolute values are given since control values of normal plasma amino acids vary slightly among several authors. Therefore descriptive terms (normal, low, elevated) are employed designating the deviation of values from control values used in each particular experiment. Terms are put in parentheses when the deviation from normal was only slight. Plasma amino acids Alanine y-Aminobutyric acid Arginine Aspartic acid Asparagine Citrulline Cystine Glutamic acid Glutamine Glycine Histidine Isoleucine Leucine Lysine Methionine (alloleucine) Ornithine Phenylalanine Proline Serine Taurine Threonine Tyrosine Valine Knox, 1960 Refcrenccr p. 209-215

Phenyllcetonuria’ low low low

syrup disease‘

low

normal

-

-

low low (low) low (low) elevated elevated elevated elevated

-

normal low low

-

-

low low normal elevated low low (low) (low) (low) elevated low (low) (low) low low

low low low normal normal elevated elevated normal elevated normal normal (low) low low low normal elevated

-

Dancis et al., 1959

Delayed maturation of tyrosine metabolism3

-

elevated elevated (elevated) elevated elevated elevated

Auerbach er al., 1963

Citrullinuria4

normal elevated low normal elevated low normal

-

low elevated low low elevated elevated low normal (low) low normal normal low McMurray ei al., 1963

198

H. H. BERLET

common pattern of plasma amino acids cannot be expected in these conditions the table nevertheless discloses that an elevation of one or several amino acids is accompanied in all four instances by the depression of several other plasma amino acids. None of the amino acids is affected in the same way in all four syndromes. It is of interest that the total a-amino acid nitrogen of the plasma in the case of phenylketonuria was normal, the elevation of one plasma amino acid being counterbalanced by a depression of other amino acids. There appears to be a mechanism for the homoeostatic control of the total amino acid nitrogen of plasma which is operating in the amino acidopathies. It would seem, therefore, useful to study plasma amino acid patterns under the various conditions of amino acid administration to demonstrate whether there is such a common mechanism for the maintenance of total a-amino acid nitrogen in amino acidopathies. Evidence for such a regulatory mechanism was obtained previously by Knauff et al. (1 960, 1963) in animal experiments upon chronic poisoning with either carbon tetrachloride or thioacetamide. The values for total a-amino acid nitrogen remained practically constant, while the overall plasma amino pattern was severely disturbed. An amino acid imbalance can be particularly disastrous by causing a poor supply of important nutrients to the developing brain (Udenfriend, 1963). It is well established that the blood-brain barrier system for amino acids begins to function during the neonatal period (Himwich and Himwich, 1954; Himwich et al., 1957; Guroff and Udenfriend, 1964) and the competitive inhibitory function of an elevated amino acid thus is able to affect the brain throughout the postnatal development and the maturation beginning virtually at birth. It is interesting to note, however, that the central nervous system can be apparently injured also during the prenatal growth by an extraneous elevation of phenylalanine. Mabry et al. (1963) reported recently on mentally retarded children without the phenylketonuric syndrome born to phenylketonuric mothers, confirming a previous observation by Dent (1957) of non-phenylketonuric mentally retarded children of phenylketonuric mothers. These observations indicate that during the fetal period phenylalanine or other factors present in the maternal plasma enter the fetal circulation to exert their effect on the growing brain at a time when it does not have the protective action of the brain barrier. I N T E R A C T I O N S O F P H E N Y L A L A N I N E W I T H THE METABOLISM O F T Y R O S I N E

Phenylalanine may alter the metabolism of tyrosine and dopa, and affect the formation of catecholamines by a mechanism of a competitive inhibition similar to that described for the hydroxylation and decarboxylation of tryptophan (Fig. 3). As early as 1954, Cawte reported on 2 phenylketonuric patients who were more sensitive to injected epinephrine than controls. Weil-Malherbe (1955) found low plasma levels for norepinephrine and epinephrine in phenylketonuria. Nadler and Hsia (1 961) found in addition a low urinary excretion of dopamine, norepinephrine and epinephrine in their phenylketonuric patients but normal plasma levels of tyrosine were present.

A M I N O ACID METABOLISM I N A M I N O A C I D O P A T H I E S

I99

1YROSINE -

,

O C l i - $ H - C O O H

.no

-

NH2

ADRENALINE NORADRENALINE

( URfNE )

= ENZYME ITJHIBITION

= PRESUMPTIVE ENZYME lNHl0lTlON

Fig. 3. Effect of phenylalanine and its derivatives on the metabolism of tyrosine. Enzyme reactions: ( I ) Tyrosinase system; (2) tyrosine hydroxylase; (3) Dopa-decarboxylase; (4)Dopamine-b-oxidase.

Boylen and Quastel (1961) showed that the synthesis of epinephrine from tyrosine was inhibited by sodium phenylpyruvate in vitro. Slices of guinea pig adrenal medulla were incubated with [“C]-labelled tyrosine and the formation of [14C]-labelled epinephrine was measured. Concentrations of sodium phenylpyruvate comparable to those occurring in the plasma of phenylketonurics resulted in a 50% inhibition of epinephrine formation accompanied by an accumulation of dopa which did not occur in the controls. Phenylalanine had no inhibitory effect. The in vitro evidence provides an explanation for the alterations of catecholamine levels through an inhibiton of dopa decarboxylase in the presence of increased amounts of phenylalanine derivatives. The assumption that phenylalanine interferes with the formation of catecholamines can be correlated with other previous in vitro experiments. Dopa decarboxylase from hog kidney cortex (Hartman et af.,1955) and from the adrenal medulla (Fellman, 1956) were both demonstrated to be inhibited in vitro by phenylpyruvic acid, phenyllactic acid and phenylacetic acid but not by phenylalanine (see also tryptophan decarboxylase). The reduced urinary excretion of vanilmandelic acid and dopamine is then merely reflecting the reduction of plasma levels of norepinephrine, epinephrine and dopamine. It also may explain why normal levels of plasma tyrosine were found in cases of depressed plasma levels of catecholamines, the conversion of tyrosine to dopa not being sufficiently affected to raise plasma tyrosine levels. Melanin: PA has not only an effect on catecholamines but it also affects the biosynthesis of melanin from tyrosine (Snyderman P t al., 1955; Armstrong and Tyler, 1955). In phenylketonuric subjects there is a reversible decolorization of the skin and hair, due to a lack of the pigment melanin which can be restored by adding tyrosine or by eliminating phenylalanine from the diet. Both the mushroom tyrosinase system (Dancis and Bolis, 1955) and the mammalian tyrosinase system (Miyamoto and Fitzpatrick, 1957) can be inhibited by phenylalanine in vitro. A number of unusual metabolites may arise from the inhibition of the tyrosineReferences p . 209-215

200

H. H . B E R L E T

hydroxylation and of dopa decarboxylation and it may be worth interest to consider briefly the possible consequenceswith regard to alternative metabolic pathways of dopa. The tyrosinase system normally converts tyrosine to dopa and initiates the chain of reactions leading to melanin. The conversion of tyrosine to melanin is a complex reaction involving the enzymatic formation of a number of intermediates : tyrosine, dopa, dopaquinone, 5,6-dihydroxydihydroindole-2-carboxylic acid, dopachrome, 5,6-dihydroxyindole, indole-5,6-quinone and melanin (Raper, 1928). Axelrod and Lerner (1963) have shown in v i m alternative pathways to exist for dopa, 5,6-dihydroxydihydroindole-2-carboxylk acid and 5,6-dihydroxyindole through O-methylation by COMT and hydroxyindole-0-methyltransferasefrom pineal glands respectively during several of the enzymatic steps involved. The accumulation of dopa in PKU makes these alternative pathways even more likely. Although the inhibition of the tyrosinase system is believed to take place mainly during the conversion of tyrosine to dopa, and increased levels of p-hydroxylated phenolic acids were found in phenylketonuric urines, normal tyrosine levels in PKU seem to imply that other enzymatic steps are involved besides the hydroxylation of tyrosine. Inhibition of one or several others of these enzymatic reactions however by phenylalanine in PKU is likely to result in an accumulation of intermediary compounds arising from the conversion of dopa to melanin which then might be metabolized via an 0-methylation in the 3-, 5-, 6-position and excreted as inethylated dopa and indole derivatives. Dopa, unless utilized for the biosynthesis of melanin through the initial catalytic action of the tyrosinase system is normally decarboxylated to form dopamine or contributes to homovanillic acid. Homovanillic acid is the chief urinary excretion product of the catabolism of dopa as well as of dopamine (Shaw et al., 1957). When there is an inhibition of dopa decarboxylase it is likely that a portion of dopa is excreted as such. In fact, dopa could be recovered from urine upon the oral administration of tyrosine and dopa in a case of an enzymatic defect in the conversion of tyrosine to homogentisic acid (Medes, 1932). Another possibility is, by analogy to the formation of phenolic pyruvic and lactic acids from phenylalanine and tyrosine in phenylketonuria, that dopa undergoes a transamination or oxidative deamination to form dihydroxyphenylpyruvic or -lactic acid. Formation of the former compound is theoretically possible (Shaw et al., 1957) due to its labile nature, however, it has not as yet been identified in vivo. Dopa is readily 0-methylated by catechol-0-methyltransferase as it was shown by Axelrod and Tomchick (1958) who found dopa to be a very good substrate for this enzymc as compared t o the 0-methylation of the regular substrates epinephrine or norepinephrine. The 0-methylation of dopa i n 3-position would yield 3-methoxytyrosine, which is not metabolized to homovanillic acid in appreciable amounts (Shaw et al., 1957). Recently urinary 3-methoxytyrosine was regularly found when dopa and dopamine were administered orally to humans and rats (Takesada et al., 1963). Brain (atecholamines: In contrast to the depression of plasma catecholamines, brain catecholamines do not seem to be depressed by phenylalanine. Green et ul. (1962) observed that there was essentially no change or even a slight increase of brain catecholamines in weanling rats fed 9 times the amount of phenylalanine contained

A M I N O A C I D METABOLISM I N A M I N O A C I D O P A T H I E S

20 1

i n the basal diet. There was a 3-4 fold increase of dopamine under these conditions which was further elevated when the tryptophan content of the diet was reduced. Brain serotonin was effectively reduced as was to be expected. The discrepancy between the reaction of plasma and brain catecholamines to high plasma levels of phenylalanine requires further evaluation. Our present knowledge limits PA-hydroxylase to the liver and does not suggest PA as a precursor of brain dopa and dopamine. The accumulation of dopamine indicates that the reaction leading to norepinephrine which is mediated by dopamine-/?-oxidase, a phenylethylamine-/?-hydroxylasesystem, is inhibited. In this connection it is noteworthy that phenylethylamine, an increased intermediate of PA in PKU, is also hydroxylated by the dopamine /S-oxidase to form the corresponding @-alcoholderivatives (Levin and Kaufman, 1961) and a competitive inhibition between dopamine and phenylethylamine concerning the dopamine @-oxidasebecomes possible. The presence of normal levels of norepinephrine, however, does not support this view as the only possible explanation. Evidence has been obtained, that dopamine might also serve as precursor of the melanin synthesis in certain brain areas (Van der Wende and Spoerlein, 1963). A possible inhibition of the conversion of dopamine to melanin by phenylalanine derivatives might therefore contribute to an elevation of brain dopamine. It is also of interest that an in vivo conversion of tyrosine to dopa and to catecholamines in cat brain was reported recently (Masuoka et al., 1963; McGeer et al., 1963). In confirmation Nagatsu et al. (1964) were able to obtain a tyrosine hydroxylase activity from cell-free preparations of brain and sympathetically innervated tissue. Since dopa has never been detected in the bloodstream under normal conditions it was suggested that tyrosine may represent the physiological source of brain catecholamines and the brain is apparently able to biosynthesize its own catecholamines in a fashion similar to its recently demonstrated ability to hydroxylate tryptophan (Gal el al., 1963, 1964) without depending on the dopa-formation by the tyrosinase system which is limited to melanocytes. It is not yet known whether PA acts upon this hydroxylase system in a fashion similar to the inhibition of the tyrosinase. P H E N Y L A L A N I N EMETABOLITES W I T H PHARMACOLOGICAL ACTIONS I N

PKU

Little can be said as yet concerning the secondary effects of phenylalanine on the central nervous system. These effects are of great importance, though, since in both inherited and experimental phenylketonuria the secondary biochemical and organic defects due to an elevated plasma PA level probably account for the development of mental deficiency. The number of different compounds occurring in phenylketonuria has raised the question of a specific ‘toxic’ agent causing the secondary symptoms of this entity especially those of mental retardation. Its nature has yet to be finally proven and the postulate of a toxic agent has therefore not remained unchallenged. However, it could be shown in dietary experiments on phenylketonuric infants that in contrast to deaminated and decarboxylated PA-metabolites such as phenylpyruvic acid, phenylacetic acid, only phenylalanine could definitely reverse the beneficial effects References p . 209-215

202

H. H. BERLET

which were brought about by a low phenylalanine diet (Bickel et al., 1954; Armstrong and Tyler, 1955). One can assume at least, that if not phenylalanine itself, a derivative of phenylalanine could be named the neurotoxic agent. Several compounds such as phenylethylamine and o-tyramine for example, which arise primarily or secondarily from the phenylalanine disorder exert pharmacological actions upon the central nervous system and deserve a more detailed consideration. A finding of interest in this connection which may reflect the fate of increased brain phenylalanine and its effect on the central nervous system is that abnormal metabolites such as phenylethylamine (Jepson et al., 1960) or hypothetically an accumulation of o-tyramine (Mitoma et al., 1957) may play the role of a neurotoxic agent. When a monoamine oxidase inhibitor was given to phenylketonuric patients the urinary excretion of phenylethylamine increased considerably (Jepson et al., 1960). The addition of a-methyldopa, an inhibitor of the aromatic L-amino acid decarboxylase restored the excretion of phenylethylamine to normal (Oates et al., 1963). In contrast, normal individuals showed only a small increase of phenylethylamine as a rcaction to the monoamine oxidase inhibitor. It was concluded that phenylethylamine is a n important intermediate in phenylketonuric metabolism arising from the decarboxylation of PA occurring at markedly higher tissue levels than in normal individuals. Furthermore, phenylethylamine and dopamine are p-hydroxylated by the same enzyme, namely, a phenylethylamine-/3 hydroxylase (Levin and Kaufman, 1961) which acts upon a number of substituted phenylethylamines such as tyramine, mtyramine and 3-methoxy-4-hydroxyphenylethylamineor pharmacologically active amines such as a-methyltyramine and a-methyl-m-tyramine (Creveling et al., 1962). Therefore, phenylethylamine may act in vivo as a pharmacologically active agent, possibly as a competitive or noncompetitive enzyme inhibitor of the dopamine-phydroxylase. Indeed phenylethylaniine is known to have vasoactive and convulsive properties (Chen, 1927). Lt is also a potent releaser of norepinephrine and adrenaline from binding sites displaying in vitro a tyramine-like action in releasing catecholamines from medullary granules isolated from adrenal glands (Schumann and Philippu, 1962). It may be of interest in this connection to recall that Green et al. (1962) found a marked elevation of brain dopamine in rats fed a high phenylalanine diet. Norepinephrine levels remained unchanged. If phenylalanine were to act upon catecholamines through the releasing action of phenylethylamine one would certainly expect a depression of norepinephrine. Lnstead the accumulation of dopamine in the presence of a normal nsrepinephriiie levcl is suggestive of an increased formation of dopamine or at least a slow turnover of dopamine to norepinephrine. Mitoma rt al. (I 957) postulated o-tyramine as an intermediate during the increased degradation of phenylalanine via o-tyrosine to o-hydroxyphenylacetic acid in plienylketonurics (Fig. 4). The conversion of phenylalanine to o-tyrosine was found to be possible i n mammalian tissue. o-Tyrosiiie can be further decarboxylated to the corresponding amine. o-Tyraminc is known to have an D-amphetamine like action and a hypothetical role in PKU was therefore suggested. In contrast to these previous findings by Mitoma the intermediate compound of the degradation of phenylalanine

AMINO A C I D METABOLISM I N A M I N O A C I D O P A T H I E S

203

P H E NYLALAN INE

PHENYLPYRUVIC

ACID

0-TYROSINE

1

0-HYDROXYPHENYLPY RUVIC

0-TYRAMINE

0-HYDROXYPHENY LACETIC ACID

Fig. 4. Conversion of phenylalanine to o-hydroxyphenylaceticacid.

to o-hydroxyphenylacetic acid was more recently found to be o-hydroxyphen ylpyruvic acid, formed enzymatically from phenylpyruvic acid (Taniguchi and Armstrong, 1963). An extract from mammalian liver tissue used in this investigation, exhibited a specific phenylpyruvate oxidase activity. Other substrates like phenylalanine, phenylacetic acid, o-hydroxyphenylpyruvic acid did not yield other phenolic compounds; p-hydroxyphenylpyruvic acid, supposedly derived from the naturally occurring isomer of tyrosine, was an excellent substrate for this enzyme system but yielded homogentisic acid. o-Tyramine remains therefore a hypothetical intermediate and it becomes questionable whether the pathway which was demonstrated by Mitoma et al. (1957) is existent for phenylalanine. In conclusion it can be stated that there remain two major hypotheses which result from the experimental approach to phenylketonuria. One is that there is probably an accumulation of a neurotoxic agent in the brain, derived from phenylalanine which acts upon a specific biochemical entity or entities of the brain. The specific nature of this agent is not yet known. A second possibility is that phenylalanine interferes profoundly with active transport mechanism across the blood- brain barrier as well as across the intestines. This interference may not be restricted to amino acids but may affect other blood constituents which serve as mandatory nutrients or cofactors for the maturation of the developing brain. The increasing amount of information on amino acids other than phenylalanine, which was in part discussed in the preceding paragraphs, permits a more general application of the conclusions drawn from PA to other conditions which are associated with an elevation of plasma amino acids due to an enzyme defect. B E H A V I O R A L A N D S T R U C T U R A L EFFECTS O F P H E N Y L A L A N I N E

Behavioral changes in experimental PKU are of great interest since they indicate that damage to the CNS has occurred which makes it possible to correlate behavioral, anatomical, histological and biochemical data. This approach is in fact the first experimental one t o the study of mental retardation. In phenylketonuric rats a marked retardation in temporal discrimination learning was observed (Auerbach et al., 1958) and poor performance in maze learning. Similar observations were reported by other authors (Yuwiler and Louttit, 1961; Louttit, References p. 309-215

204

H. H. RERLET

1962). In infant monkeys Waisman et al. (1962) found a poor performance in delayed reaction tests, in the Hebb Williams maze, and in visual discrimination set learning. In a number of these behavioral experiments attempts were made to relate the behavioral pattern to the lowering of brain serotonin levels in PKU because of the hypothetical view proposed by Woolley and Shaw (1954) of the implications of a low brain serotonin content on intellectual and behavioral functions of the mind. This view was taken up by Pare et al. (1957) and applied to the low levels of brain serotonin i n PKU as a possible cause of the mental retardation. In phenylketonuric animals, however, it was found that there was no correlation between brain serotonin and performance of these animals in the various tests applied (Yuwiler and Louttit, 1961; Louttit, 1962). In addition, the restoration by a moiioamine oxidase inhibitor (isocarboxazid) of the depleted brain serotonin failed to improve the performance of these animals. Thus the significance of low brain serotonin levels with respect to PKU remains debatable. In fact, Woolley and Van der Hoeven (1963) observed, in contrast to the original concept, that an excess of brain serotonin in mice had also an adverse effect on the maze learning abilities of his animals while a decrease of serotonin and brain catecholamines seemed to enhance the learning ability slightly. However, the latter results were obtained with adult animals and the authors point out that a depression of brain serotonin will have a consistently deleterious effect on the developing CNS of newborn and infant animals. If altered levels of a neurohormone do not seem to account for an impaired functioning of the central nervous system, histological changes due to the prolonged exposure to toxic influences should yield vajuable information as to the site of damage of certain brain areas. Akert et al. (1961) found histological alterations of glial mitochondria and evidence of incomplete myelinations of different parts of the brain of phenylketonuric rats and monkeys and, interestingly enough, also in the brain biopsy of a phenylketonuric infant. In a comprehensive review on PKU and histological post-mortem findings in brains of phenylketonuric subjects the point is stressed that among a number of other less systematic histological findings an abnormal myelination of parts of the nervous system was found most frequently (Knox, 1960). It seems possible that there is a relation between phenylalanine and myelination. This point gains importance when we remember that Halstead during the Hixon symposium in 1951 speculated about a possible relation between myelination and the development of the biological substrate of intelligence. P H E N Y L A L A N I N E A N D OTHER M E N T A L DISEASES

Phenylalanine and related metabolites of the catecholamine pathway have been implicated from two different points of view as playing an etiological role in mental diseases such as endogenous psychoses. One view is that abnormalities of the PA metabolism and a higher incidence of mental illness were found in heterozygous relatives of phenylketonuric subjects. The other view was propagated and tested by Hoffer and his group (1954) who pointed out that epinephrine is metabolized by schizophrenic patients differently from normal subjects.

A M I N O A C I D METABOLISM I N AMINO A C I D O P A T H I E S

205

Abnormalities of the phenylalanine metabolism in heterozygous individuals with regard to phenylketonuria were first demonstrated by Hsia et al. (1956). Heterozygotes in contrast to normals, could handle a phenylalanine load less well having a higher and longer sustained rise of plasma phenylalanine. Knox and Messinger (1 958) went one step further and found that the statistical distribution of mean fasting plasma phenylalanine levels of heterozygotes were distinctly higher than that of a normal group. It is, therefore, of interest to note that a survey on the incidence of mental illness among phenylketonuric heterozygotes seems to indicate a higher rate than can be expected in a normal population (Thompson, 1957). On the other hand, Knox (1963) reported on the evaluation of a group of 100 hospitalized mental patients and a 15 % higher incidence of elevated plasma PA levels was found in heterozygotes than in a normal control population. Whether these results mean that heterozygotes are more susceptible to mental illness and thus represent a substantial percentage of hospitalized mental patients or not is not yet definitely answered. It would be, therefore, premature to be more than speculative in establishing a relationship between the underlying cause of mental illness in heterozygous PKU and endogenous psychoses by means of a metabolic disorder of an amino acid as a common denominator. However, the amino acid metabolism in schizophrenia has gained increasing interest during recent years and investigations along these lines may very well profit from results already obtained in other fields. For example, a similar test for the detection of heterozygotes with respect to Maple syrup urine disease was devised by Linneweh and Ehrlich (1963) who were successful in demonstrating both an elevation of alloisoleucine of the fasting plasma as well as a sustained elevation of plasma-isoleucine and allo-isoleucine upon the oral administration of leucine, isoleucine and valine. Knox (1 963) emphasizes the surprisingly high incidence of heterozygotes for rare inherited diseases which are carried on as recessive traits, as based on the HardyWeinberg law, describing incidence and relevance of genotypes in a random-mating population. The number of latent ‘amino acidopathic’ individuals in our population may be therefore considerably higher than it was formerly realized and the pathogenetic potentialities of the heterozygous condition should become increasingly important with respect to mental disorders. An interesting question therefore would be whether the slight elevation of certain amino acids in heterozygotes of amino acidopathies and their inability to metabolize certain amino acids as well as normals do, render them more susceptible to mental disorders. Another hypothesis was propagated by Hoffer et al. concerning the schizophrenics and a possible abnormality in the conversion of adrenochrome to adrenolutin which is hallucinogenic when tested pharmacologically. Attempts were made to test this hypothesis clinically in schizophrenic patients by eliminating precursors of adrenaline from the diet. Diets deficient in phenylalanine, tyrosine and tryptophan were given to two groups of schizophrenic patients for a period of time up to six weeks (McGeer et al., 1956; Bogosch, 1957). N o beneficial effects were seen from this diet. In fact the diet was felt to have a ‘psychotoxic’ effect. The reason for the latter observation is unknown, however, in the experiments with diets deficient in amino acids an endoReferences p . 209-215

206

H. H. B E R L E T

genous liberation of amino acids may have occurred accounting either for the psychotoxic effect or for the failure to elicit beneficial effects. The original proposition, however, concerning the role of abnormal reaction products of adrenaline also had to be revised since some of the postulated abnormal compounds were found t o be normal metabolites during the inactivation and breakdown of adrenaline and noradrenaline. The possibility remains that an abnormal methylation of catecholamines is characteristic of the schizophrenias. Additional data which support this assumption have been obtained only recently by several groups of investigators. The presence of Nmethylmetanephrine was demonstrated in urines of juvenile psychotics (Perry, 1963). Although this compound may occur also in urines of non-psychotic subjects (Itoh et al., 1962) and its precursor N-methyladrenaline was found in adrenal glands of the rat, rabbit, guinea pig and monkey (Axelrod, 1960) the amount of N-methyl-metanephrine excreted by psychotics was considerably higher than normal when the excretion ratio between N-methyl-metanephrine and metanephrine of I : 10 was employed for comparison (Perry and Schroeder, 1963). The same group of psychotics did not excrete detectable amounts of another methylated catecholamine, 3,4-dimethoxyphenylethylamine. This compound which is structurally closely related to catecholamines was first found in urines from schizophrenic patients only (Friedhoff and Van Winkle, 1962). It was shown later that labelled 3,4-dimethoxyphenylacetic acid was recovered from the urine of schizophrenic patients upon the intravenous infusion of labelled dopamine (Friedhoff and Van Winkle, 1963) and the incubation of dopamine with liver tissue obtained from a schizophrenic patient resulted in an O-methylation of dopamine in the 3- as well as in the 4-position in v i m . These results seemed to indicate strongly two things : first, 3,4-dimethoxyphenylethylamineis a specific metabolite in the urine of schizophrenic patients and second, dopamine is a precursor for the formation of this compound. A reevaluation, however, of the occurrence of 3,4-dimethoxyphenylethylamine in a large number of urines could not confirm the exclusiveness of this compound for the schizophrenias (Takesada et al., 1963). In a total of 67 normal or psychoneurotic subjects 3,4-dimethoxyphenylethylaminewas found in 35 or roughly 50 % of the cases. However, the same authors found it in the urine of 70 out of 78 schizophrenic patients. Therefore the incidence of the occurrence of 3,4-dimethoxyphenylethylamine is strikingly higher in schizophrenic individuals than in normal or psychoneurotic subjects. In addition to dopamine 3-methoxytyramine was suggested as another precursor of 3,4-dimethoxyphenylethylamine,since the oral administration of L-dopa and dopamine consistently yielded 3-methoxytyramine in urines from healthy humans and rats. In the meantime the presence of 3,4-dimethoxyphenylethylamine in the urine of schizophrenic patients was confirmed by another group of investigators (Sen and McGeer, 1964). In addition they were able to identify by means of gas chromatography another so far unknown 0-methylated 4-hydroxybenzene, namely 4-methoxyphenylethylamine (methoxytyramine). This compound was found in 10 out of 15

A M I N O A C I D METABOLISM I N A M I N O A C I D O P A T H I E S

207

urines from acutely ill schizophrenic patients. A comparison with normal urines was not made on a basis large enough to prove that 4-methoxyphenylethylamine is a unique excretion product in the schizophrenias. The presence of these new urinary metabolites arising from the various intermediate steps in the biosynthesis and inactivation of catecholamines provides strong evidence that alternate pathways exist for catecholamines either normally or under certain disease conditions. Fig. 5 gives a tentative outline for the probable origin of abnormal ORIGINAL COMPOUND TYROSINE

.1

1

DOPA

DOPAMINE

INTERMEDIATE

ABNORMAL

( HYPOTHETICAL)

).

Methoxytyrosine

),

3 -me thoxytyr o

6

).

4 -rnethoxyphenylethylarnine

- Methoxytyramine -

ine

,

).

3-methoxy

METABOLITE

3, 4-dimethoxyphenylethylarnine

- 4 - h yd r ox y p h e n y l e t h y l a m i n e

J. -1

NO R E P I N E P H R I N E

EPINEPHRINE

.1 1

METANEPHRINE

>,

N -rn e t h y l m e t a n e p h r i n e

VANILMANDE LIC ACID

Fig. 5. Origin of phenylalanine metabolites in cases of mental disease (schizophrenia).

methylated catecholamines and intermediary reaction products during the formation. Several of the enzymatic steps involved have been previously shown possible by in vitro experiments. Dopa, dopamine and a number of other catecholamine derivatives serve as good substrates of the catechol-0-methyltransferase in vitro (Axelrod and Tomchick, 1958). The 0-methylation of dopa in 3-position to form 0-methyl-dopa (3-methoxytyrosine) was again demonstrated by Axelrod and Lerner (1963) in vitro during the initial steps of the biosynthesis of melanin as an alternative pathway for dopa. As for the N-methylation of metanephrine it was shown repeatedly that a number of enzyme systems like phenylethylamine-N-methyltransferase(Kirshner and Goodall, 1957), imidazole-N-methyltransferase (Brown et al., 1959) and an enzyme system isolated from rabbit lung (Axelrod, 1962) are capable of N-methylation of amines. Whether the formation of these unusual methylated amines is normal or indicates a temporary or permanent aberration of normal pathways in mental disease remains to be answered. Considering a temporary impairment of enzymatic steps as an explanation one would have to assume a partial or complete block of enzyme reactions under certain conditions resulting in the enhanced utilization of alternate pathways. This view is supported by the common observation that the inhibition of monoamine References p 209-215

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oxidase greatly facilitates the demonstration and identification of biogenic aniines in body fluids and tissues. The formation of these amines may also reflect a ‘spilling over’ due to a rate limiting enzymic reaction such as the hydroxylation of tyrosine in the course of the biosynthesis of catecholamines. The latter explanation could hold true especially in cases with an increased amount of amine precursors to be handled or during an increased turnover and release of catecholamines in states of increased anxiety and increased psychotic activity accompanied by agitation and motor hyperactivity. Pharmacological actions are known for some of the niethylated amines and it is well to remember that the close resemblance of the 0-niethylated catecholamines with mescaline (3,4,5trimethoxyphenylethylamine), has consistently attracted the attention of investigators. However it should be also kept i n mind that essentially minor rearrangements of the chemical configuration of a compound and especially of ring substituents may alter the properties of a compound profoundly and even abolish entirely certain effects (Jacobsen, 1963). In addition, the amounts of the amines recovered from patient urines are very small as compared with those quantities which are required in human beings to biing about definite pharmacological actions. The physiological role of these amines remains doubiful, therefore, at present and the implications of their presence in the urine of mental patients await further clarification. SUMMARY

This discussion was intended to describe mutual interactions between amino bcids and the effects of amino acids on unrelated enzymatic reactions with main emphasis on phenylketonuria in order to indicate common principles for the reaction patterns of amino acids in the primary amino acidopathies which are accompanied by mental retardation or mental symptoms. ( I ) Primary amino acidopathies accompanied by mental retardation are characterized by the elevation of one or several plasma amino acids in contrast to primary amino acidopathies not accompanied by mental defects. The increase of the plasma level of one amino acid level interferes with transport and metabolism of other amino acids in several ways as described below. (2) A number of aromatic and aliphatic amino acids are capable of inhibiting the intestinal absorption of amino acids creating an imbalance of the plasma amino acid patterns, one or several amino acids being elevated, others being depressed. There is evidence that the total plasma amino acid content (nitrogen) is maintained by a regulatory mechanism even in the presence of an excess of one or several plasma amino acids, (3) The amino acid uptake by the brain is also inhibited by an elevation of aromatic and aliphatic amino acids in the plasma. The increase of an amino acid in the bloodstream is reflected by a similar increase in the brain although to a somewhat lesser extent than in the blood. It is concluded that a disturbance of brain amino acids and amines similar to that in phenylketonuria, is likely to exist in other conditions accompanied by elevations of plasma amino acids.

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(4) Phenylalanine and its derivatives affect the enzymatic hydroxylation of tryptophan, the decarboxylation of 5-HTP, the hydroxylation of tyrosine and the decarboxylation of dopa. Phenylethylamine may interfere with the b-oxidation of dopamine. The indole defect in phenylketonuria is believed to be the combined effect of enzyme inhibition (hydroxylation and decarboxylation) and transport inhibition, intestinally and across the blood-brain barrier. Inhibitory interactions between amino acids and enzyme reactions concerning unrelated amino acids seem to exist for other amino acids than phenylalanine, tryptophan and dopa (leucinetryptophan). (5) A number of derivatives with pharmacological actions arise from an abnormal metabolism of phenylalanine. Phenylethylamine and o-tyramine occur in phenylketonuria. Other amines such as 4-methoxyphenylethylamine, 3,4-dimethoxyphenylethylamine and N-methylmetanephrine, arising from an unusual methylation of tyrosine or tyramine respectively, dopa or dopamine and metanephrine were recovered from urines of schizophrenic patients. The implications of these amines with respect to primary amino acidopathies with mental retardation and mental disease are not at all clear. (6) In trying to elicit a common denominator in primary amino acidopathies with mental defects it can be pointed out that a disturbed plasma amino acid pattern may occur with the elevation of any amino acid. The inultilateral effects of an elevated plasma amino acid on enzyme reactions and absorptions and transports of other amino acids especially during the development and maturation of an organism appears to be an important feature of amino acidopathies. This particular aspect may therefore further the understanding and future approach to mental disease and mental retardation. ACKNOWLEDGEMENTS

I am indebted to Dr. W. A. Himwich for her encouragement to complete this work as well as for her generous help. I am also grateful to Dr. G. R. Pscheidt for valuable advice. REFERENCES

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by phenylalanine and its relationship t o hair pigmentation in phenylketonuria. Nature (Lond.), 179, 199-200. NADLER, H. L., AND HSIA, D. YI-YUNG,(1961); Epinephrine metabolism in phenylketonuria. Proc. Soc. exp. Biol., 107, 721-723. NAGATSU, T., LEVITT,M., AND UDENFRIEND, S., (1964); Conversion of L-tyrosifie to 3,4-dihydroxyphenylalanine by cell-free preparations of brain and sympathetically innervated tissues. Biochem. biophys. Res. Coninirm., 14, 543-549. NEILL,K . C., AND LANGFORD, H. G . ,(1961); Studies on the production of phenylketonuria in the rat. Fed. Proc., 20, 2. OATES,J . A . , NIRENBERG, P. Z., JEPSON, J. B., SJOERDSMA, A , , A N D UDENFRIEND, S., (1963); Conversion of phenylalanine t o phenylethylamine in patients with phenylketonuria. Proc. Soc. exp. Biol., 112, 1078-1081. PAINE,R. S., (1960); Evaluation of familial biochemically determined mental retardation in children with special reference t o aminoaciduria. New Engl. J . Metl., 262, 658-665. PARE,C. M. B., SANDLER, M., AND STACEY,R. S., (1957); 5-Hydroxytryptamine deficiency in phenylketonuria. Lancet, i, 551-553. PAR^, C. M. B., SANDLER, M., A N D STACEY, R. S., (1958); Decreased 5-hydroxytryptophan decarboxylase activity in phenylketonuria. Lancet, ii, 1099-1101. PARE,C. M. B., SANDLER, M., A N D STACEY,R. S., (1959); The relationship between decreased 5hydroxyindole metabolism and mental defect in phenylketonuria. Arch. Dis. Childh., 34,422425. PERRY, T . L., (1963); N-Methylnietanephrinc: Excretion by juvenile psychotics. Science, 139,587-589. PERRY, T. L., HANSEN,S., TISCHLER, B., AND HESTRIN, M., (1964); Defective 5-hydroxylation of tryptophan in phenylketonuria. Proc. SOC.exp. Biol., 115, 118-123. PERRY, T. L., AND SCHKOEUER, W. A., (1963); The occurrence of aniines in human urine: determination by combined ion exchange and paper chromatography. J . Chroniatog., 12, 358-373. POLLIN,W., CARDON, P. V., AND KETY,S. S., (1961); Effects of amino acid feedings in schizophrcnic patients treated with iproniazid. Science, 133, 104-105. R A P ~ RH., S., (1928); The aerobic oxidases. Physiol. Rev., 8, 245-282. RAPPORT, M. M., GREEN, A. A., AND PAGE,I. H . , (1948); Krystalline serotonin. Science, 108,329-330. RENSON,J . , WEIWACH, H., AND UDENFRIEND, S., (1962); Hydroxylation of tryptophan by phenylalanine hydroxylase. J. biol. Chenr., 237, 2261-2264. RUSSELL, A., LEVIN,B., OBERHOLZER, V. G., A N D SINCLAIR, L., (1962); Hyperammonaemia. A new instance of an inborn enzymatic defect of the biosynthesis of urea. Lancer, ii, 699-700. SAKAMI, W., AND HARRINGTON, H., (1963); Amino acid metabolism. Ann. Rev. Biochern., 32, 355-398. SAMIY, A. H., AND SPENCER, R. P., (1961); Accumulation of 1-phenylalanine by segments of small intestine. Anier. J. Physiol., 200, 505-507. SCHAFER, I. A., SCRIVER, C. R., AND EFRON,M. L., (1962); Familial hyperprolinemia, cerebral dysfunction and renal anomalies occurring in a family with hereditary nephropathy and deafness. New Engl. J . Med., 267, 51-60. SCHANBERG, S. M., (1963); A study of the transport of 5-hydroxytryptophan and 5-hydroxytryptamine (scrotonin) into brain. J . fliurniacol. exp. Ther., 139, 191-200. SCHUMANN, H. J., A N D PHILIPPU, A., (1962); Release of catechol amines from isolated medullary granules by sympathomimctic aniines. Nafure (Lond.), 193, 890-89 I . SEN, N. P., AND M c G E ~ RP. , L., (1964); 4-Methoxyphenylethylamine and 3,4-dimethoxyphenylethylamine in human urine. Biochenr. hiophys. Res. Conitnun., 14, 227-232. SHAW,K. N. F., MCMILLAN, A., AND ARMSTRONG, M. D., (1 957); The metabolism of 3,4-dihydroxyphenylalanine. J . biol. Chem., 226, 255-266. SNYDERMAN, S. E., NORTON, P., AND HOLT,L. E., (1955); EfTect of tyrosine administration in phenylketonuria. Fed. Proc., 14, 450-451. SOURKES, T. L., (1962); Biochemical abnormalities in mental disease. Neurochemistry. K.A.C. Elliott, I. H. Page and J. H. Quastel, Editors. Springfield, Charles C. Thomas (p. 990-1011). SPENCER, R. P., A N D SAMIY, A. H., (1961); Intestinal absorption of L-phenylalanine in vituo. Amer. J. Plzysio/., 200, 501-504. SPRINCE, H., (1961); Indole metabolism in mental illness. Clin. Chem., 7, 203-230. STANBURY, J. B., (1960); Familial goiter. The metabolic Basis of inherited Disease. J. B. Stanbury, J. 5 . Wyngaarden and D. S. Fredrickson, Editors. New York, McGraw-Hill Book Company, Tnc. (p. 273-320). TADA,K., ITO,H., WADA,Y., AND ARAKAWA, T., (1963); Congenitaltryptophanuriawith Dwarfism. Tohoku J. exp. Med., 80, 118-134.

AMINO ACID METABOLISM I N AMINO ACIDOPATHIES

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216

Energy Flow in Brain F R E D E R I C K E. S A M S O N , J R . Department of Comparative Biocheniistry and Physiology, The University of Kansas, Lawrence, Kan. ( U . S . A . )

The energy requirement of the human brain is equal to that of a dim light, something like a 25-watt bulb (Kety and Schmidt, 1948), yet the statement is often made that the brain has a large requirement for energy. In fact, the energy requirements for functioning of several other organs are as great as that of the brain and certainly the energy utilization of a large, vigorously working muscle exceeds that of brain, on a weight basis as well as totally. None the less, we are impressed by the energy needs of the brain, not because of the amount but because the smallest interruption or even reduction of the rate of energy supply on the brain is almost immediately followed by functional failure. When we consider the great dependence of the other organs and, indeed, the entire organism upon the functioning of the brain, the energy requirement, although small, looms large. The name of Harold Himwich is almost synonymous with cerebral energy metabolism (Himwich, 1951), and it is appropriate, as well as a privilege to review the results of some of our experiments on brain energy metabolism in this volume dedicated to him on the occasion of his 70th birthday. Our studies have been very much influenced by the work and thinking of Dr. Himwich. The work presented here is based on the experiments and ideas of my colleagues as well as myself, in particular, Drs. William M. Balfour, Nancy A. Dahl and Dennis R. Dahl. Although some have questioned the restriction of brain energy metabolism to glucose (Geiger, 1962) - the almost continuous need for oxygen was recognized a long time ago and is not challenged. Yet in one sense, the brain energy metabolism does not have a need for the oxygen itself - more strictly speaking, it has a need to dispose of the electrons, spent after their journey falling down the electron transport system. Oxygen serves as an excellent acceptor since its supply is usually abundant and the product is a reasonably safe compound, water. In addition, of course, the necessary enzymes and other accoutrements to achieve this event are present. However, the oxygen supply does not have to be as continuous as the energy supply must be, as illustrated in the fact that pyruvate may replace oxygen as an electron acceptor for a period of time; glycolysis supplying energy rapidly enough to meet the needs of the adult mammalian brain for about a minute and in special instances such as the hypothermic newborn rat for more than an hour (Samson and Dahl, 1957).

ENERGY FLOW I N BRAIN

217

But if the energy supply is further reduced by inhibiting glycolysis, failure of the brain is almost immediate. The requirement for energy by the brain, then, is critical, indeed, there is a minimal rate of flow for activity and, since the dimensions ‘energy per unit time’ are equivalent to ‘power’, what we are really interested in is the ‘power consumption’. Himwich has discussed the survival of animals in anoxia and concludes from a sizable volume of work that the brain is the limiting organ in tolerance to anoxia (Himwich, 1951). Thus, under appropriate experimental circumstances, the survival time of an animal can give information about the state of the brain. The importance of glycolysis in meeting the energy requirements of the brain is seen in the great reduction of the survival time of rats in nitrogen, when they are injected with the glycolytic inhibitor, sodium iodoacetate (IAA) (Fig. 1).

Sec In nitrogen

Fig. I . Effect of IAA on survival time in nitrogen. IAA curve refers to rats injected with 1 pM/g body weight of NaIAA 5 min prior to anoxia; body temp. 35-38”; 21-day-old. Taken from Samson, Balfour and Dahl (1959).

Although the question has been raised about ATP as the direct source of energy, for example, in muscle contraction, it is a more difficult question in the case of brain largely because of the heterogeneous composition and the fact that there is not a single event with the discreteness of a muscle contraction to be considered, unless it be the action potential of nerve fibers. The experiments of Keynes show a direct connection between high energy phosphates and the pumping of Na+ in the squid axon (Keynes, 1960). We have found that the fall in the spike height and the conduction velocity in chicken vagus nerve when the energy supply is cut off by anoxia and a glycolytic inhibitor is very closely related to the fall in the ATP concentration (Dahl et a]., 1963). The question ‘is ATP directly in the flow of energy in any organ?’ is exceedingly difficult to answer with a conclusive ‘yes’, in view of the myriad of reactions involving ATP. That is, because of the large variety of paths available to ATP, the demonstration’of its participation in a main path, the route of the greatest amount of flow, would be:o bscured. The weight of the evidence from our experiments supports the idea that in brain Referenres p . 2271228

F. E. S A M S O N , JR.

218

TABLE I HIGH ENERGY PHOSPHATES A N D RELATED COMPOUNDS D U R I N G MATURATION

Average values on acid-soluble fractions of brain taken from Lolley et al. (1961) Age in days (pcM per g frozen brain)

ATP ADP AMP GTP GDP GMP UTP U DP UDP-Acetyl glucosamine UDP-Glucose

1

5

10

21

1.97 0.14 0.11 0.69 -< 0.01 i 0.01 0.32 < 0.01

2.10 0.20 0.14 1.10 '.c 0.01 * :. 0.0 1 0.43 -< 0.01

2.10 0.34 0.16 0.74 << 0.01 ~:0.01 : 0.21 0.01 :

1.99 0.39 0.17 0.66 .:' 0.01 <<0.01 0.36 c' 0.01

0.35

0.19

0.33

0.19

0.25 2.84

0.13 2.18 _ _ -

+

UMP CTP CDP ITP IDP IMP

I

. i0.01

CMP PC ___

0.17 2.99

0.17 2.63

~

t

I

"0

t

-

I

20

I

I

40

I

I t 60

I

I

80

Sec in nitrogen

Fig. 2. Cerebral ATP during anoxia. Rats at 35-38"; 21-day-old; arrows on abscissa indicate (50 survival time) from Fig. 1. Taken from Samson, Balfour and Dahl (I959).

ST50

ATP is directly in the main path of energy flow. Of the known -P compounds, ATP is the most abundant in brain (Table I). Although phosphorylcreatine (PC) is next in abundance and has been shown by many investigators to be the first of the -P compounds to fall when the energy supply is cut off (Lolley and Samson, 1962), the generally accepted idea is that the PC functions only as a reserve, regenerating ATP from ADP. It is clear that the cerebral ATP falls steadily when an animal is subjected

219

ENERGY FLOW I N B R A I N

to an atmosphere completely free of 0 2 . If, in addition, glycolysis is inhibited by the injection of IAA, the ATP disappears faster (Fig. 2). Under these conditions, the generation of ATP is almost completely stopped, especially after the first few seconds during which the PC has been exhausted as a reserve, and the rate of the disappearance should be a good measure of the rate of utilization. It is possible to estimate the rate of utilization by taking the slope of the line at its steepest point as has been done in Fig. 3. When this is compared with measurements of the 0 2 consumption which would be necessary to meet this utilization, we find a remarkably good agreement. More specifically, from the steep part of the curve describing the rate of ATP disappearance, we calculate the ATP utilization of the 21-day-old rat brain as 0.25 pM/g/ sec whereas the 0 2 utilization of electrically stimulated in vitro rat brain of 21-day-old rats was reported by Greengard and Mcllwain (1955) as 120 ,uM/g/h, which, at 6 -P generated per mole of 0 2 , gives a rate of 0.20 ,uM/g/sec.

t

I

I 1

I 2

I

1

I

3

Min in nitrogen

Fig. 3. Calculation of - P utilization from the slope of the ATP disappearance in the most rapid phases. The extrapolation value is 3.5 p M / g and, if one ATP contributes 2 - P to the pool, it would mean a total effective high energy pool of 7.0 ILM P per g. Taken from Samson, Balfour and Dahl (1960).

-

It is important to note that the ‘survival time’ of the rats in N2 corresponds to the same concentration of cerebral ATP with and without the IAA, although the length of the survival time is shortened by a factor of 6 by the IAA (see arrows in Fig. 2). A considerable amount of evidence favors ATP as the intimate substrate for many transport processes (see review by Edelman, 1961). It seems very likely that the energy flow in brain is largely directed to the maintenance of concentration gradients and those of Naf and Kf would be most prominent. Studies on transport mechanisms of those ions in other tissues, in kidney and erythrocytes, for example, have a direct bearing on the energy flow in brain and the converse is also true. On the other hand there is some evidence that -P is not on the direct path for the active transport of Na+ and this might apply to brain. More specifically, Conway (1963) has proposed a ‘redox’ energy supply for the Naf pump in muscle. In this theory, the flow of electrons through a special path, not including ATP, supplies References p . 227/228

220

F. E. S A M S O N , JK.

the energy for the Na+ transport. Recent experiments supporting this theory demonstrated an incrcase in the Na+ secretion in muscle following treatment with dinitrophenol (DNP). In these experiments, the flow of electrons was increased as seen in the increased 0 2 uptake or lactate production under anaerobia, and at the same time there were indications that the Na+ pump increased significantly. We wondered if a similar experiment on brain would give evidence about the ‘redox theory’ in brain. Mice were subjected to complete anoxia, and the survival time and the changes in the cerebral ATP concentration during the anoxia were determined. In the experimental mice, 2,4-DNP (30 mgikg) was injected and after a suitable period of time for absorption, they were subjected to anoxia and the survival time and the ATP changes determined. It was found that the average survival time decreased from 12 sec in the controls to about 6 sec after DNP and that the ATP concentration fell faster in the DNP injected rats. It was interesting that the cerebral ATP had fallen to the same concentration, 1.83 pM/g average in controls, and 1.86 pM/g average for experimentals at the time of the two different survival times, and both groups had about the same starting concentrations, 2.17 pM/g average for controls, and 2.25 pMig average for experimentals. The DNP had brought about an increased flow of electrons as evidenced by a small increase in the body temperature of the injected mice. However, the survival of the animal in the anoxia, which possibly is related to the operation of the cation pumps, corresponded closely to the ATP level and not to the increased electron flow. This evidence does not support a ‘redox theory’ in brain. The question might be raised : Is the power requirement related to the area of the active surfaces? If the energy utilization by the brain is considered to be directed to the maintenance of concentration gradients and if the events demonstrated in the squid axon are representative of those in the neuronal processes in brain, then the major gradients to be sustained are those of Na+ and K+ (Hodgkin, 1958). The very small extracellular space of brain (Dobbing, 1961), and the enormous ramifications of the minute extensions would mean that the movement of even small quantities of Na+ and K+ would produce sizable changes in gradients. This is illustrated in a rough mathematical calculation of the concentration changes : Suppose the extracellular space is composed of spaces of about 15 mp in diameter, the space volume associated with a 1 cm2 surface would be (I cm2) x (1.5)( 1O-fi cm) = (1.5)( 1O-fi) cm3. If the movement of Na+ and K t through an active surface is about the same as for the squid axon, namely, (4.5)(10-12 moles) cm2, then

(4.5)(10-12)(103) ..~ = (3)(10V) moles of Na+ or (I .5)( 10V)

K + per liter concentration change upon one impulse, With the salt concentration of mammalian extracellular fluids approximately 0.15 M , there would be a depletion of the total Na+ from the space adjoining the active surface with 50 impulses. It is just this very condition, it seems, that is the cause of the high susceptibility of the brain to an interruption of power supply. If the reasoning is correct we should expect a relationship between susceptibility to the interruption of the power supply (and the rate of energy flow) and the amount of

22 1

E N E R G Y FLOW I N B R A I N Survival of rots injected with I A A

Fig. 4. Survival time of different aged rats in a nitrogen atmosphere and after injection of IAA. Points represent maximal time survived. The inter-relationship of body temperature with age is also shown. Taken from Samson and Dahl(l957).

2.0

m

a

k

I

I

I

10

I

20

I

I

30

Sec in nitrogen

Fig. 5. Effect of age on cerebral ATP utilization. Rats of indicated age injected with 1.0 p M / g body weight of NaIAA 5 min prior to anoxia; body temp. 35-37". Taken from Samson, Balfour and Dahl (1960).

active surface. The brain of the rat in various stages of neonatal development seems to offer an opportunity to test this. The maturation from birth to early adulthood is associated with a development of increasing surface to volume relationships. The quantitative demonstration of this in the rabbit cerebral cortex has been demonstrated by Schadt and Baxter (1960). The dramatic tolerance of newborn animals t o anoxia has been known for a long time and Himwich and his colleagues related it to the energy metabolism of the brain (Himwich, 1951). Thus it is well-documented that there is a steady increase in the 0 2 consumption and glycolysis during the maturation of the rat (Himwich, 1951), and the increased tolerance of newborn rats is not a consequence of a greater glycolysis. This is illustrated in vivo by the increased tolerReferences p . 2271228

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F. E. S A M S O N , JR.

ance of newborn rats to anoxia even after glycolysis is inhibited by IAA (Fig. 4). Also it is not a consequence of a greater reserve of high energy phosphates, since these seem to be maintained at about the same concentration (Table I). The direct demonstration of the increased ATP utilization with maturation is seen in experiments measuring the rate of disappearance of ATP in brain under conditions of anoxia and glycolytic poisoning with IAA (Fig. 5 ) . These findings neatly fit the picture that the power requirement is directly related to the area of the active surfaces. It is important to note that the Naf-K+ stimulated Mg-ATPase which is believed to be involved in active cation transport (McIlwain, 1963), increases considerably during neonatal maturation in rat brain (Samson et al., 1964). There appears to be very little of this enzyme system in the 1-day-old rat and the system grows primarily in the 10-day-old to 21-day-old period. The increased ATP utilization during maturation suggested to us that the number or average size, or both, of mitochondria might also increase. We found, indeed, that the size of the mitochondria1 fraction obtained by differential centrifugation (Fig. 6),

C .(u +

h

-.-

Concentration of mitochondria1 protein per 100 g broin

2-

0

’D L

:’-/*

8 E

I

I

I

20

I

40

I

Age (days)

Fig. 6. ‘Mitochondria1fraction’ protein with age. Each point is average of 6 determinations on fraction prepared by differential centrifugation in 0.25 M sucrose. Taken from Dahl and Samson (1 959).

;t.L-L-L %

10

30 40 Age (days)

20

50

Fig. 7. Number of mitochondria with age. Mitochondria in homogenates counted under phase contrast microscopy (see text). Dotted lines represent the limits with a 0.95 probability. Calculated from data given in Samson, Balfour and Jacobs (1960).

223

ENERGY FLOW I N B R A I N

E“

I

OO L

20 t Age (days)

40

I

Fig. 8. ‘Mitrochondrial fraction’ protein per cell. Estimated from protein in differential centrifugation preparation of mitochondrial fraction and the DNA content of the brain. DNA assumed to be 6.1 pg/cell. Taken from Dahl and Samson (1959).

and the number of mitochondria counted with phase contrast microscopy (Fig. 7), increased during maturation. Particularly interesting is the increase of the average number of mitochondria per cell (Fig. 8). However, the average mass of the mitochondria calculated from these data suggests that the additional mitochondria which appear are smaller (i.e. average calculated mass falls from 0.4 pg to 0.3 pg in the I -day-old to 50-day-old rat). Although these experiments are strongly suggestive, they have the weakness that the ‘mitochondrial fraction’ of the brain is not composed entirely of mitochondria (Whittaker, 1963; De Duve, 1963), and phase contrast microscopy does not reveal enough about the structures to make mitochondrial identification certain. We have found the same kind of evidence for differences in the number of mitochondria in different species. The mitochondrial counts with phase contrast microscopy identification increase in the order frog, turtle, chicken and rat (Wahbe et al., 1961). However, the ratio of mitochondria to rate of energy flow is higher in the newborn (Samson et al., 1960). In other words, the ‘powerhouses’ seem to appear sooner in development than the ‘surfaces’ which will consume the power (Table 11). Is the rate of energy flow directly related to the activity of the brain? If the activity T A B L E I1 - POWER UTILIZATION COMPARISON Taken from Samson, Balfour and Dahl(l960)

POWER PRODUCTION

___

days

‘Mitochondria1 fraction’ protein mglg brain

1 21

11.0 24.4

Age in

Rate of ATP utilization iuMlglsec

ATP ratelmito. protein p M ATPlglmito. protein

-

0.02

1.8 10

0.25 ..

References p . 2271228

F. E. S A M S O N , JR.

224

of the brain is directly tied to the energy flow, and in particular, the flow through ATP, there should be a pronounced change in the energy requirements and rate of utilization of ATP with changes in the activity of the brain. In our experience, this is certainly the case. There is a definite reduction o f the energy requirement when the metabolism of the animal is slowed by reduction of the body temperature (Samson et a/., 1958) as illustrated by the greatly prolonged survival time in anoxia under these conditions (Fig. 4). The rate o f ATP utilization in the brain is also greitly slowed as the body temperature and hence the cerebral activity are reduced (Fig. 9). Some drugs which produce unconsciousness in animals have a similar effect. For example, ether and paraldehyde will greatly extend (Fig. 10) the survival time of rats in anoxia (Balfour et al., 1959) and also cause a reduction of the rate of ATP

t ot,

I

I

1

I

I

2 Min in nitrogen

'+

Fig. 9. Effect of temperature on ATP utilization. 21-day-old rats, cooled by immersion in cold water, injected with IAA and placed in an atmosphere of nitrogen. Slope lines are sketched in from the phase of rapid ATP disappearance. See Fig. 3 also. Taken from Samson, Balfour and Dahl (1960).

Fig. 10. Effect of ether, paraldehyde and alcohol on survival time. 21-day-old rats injected with I A A to block glycolysis and placed in Nz. All animals were unconscious from drug at the time of exposure to NZ (Balfour, Samson and Dahl, 1959).

225

E N E R G Y F L O W IN B R A I N

0

Control

u

00

10

20

30

Sec in nitrogen

Fig. 11. Effect of ether, paraldehyde and alcohol on ATP utilization. 21-day-old rats injected with IAA and placed in Nz while unconscious frdm the drug. Rats frozen immediately in liquidnitrogen and ATP in brain determined (Balfour, Samson and Dahl, 1959).

utilization (Fig. 11). On the other hand, ethanol in amounts which cause unconsciousness in rats neither extends the survival time or changes significantly the utilization rate of ATP. By and large, however, it seems that most of the drugs which depress cerebral activity also cause a reduction in the -P utilization rate; this is the result of reduced cerebral activity rather than its cause (Mcllwain, 1959); the higher than normal levels of -P in the depressed animals is evidence for this. A direct demonstration that connects ATP to activity in nerves is seen in the experiments of Greengard and Straub (1959) in which it was shown that stimulation of the rabbit vagus resulted in a drop in the ATP and other energy related intermediates. It seemed to us that a clearer demonstration of this could be shown if the generation of ATP were prevented, by studying nerves under anoxia and glycolytic inhibition. Experiments were carried out on the chicken vagus which because of its many non-niyelinated fibers should be permeable to IAA and also with many small fibers has a large amount of active surfaces per unit weight. The results showed a continual utilization of ATP even at ‘rest’ which was greatly increased when the nerve was stimulated. An increase in the rate of stimulation caused an increase in the disappearance rate of the ATP and, in addition, a more rapid failure of the nerve to conduct impulses (Dahl et al., 1964). It was found that the ‘cost’ of an impulse along the chicken vagus was about (5.40)(10-10 M ) of ATP per gram of nerve. This is about 10 times the calculated value for sciatic nerves composed largely of myelinated A fibers (Cheng, 1961). Although it appears from this that the energy cost of conduction in myelinated A fibers is much less than in non-myelinated C fibers, it must be pointed out that the number of fibers and active surfaces in a gram of vagus nerve far exceeds that in an equal weight of a nerve composed of large, myelinated fibers and as a result the cost per bit of information is probably less in the small nonmyelinated fibers (Dahl et a/., 1964). A very prominent and distinguishing aspect of brain metabolism is the role of glutamic acid as a fuel (Waelsch, 1962). It has been shown by Baxter et aZ. (1960) References p . 2271228

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that glutamic acid decarboxylase increases parallel to the surface area of the dendrites during maturation of the rabbit cerebral cortex. We can add further support to the general notion that glutamate metabolism increases with increasing complexity of the brain with a reaction of another kind. We found that the glutamic oxaloacetatetransaminase (GOT), both ‘soluble’ and particulate forms, increases several fold with the maturation of the rat brain (Samson et a]., 1962). Also, we have some evidence that a large part of the GOT is in a non-mitochondria1 particle. Among several animals the cerebral GOT per unit weight increases in the order: necturus, turtle, frog, chicken and rat, whereas, the lactate dehydrogenase both in ‘soluble’ and ‘particulate’ form is about the same in all of these animals (Dahl and Samson, 1963). Clearly, there is much work, both of a routine and of an imaginative kind, left to do in the study of energy flow in brain. In particular, the subcellular localization of various enzymes and substrates needs further study. The enormous diversity of the cellular processes and intracellular structures in brain tissue, many of wh’ch are not present in other tissues, makes extrapolation from studies on other organs especially inadequate. The most important question at the present time in the study of the cerebral energy flow appears to be the nature of the ionic pumps which represent the last major event of the energy flow in brain, the energy then leaving the brain in the form of heat. All the other metabolic steps are preparatory to this one, and this completed, the energy, now heat, is no longer useful to the brain. SUMMARY

Experiments relating nervous tissue activity and ATP utilization are presented and the changes in ATP utilization under a variety of experimental conditions are discussed. The survival of rats in anoxia was found to decrease with increasing neonatal maturation, and to be closely related to the decrease in cerebral ATP concentration. Inhibition of glycolysis by iodoacetate was shown to greatly decrease the survival time of rats in anoxia. In experiments with chicken vagus nerve, the spike height and conduction rate of non-myelinated fibers were found to be altered with changes in the ATP concentration. A calculation of the ‘energy cost’ for activity reveals a greater cost in the vagus nerve than in the frog sciatic (largely myelinated fibers), however, it is noted that the much greater number of small fibers in the vagus could mean a smaller energy cost per bit of information. The cerebral energy flow, in particular the ATP utilization, is shown to be greatly slowed with the reduction of brain ‘activity’ by hypothermia and by certain drugs; and in nerve fibers to increase directly with the rate of stimulation. The experiments discussed support the idea that ATP is directly in the path of energy flow in nervous tissue and further that the power (i.e. energy per unit time) requirement is related to the area of the active surfaces per unit weight of tissue. it is concluded that the major flow of energy in the brain passes through ATP and very likely is directed toward re-establ‘shing the ionic changes resulting from nervous activity, by way of a Nai-K+ stimulated Mg++ dependent ATPase system.

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ACKNOWLEDGEMENT

The work reviewed here was aided by grants B-1151 and B-3070 from the National institute of Neurological Diseases and Blindness, U.S. Public Health Service. REFERENCES

W. M., SAMSON, F. E., A N D DAHL,N. A., (1959); Effect of certain central nervous system BALFOUR, depressants on cerebral energy metabolism. Physiologisr, 2 , Nr. 3. E., (1960); Maturational changes in cerebral cortex. 11. BAXTER, C. F., SCHADE,J . P., A N D ROBERTS, Levels of glutamic acid decarboxylase, y-aminobutyric acid and some related amino acids. Znhibition in the Nervous System and y-Aminobutyric Acid. E. Roberts, Editor. Oxford, Pergamon Press (p. 214). CHENG,S. C . , (1961); Metabolism of frog nerve during activity and recovery. J . Neurochem., 7, 278-288. CONWAY, E. J., (1963); Significance of various factors including lactic dehydrogenase on the active transport of sodium ions in skeletal muscle. Nature, 198, 760-763. F. E., (1959); Metabolism of rat brain mitochondria during postnatal DAHL,D. R., AND SAMSON, development. Amer. J . Physiol., 196, 470-472. DAHL,D. R., A N D SAMSON,F. E., (1963); Particulate glutamate-oxaloacetate transaminase and lactate dehydrogenase in brain. Comparative distribution among vertebrates. Fed Proc., 22, 2821. F. E., A N D BALFOUR, W. M., (1964); ATP and electrical activity. Amer. J . DAHL,N. A., SAMSON, Physiol., 206, 8 18 --222. DE DUVE,C., (1963); The scope and limitations of cell fractionation. Methods of Separation of Subcellular Str~icturalComponents. Biochemical Society Symposia No. 23. Cambridge, University Press (p. 1). J., (1961); The blood-brain barrier. Physiol. Rev., 41, 130-188. DOBBING, I. S., (1961); Transport through biological membranes. Ann. Rev. Physiol., 23, 37-70. EDELMAN, A,, (1962); Metabolism and function in the brain. Neurochemistry. K. A. C. Elliott, I. Page GEIGER, and .I. H. Quastel, Editors. 2nd Edition. Springfield, C. Thomas (p. 128). H., (1955); Metabolic response to electrical pulses in mammalian GREENGARD, P., A N D MCILWAIN, cerebral tissues during development. Biochemistry of the developing Nervous System. H. Waelsch, Editor. New York, Academic Press (p. 251). R. W., (1959); Effect of frequency of electrical stimulation on the GREENGARD, P., AND STRAUB, concentration of intermediary metabolites in mammalian nonmyelinated fibers. J . Physiol., 148, 353-361. HIMWICH,H . E., (1951); Brain MetaDolism and cerebral Disorders. Baltimore, The Williams and Wilkins Co. A. L., (1958); Ionic movements and electrical activity in giant nerve fibers. Proc. roy. HODGKIN, SOC.B , 148, 1-37. KETY,S. S., AND SCHMIDT, C. F., (1948); The nitrous oxide method for the quantitative determination of cerebral blood flow in man: theory, procedure and normal values. J . clin. Invest., 27, 476-583. KEYNES, R. D., (1960); Regulation of the inorganic ion content of cells. Ciba S,vmp. Study Group No. 5. Boston, Little, Brown (p. 77). LOLLEY, R. N., BALFOUR, W. M., AND SAMSON, F. E., (1961); The high-energy phosphates in developing brain. J . Neurochem., 7,289-297. F. E., (1962); Cerebral high-energy compounds: Changes in anoxia. LOLLEY, R. N., AND SAMSON, Amer. J . Physiol., 202, 77-79. H., (I 959); Biochemistry and the Central Nervous System. 2nd Edition. Boston, Little, MCTLWAIN, Brown. MC~LWAIN, H., (1963); Chemical Exploration of the Brain. Amsterdam, Elsevier. F. E., BALFOUR, W. M., A N D DAHL,N. A,, (1958); The effect of age and temperature on the SAMSON, cerebral energy requirement in the rat. J . Geront., 13, 248-251. SAMSON, F. E., BALFOUR, W. M., A N D DAHL,N. A., (1959); Cerebral free energy and viability: ATP in rats under nitrogen and iodoacetate with the effects of temperature. Amer. J . Physiol., 196, 325-326.

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SAMSON, F. E., BALFOUR, W. M., AND DAHL, N. A., (1960); Rate of cerebral ATP utilization in rats. Amer. J . Physiol. 198, 213-216. SAMSON,F. E., BALFOUR, W. M., AND JACOBS,R. J., (1960); Mitochondria1 changes in developing rat brain. Amer. J . Physiol., 199, 693-696. SAMSON,F. E., A N D DAHL, N. A., (1957); Ccrcbral energy requirement of neonatal rats. An7er. J. Physiol., 188,277-280. SAMSON, F. E., DAHL,D . R., AND FULLER, T. C., (1962); Enzyme distribution in brain subcellular particles, XXII Int. Congr. Physiol. Sci., Abstract No. 1125. SAMSON, F. E., DICK, H., AND BALFOUR, W. M., (1964); The cerebral Na+-K+stimulated Mg++-ATPase during maturation of the rat. Lije x i . , 3, 511-515. S C H A DJ.~ , P., AND BAXTER,C. F., (1960); Maturational changes in cerebral cortex. 1. Volume and surface determinations of nerve cell components. Inhibition in the Nervous System and y-Aminobutyric Acid. E. Roberts, Editor. Oxford, Pergamon Press (pp. 207-213). WAELSCH, H., (1962); Amino acid and protein metabolism. Neurochemistry. K. A. C . Elliott, I. Page and J. H. Quastel, Editors. 2nd Edition. Springfield, C. Thomas (p. 288). WAHBE,V. G., BALFOUR, W. M., AND SAMSON,F. E., (1961); A comparative study on vertebrate brain mitochondria. J . comp. Biochem. Physiol., 3, 199-205. W H I r r A K m , V. P.,(1963); The separation of subcellular structures from brain tissue. Methods of Separation of subcellular Atructural Components. Biochemical Society Symposia No. 23. Carnbridge, University Press (p. 109).

229

Direct Action of Atropine on the Cerebral Cortex of the Rabbit F. R I N A L D I Neuropsychiafric Institute of ihe Universiiy of Naples, Naples (Italy)

In an earlier study (Rinaldi and Himwich, 1955) we described how atropine inhibits electrographic alerting responses produced by physiological stimuli and by direct stimulation of the midbrain reticular formation. This explained how the EEG of atropinized animals resembles that occurring during natural sleep. Cholinergic drugs, on the contrary, evoke an EEG pattern of permanent alertness. We interpreted these findings with the hypothesis that a cholinergic mechanism was involved in that function of the reticular system subserving desynchronization of cortical activity in the ‘alerting’ or ‘activating’ responses. In a later study (Rinaldi and Himwich, 1955b) we observed that the effects of atropine and cholinergic drugs remained after transection of the brain stem at the midbrain level. This was taken as an indication that the site of action of these drugs in so far as they affect the electrical activity of the brain - had to be higher than the lower midbrain. Furthermore, we observed that the electrical activity of a cerebral hemisphere surgically separated from its neural connections with the rest of the brain (isolated hemisphere) could not be modified by atropine and cholinergic drugs, at least in acute preparations. We therefore extended our hypothesis by stating that the site of action of these drugs had to be located at the mesodiencephalic level. We thought that the cholinergic portion of the reticular system responsible for the desynchronization of cortical activity should be searched for at the diencephalic or upper midbrain level. In a later investigation (Rinaldi, 1959) we again studied the effect of atropine and cholinergic drugs in isolated rabbit hemispheres, using chronic preparations rather than experimenting shortly after surgery, as we had done before. In these experiments it was observed that the electrical activity of the isolated cortex can indeed be modified by atropine and cholinergic drugs, although the typical opposite electrical patterns (sleep-like with atropine, alert-like with cholinergic drugs) cannot be recognized as in the intact cortex. This fact was sufficient to consider again the cortex as a possible site for the action of atropine and cholinergic drugs. Perhaps the atropine-induced blockade of ‘alerting’ responses, from stimulation of reticular system, is exerted at the cortical level. Either the cholinergic neurone is the reticulo-cortical path and atropine blocks its References p. 2431244

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termination, or a cholinergic function resides in an intracortical mechanism without which the reticular desynchronizing effect cannot be brought about". It became apparent, therefore, that data should be obtained as to the direct action of atropine on the cerebral cortex itself. The present report concerns an experimental attempt to study the effect of atropine on the spontaneous electrical activity of the cerebral cortex, in conditions ensuring that the effect of atropine is exerted only 01 predominantly on the cerebral cortex, in an experimental preparation that maintained intact all connections of the cortex with the rest of the brain. These conditions were realized by two techniques. One was the classical method of applying atropine directly to the surface of the cortex. The other was the infusion of atropine directly into the cortical circulation by means of a microcannula inserted in a cortical artery. METHODS

( A ) General techniques

The data reported in this paper were obtained in 22 albino rabbits ranging in weight from 1.8 to 3.5 kg. All experiments were performed in curarized animals and artificial respiration was maintained by positive pressure through a tracheal cannula. The head was immobilized in a stereotaxic instrument of the type described by Sawyer et al. (1954). Recording of the electrical activity of the cerebral cortex and other brain structures was effected by means of an inkwriting electroencephalographic instrument. Electrical activity of all sites under study was recorded against a common reference electrode: a stainless steel screw was inserted in the bone at the root of the nose. Cortical electrodes were made of 1.5 mm wide solder discs at the extremity of vinyljacketed lead-off wires. Subcortical electrodes consisted of pairs of Formvar-coated nichrome wire, 200 p thick, twisted together to form a single bipolar electrode. Its tip was cut in a slanted plane so that the bare points of the two poles were vertically separated by a distance of not more than 1 mm. For placement of subcortical electrodes, the coordinates given in the atlas of Sawyer and others (1954) were followed. Actual positioning was ascertained postmortem by a histological method. Subcortical electrodes and cortical electrodes, for sites not underlying craniectomy openings, were inserted through 2 mm wide drill holes in the skull. The electrodes were fixed to the skull with acrylic cement. Two Grass S-4 stimulators witha stimulus isolation unit were used for electrical stimulation of brain centres. The data reported here refer only to stimulation of the midbrain reticular formation to elicit 'alerting' or desynchronization of spontaneous cortical activity. For this purpose trains of pulses of 0.1 msec duration at a frequency of 300/sec were employed. The intensity of the stimulation was varied as necessary to produce the above-mentioned effect.

* Other workers have furnished data relating to this subject. They will be discussed later, together with the findings of the experiments described in the present paper.

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23 1

The selected target site for the tip of the stimulating electrodes was the lateral reticular formation of the upper midbrain at the level of plane P-8 of Sawyer’s coordinates. ( B ) Topical application of atropine A first group of 16 animals was used for studies of the action of atropine topically applied to the surface of the cerebral cortex. In these experiments the sensorimotor cortex of the right side was exposed through a craniectomy 5-8 mm wide and a star-like dural incision of the same size. Atropine sulphate (0.5 % solution in saline) was applied to the cortical surface by means of squares of filter paper 3 mm wide. The following method was used in all animals, in order to ensure application of paper squares always to the same cortical site and in the same spatial relation to the recording electrodes. The point of the cortical surface, where movements of the contralateral forepaw were obtained with threshold stimulus intensity, was determined before curarization. On this point a recording electrode was placed and its lead-off wire was cemented to the bony edge of the craniectomy. The atropine-soaked squares were placed 2 mm medially to the recording electrode, in order that an eventual effusion of atropine solution from the paper would run by gravity toward the electrode site. ( C ) Atropine infusion in a cortical artery

A second group of animals was used for the study of the effects of atropine infused in a cortical artery. The number of animals in which this method was tried was, in fact, 21. The technical difficulties were so great, however, that many preparations had to be considered faulty for one reason or another. Therefore, our observations are limited to only six animals that were acceptable as adequately prepared. The infusion was performed by means of a glass microcannula obtained from a haematocrit capillary glass tube by the technique currently used for preparing micropipettes. The diameter of the opening of the cannula tip was about 100 p. The cannula was very short (about 3-4 mm) for minimal weight and its tip was as short as possible for maximal resistance to breaking during insertion. The glass microcannula was fitted to the end of a light and very flexible vinyl plastic tubing of 0.25 mm inner diameter and 0.5 mm outer diameter. The tubing, 15 cm long, was connected to a chamber of 1 ml capacity made of self-sealing rubber. This was the injection chamber. The chamber was in turn connected by 25 mm of tubing to a Murphy drip with flow characteristic of 60 drops per ml. The Murphy drip was fed through a larger rubber tubing, 3 m long, attached to an 1.V.-infusion bottle suspended from the ceiling by a pulley mechanism allowing regulation of height and therefore of pressure and flow rate.

( I ) Artery selection In the rabbit, practically the entire convexity of the cerebral hemisphere is fed through branches of the middle cerebral artery. Exception is to be made for a narrow strip of cortex along the interhemispheric fissure, which is fed by the terminal arborizations References p . 2431244

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of the anterior cerebral artery, branching along the mesial surface ofthe hemisphere. The demarcation between the two territories, that of the middle cerebral and that of the anterior cerebral, is marked by a longitudinal ideal line running in the only shallow sulcus recognizable on the rabbit brain convexity : the lateral sulcus. The middle cerebral artery is the direct continuation of the internal carotid and is usually very short or inexistent as a main trunk because it divides very soon after the origins of the anterior cerebral and posterior communicating arteries. The primary branches of the middle cerebral artery are usually 3 or 4, rarely 5 . A first and most posterior branch runs backward and is distributed to the posterior part of the pyriform lobe. The second one is distributed also to the pyriform lobe, but its terminal branches cross the rhinal fissure in its posterior third and feed posterior neocortical areas of the basal and lateral surface of the hemisphere, without appearing on the convexity. The third and fourth primary branches are the most anterior ones and often arise as a single trunk that does not divide until it has reached or crossed laterally the rhinal fissure. These primary branches give origin to 4-6 secondary branches that pass over to the convexity. The larger and more constant of these branches - visible on the convexity - is, counting from back to front, the third one. It usually divides into two terminal branches in a Y-shaped configuration, just laterally to the anterior end of the lateral sulcus. The angle formed by the short arms of the Y opens backward and slightly upward. This branch was the one selected for cannulation and the cannula was inserted at the point of division, entering the main trunk against the direction of the blood flow. ( 2 ) Cannulation of the artery The two common carotid arteries were prepared and the right one, ipsilateral to the side of cannulation, was ligated. Around the other one, a tie was passed in order that a traction on the tie could occlude temporarily also the left carotid to ensure reduction of blood pressure and flow in the cerebral territory. A large craniectomy was performed to expose as much as possible of the convexity of the right hemisphere; the cortical electrodes were put in place on the surface and their lead-off wires secured to the edges of the craniectomy. An assistant was ready to operate the carotid tie, when necessary, and to maneuver a gentle flow of warm saline to wash away blood that might obscure the operation field. The perfusion system was filled with heparinized (10 mg/100 ml) Ringer’s solution and the pressure adjusted to cause rapid drop formation at the tip of the microcannula. A dissection microscope was focused over the Y-shaped division of the large arterial branch. With a very sharp steel point (a dissection needle electrolytically sharpened) the artery wall was punctured at the division point, to make at way for the glass cannula. Bleeding was controlled but not stopped by traction on the carotid tie. Then, while blood was washed off by the flow of saline, the microcannula was inserted in the artery in a direction parallel to the vessel and rested flat on the cortical surface.

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The cannula was handled by its plastic tubing and carefully pushed into the artery until the leakage of blood stopped. This was the most critical point of the operation, since breaking of the cannula tip or the artery wall or tearing of the collaterals may easily happen. The perfusion bottle was raised until the blood that had run up inside the cannula and the tubing ran back into the artery and fluid flowed through the Murphy drip at the rate of about 20 drops per min. Paraffin of low melting point (42") was then dripped over the cannula, the artery and the end of the tubing, to secure stability of the connection. Then the entire portion of tubing running over the cortex was imbedded and the entire posterior half of the exposed brain surface was covered as far as the bone edges by paraffin. The paraffin solidifies sufficiently to maintain the cannula in position and yet it is sufficiently elastic to follow the slight movements of the cortical surface caused by respiratory acts. ( 3 ) Causes of .failure of the preparation Because of the smallness of the vessel (just about the size of the cannula tip when filled with blood) and of the fragility of the cortical tissue, the technique can fail very easily. Most commonly traction on the artery causes tearing of cortical penetrating branches with intracortical or subpial bleedings. Often the tip of the cannula breaks or slips out of the hole in the vessel wall so that bleeding resumes and perfusion fluid runs out instesd of into the artery. Less commonly the cannula or the artery becomes clogged. Some of these failures can be recognized during the preparation stage. Later on, however, as the field is covered by paraffin, one cannot be sure that the perfusion proceeds correctly, even when the entire experiment runs smoothly. Even the experimentally induced changes of cortical activity do not help to detect faulty perfusions. In fact, the effect of atropine infused through the cortical circulation is in many respects similar to that of atropine applied to the cortical surface. To have a definitive check, all experiments were ended by injecting into the perfusion system, while the animal was living, a dye solution (toluidine blue) to ascertain whether the flow was occurring only and entirely into the arterial system. The faulty experiments were very numerous, in fact two out of three experiments had to be eliminated from consideration. The dye perfusion tests have shown that, in successful experiments, staining of vessels involved retrogradely the perfused branch and the other cortical branches of the middle cerebral artery as far anteriorly as the sensorimotor area. ( 4 ) Experimental infusion of atropine

This was effected by injecting into the rubber chamber 0.1 ml of a 0.5 solution of atropine sulphate (a dose of 0.5 mg). Considering the volume of fluid contained in the part of the perfusion system where the atropine could diffuse, we estimate that the concentration of atropine entering the cortical circulation was initially maximal, about 0.40 mg/ml, and then decreased progressively to zero. References p . 243!244

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In control recordings of the electrical brain activity, the resting or ‘sleeping’ state is characterized (Fig. 1) by random high-voltage slow activity and periodic 14/sec

Fig. 1 . Electrical activity of the rabbit brain in control conditions with features of a sleeping pattern. The leads are: 1. electrocardiogram; 2. left ventrolateral thalamic nucleus; 3. right sensorimotor cortex; 4. left sensorimotor cortex; 5. right visual cortex; 6 . left visual cortex; 7. right hippocampus.

. .

ret.1 stim.

2’;

L 1 sec

Fig. 2. An alerting response to reticular high-frequency stimulation (at the arrow), in control conditions. Leads as in Fig. I.

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spindles in cortical leads of both sides. The hippocampal recording exhibits instead 4-7 sec rhythmic waves mixed with slower wave-forms and faster activity. A high-frequency reticular stimulation (Fig. 2) of threshold intensity, causes a desynchronization of cortical activity that is simultaneous in both hemispheres and is characterized by disappearance of slow waves and 14/sec spindles and appearance of fast activity of low voltage. In the hippocampal record the ‘alerting’ response appears as an augmentation of amplitude and regularization of the 4-7 sec activity. ( A ) Infusion of atropine in a cortical artery

Shortly (3-5 min) after 0.5 mg of atropine sulphate has been injected into the rubber chamber of the perfusion system (see METHODS), a change sets in in the activity of the cortex of both sides. This occurs after enough atropine has entered the general circulation to cause an increase in heart rate. The modification of the cortical activity consists of a persisting desynchronization in the contralateral hemisphere while on the perfused side spindles and slow waves invariably predominate (Fig. 3). This asymmetry of activity pattern between the cortices of the two sides is profound and lasts for at least 20 min. That is to say, a period sufficiently long to make it possible that the fluid of the injection chamber be renewed at least six times by continuing flow of fresh Ringer’s solution. At all times, while the asymmetric pattern is present, the spindles and slow waves of the perfused side can be readily suppressed by reticular substance stimulation (Fig. 4). The threshold for this effect remains approximately equal to that observed in control conditions for an alerting response. The activity of the contralateral cortex, which was already of the ‘alert’ type, remains unchanged by reticular stimulation.

2

Fig. 3. Electrical brain activity during infusion of atropine in an arterial branch of the right cortex. The right side cortical leads are Nos. 3 and 5. Leads as in Fig. I, References p. 2431244

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Fig. 4. An alerting response (at the arrow) to reticular stimulation during infusion of atropine in an arterial branch of the right cortex. Leads as in Fig. 1.

Fig. 5. Electrical brain activity after repeated infusions (see text for dosages) of atropine in an arterial branch of the right cortex. Leads as in Fig. 1.

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Thus, while the ‘alerting’ effect lasts, the cortical activity of both sides has the characteristics of a normal ‘alert’ record ; fast activity of low voltage predominates. However, a faint asymmetry may be apparent (Fig. 4): the activity of the perfused side may include, in addition to fast activity, some low-voltage slower waves with a definitely sharp positive phase. As the alerting effect following reticular stimulation wanes, spindles and slow waves reappear in the record of the infused cortex only. Thc asymmetry between the cortical patterns of the two sides gradually disappears as more time elapses after the introduction of atropine into the infusion system. If no additional atropine is introduced, a normal situation returns with a sleep-like pattern appearing on both sides. If, instead, the introductions of atropine are repeated 2-3 times, at intervals of 10 min, to ensure that the infusion fluid is never completely free of atropine, a further modification occurs. Spindling and slow waves appear also in the tracing from the contralateral cortex. Both cortices exhibit a sleep-like pattern (Fig. 5 ) which is symmetrical as far as the 14/sec spindles are concerned. The slow wave activity, instead, is somewhat different, because several of the slower wave forms of the infused side have higher voltage and a definite spike-like appearance. This situation becomes fully developed after at least 1.5-2 mg of atropine have run through the infusion system. A reticular stimulation can well desynchronize the cortical activity of both sides, although in the record of the perfused cortex the fast low-voltage activity is less and some spiky slower waves remain present (Fig. 6). The threshold for production of the ‘alerting’ effect by reticular stimulation begins to increase when more than 1.5 mg of atropine have entered the circulation.

Fig. 6. An alerting response (at the arrow) to reticular stimulation, in condition as described for Fig. 5. Leads as in Fig. 1. References p . 243/214

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Further addition of atropine does not produce more asymmetrical effects than those described above. As the total quantity of atropine augments, the changes of the spontaneous electrical activity of both cortices and of the threshold for reticular alerting, repeat more and more closely what has been described (Rinaldi and Himwich, 1955a) when large amounts of atropine are administered by intravenous injection. ( B ) Topical application of atropine to /he cortical surface

One application of a paper square soaked with atropine solution, usually does not cause any change of the electrical activity of the cortex of either side. If the operation is repeated, with substitution of the paper squares every 3-4 min, a change appears after the second or third application. In the cortical record of the side where atropine is applied, spindles and slow waves dominate. Thus an asymmetry between the cortical activities of the two sides becomes apparent and it is of the same type as that occurring during arterial infusion. A stimulation of the reticular formation blocks spindling and slow waves present in the cortical tracings of the side where atropine was applied. Furthermore, reticular stimulation evokes bursts of high-voltage spikes in the tracing recorded from the electrode near to the site where atropine was applied (Fig. 7).

2

S

I set

Fig. 7. Electrical brain activity and response to reticular stimulation (at the arrow) after topical application of atropine to the right sensorimotor cortex (lead 3): early effects. Leads as in Fig. 1.

This local convulsive activity evoked by reticular stimulation is at first a brief burst occurring during the train of high-frequency stimuli delivered to the midbrain reticular substance. Later, when the application of atropine has been repeated several times, the bursts of high-voltage focal spikes continue after the end of reticular stimulation

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(Fig. 8). A characteristic of these spikes is that they are limited to the neighbourhood of the atropinized area, because they do not appear in the recording from the visual area of the same side.

A

r

ret. stim.

I

~?L set I

Fig. 8. Electrical brain activity and response to reticular stimulation (at the arrow) after repeated topical application of atropine to the right sensorimotor cortex (lead 3). Leads as in Fig. 1.

Eventually random focal spikes appear at the atropinized site, in a spontaneous manner, i.e. independently of reticular stimulation. DISCUSSION

( A ) Dominance of a sleep-iike pattern on the atropinized side

The asymmetry of electrical activity pattern between the cortices of the two sides is so marked that it is considered to be unequivocally existent. Also its relationship to an asymmetrical distribution of atropine can be accepted, because the difference of electrical pattern is of the same general type whether atropine enters the arterial circulation of the cortex or it is topically applied to the surface of it. Although our method does not include a direct determination of how atropine is distributed, we can reasonably assume that, when the drug enters the circulation by a cortical arterial branch, it reaches earlier a higher concentration in the blood flowing through the cortical tissue of the same side. Furthermore, we can safely assume that local activity changes following surface application of atropine are related, at least in part, to effects of the drug solution diffusing through the superficial layers of the cortex. We have seen that the electrical cortical activity of the side atropinized by either method possesses the characters pertaining to the normal pattern of sleep : spindles and slow waves. References p . 243/244

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This fact could suggest the interpretation that since general administration of atropine enforces an electrical pattern of sleep in the whole brain, concentrations of atropine sufficiently high in the cortex of only one side enforce a local synchronization of electrical activity. Therefore, the synchronizing action of atropine might be attributed to a cortical site of action. The synchronized activity induced by local infusion of atropine appears still subject to the desynchronizing action of reticular influences. Reticular stimulation can readily stop spindling and slow waves in the atropinized cortex at least as long as not enough atropine has entered the general circulation to evoke synchronization of the other hemisphere, thus acting in the same way as when generally administered. This is in accordance with observations showing that even when atropine has blocked electrocortical arousal responses from stimulation of the reticular formation, this stimulation is able to produce other important cortical effects. For instance, reticular facilitation of visual evoked cortical potentials remains (Bremer and Stoupel, 1959). Also the inhibitory effect exerted by the reticular formation upon thalamocortical recruiting is unimpaired (Loeb and collaborators, 1960). Finally, reticular stimulation can still produce a significant alteration of the spontaneous activity of the cortex, detectable by frequency analysis techniques (Martin and Eades, 1960). It appears that the synchronizing effect exerted by atropine at the cortical site is not due to a gross blockade of the termination of reticulo-cortical pathways. However, while local atropinization does not block electrocortical arousal responses, general administration of atropine definitely blocks desynchronization of cortical activity inducible by physiological alerting stimuli (Bradley and Elkes, 1953; Rinaldi and Himwich, 1955a) and by stimulation of the reticular formation (Rinaldi and Himwich, 1954a; Longo, 1955; Loeb et al., 1960). This indicates that a direct synchronizing action of atropine on the cerebral cortex does not explain entirely the effects of systemically administered atropine on the spontaneous activity of the brain and on its reactivity to reticular arousal. Other sites of action, probably at subcortical levels, must play an important role, although their identification remains uncertain at the present time. The studies with micro-injections and micro-implantation of cholinergic substances and of atropine within subcortical centres, begin to afford important data in this regard. Tn the reticular formation and in basal forebrain structures exist centres with opposite function in regulating sleep behaviour and correlated cortical activity, although they are all stimulated by cholinergic agents and inhibited by atropine. From certain sites, mainly in the medial reticular formation of the brain stem and more forward, up to the preoptic region, cholinergic stimulation induces sleep with synchronization of cortical activity, whereas atropine evokes alertness and desynchronization (Hernindez-Peon, 1962; Cordeau et al., 1962; Velluti and Hernindez-Peon, 1963; Hernindez-Peon et al., 1963). From other sites, mainly in the lateral reticular formation and in the septa1 region, it is atropine that evokes synchronization and sleep, whereas acetylcholine induces alertness (Hernandez-Peon et al.. 1963). It appears therefore that many sites exist where generally administered atropine

DIRECT ACTION OF ATROPINE O N THE CEREBRAL CORTEX

24 1

can act. Some of these (the cortical site as well as the lateral regions of the midbrain tegmentum and the septa1 areas) favour synchronization of electrocortical activity and eventually also behavioural sleep when sufficient concentrations of atropine reach them. Others, on the contrary, (medial regions of the midbrain tegmentum, hypothalamus and preoptic region) enforce, when acted upon by atropine, behavioura1 alertness and low-voltage fast activity in cortical records. When very large doses of atropine are given, the synchronizing effects on cortical activity as well as the blockade of mechanisms subserving maintenance of behavioural alertness are the prevailing result. This is the situation that probably occurs in man when atropine intoxication is used to cause a comatose state for the treatment of mental conditions (Forrer, 1951). With lower amounts of atropine the most commonly known situation occurs: the cortical activity is synchronized and unresponsive to stimuli that usually desynchronize it and cause at the same time behavioural alerting. In this stage, behavioural alertness is maintained, at least in most animal species, despite the loss of its electrocortical counterpart (Wikler, 1952; Bradley and Elkes, 1953). Our findings suggest that this situation is explainable only in part on the basis of a direct action of atropine on the cerebral cortex. Precisely it is the enforcement of a sleep-like pattern that can be due to a cortical action of atropine. The loss of the electrical arousal response cannot be explained by our findings relative to the direct action of atropine on the cortex. In fact, the sleep-like pattern, locally prevailing in the atropinized cortex, can be easily interrupted by reticular stimulation. The suprareticular and suprathalamic site of action of atropine, postulated by Loeb and collaborators, exists and seems to be located within the cortex, but it does not explain the blockade of the EEG arousal reactions. An operational description of the cortical action of atropine could be termed as a facilitation of synchronizing mechanisms without blockade of desynchronizing influences. ( B ) Desynchronization of electrical activity of the cortex contralateral to the atropinized side

The asymmetry of spontaneous electrical patterns between the two cortices is not engendered only by the prevalence of a sleep-like pattern on the atropinized side. An alert-like pattern, with low-voltage fast activity, dominates in the contralateral cortex. This is to be considered a positive finding and related to the atropinization of the other side. In control conditions, before the addition of atropine to the perfusion fluid, the cortical activity of both sides exhibits alternation of patterns, with prevalence of sleep features occasionally interrupted by ‘arousal‘ responses related to unintentional or intentional alerting stimuli reaching the animal. Such basal conditions are ensured by ‘ad hoc’ regulation of artificial respiration rate and volume, keeping in mind that underventilated animals tend to present a predominantly alert record, while in overventilated animals the sleep features prevail (Rinaldi and Himwich, 1955a). Differently from the control situation, when the asymmetrical action of atropine is present, References p . 243/244

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F. R I N A L D I

spindles and slow waves remain durably absent in the record from the non-atropinized side. If this phenomenon had occurred only with the technique of arterial infusion, where some atropine soon enters the general circulation, it could be undoubtedly attributed to an action of atropine on the contralateral cortex or other brain sites. However, the contralateral desynchronization occurs as an early change also when atropine is topically applied to the surface of one cortex. Therefore, the hypothesis may be advanced that corticifugal influences from the atropinized cortex affect directly or through cortico-reticular mechanisms the activity of the contralateral cortex, enforcing there an alert-like pattern. The direct action of atropine on the cortex seems to consist in a facilitation of intracortical synchronizing mechanisms. This fact leads to the additional hypothesis that local cortical synchronization may cause facilitation of general ‘alerting’ mechanisms. This possibility is obviously very important for understanding the function of mechanisms controlling integration and homeostatic regulation within the brain. ( C ) Convulsant efect of topically applied atropine

A late effect of topical application of atropine solution to the surface of the cortex is the appearance of focal high-voltage spikes. These discharges seem to be of a convulsive nature. In few experiments that were performed without curarization, these electrical bursts were, in fact, associated with clonic movements of the contralateral limbs and with masticatory movements. Experiments with topical application of atropine (0.2 % solution) were described by Miller et al. (1940) and of 2 x 10-5 atropine solution by Artemev and Babskii (1950) to counteract the effects of topically applied acetylcholine, eserine and prostigmine. These authors do not refer to any effect of atropine on the spontaneous activity of the cortex; it is, however, to be noted that the solutions employed were less concentrated and applied only once or repeated at long intervals. A convulsant action of atropine injected into the subarachnoidal spaces in the dog has been described by Brailovski and Ponirovski (1935). These authors defined the seizures as typically epileptic and of the type observed after local cooling of the cerebral cortex. The convulsant effects observed in our experiments and those reported by Brailovski and Ponirovski need further study to exclude aspecific mechanisms, such as osmotic changes or alterations in the concentration of electrolytes, which are to be considered whenever drug solutions are applied topically. It remains possible, however, that the direct action of atropine on the cerebral cortex may eventually evolve into a convulsive effect. A mechanism of this type may explain in part the augmentation of focal spikes in the EEG of epileptic subjects, observed after i.v. administration of atropine by Minvielle et al. (1954). An interesting aspect of the cortical convulsive effect following topical application of atropine is that the epileptic discharges are univocally intensified by high-frequency reticular stimulation.

D I R E C T A C T I O N OF A T R O P I N E O N T H E CEREBRAL CORTEX

243

This fact diversifies atropine spikes from strychnine spikes. The latter are at times inhibited and at times facilitated by reticular stimulation (Arduini and Lairy-Bounes, 1952) and the variation of effect seems to be related to the stage and intensity of cortical strychninization. On the contrary, in the case of atropine, the effect of reticular stimulation is always facilitatory : it initially evokes bursts of spikes when spontaneous discharges are not yet present and induces more intense and prolonged spiking when spontaneous discharges have appeared. From this point of view it appears not only that the atropinized cortex can still be reached by reticular influences, but that the latter have predominantly a stimulant effect on the cortex. SUMMARY AND CONCLUSIONS

Infusion of atropine into an arterial cortical vessel of one side induces a marked asymmetry of spontaneous electrical activity pattern between the cortices of the two sides. Random slow waves and I4/sec spindles, similar to those present in a normal sleeping record, prevail in the atropinized side. High-frequency stimulation of the midbrain reticular formation can readily desynchronize the activity of the atropinized cortex evoking an ‘alert’ type activity. These findings show that atropine has also a cortical site of action. The effect of atropine on the cerebral cortex can be defined as a facilitation of synchronization of spontaneous activity without blockade of desynchronizing reticulocortical influences. It can be inferred that the mechanism by which generally administered atropine blocks EEG arousal includes also subcortical sites of action in addition to a cortical one. Topical application of atropine solution to the cortical surface of one side produces two stages of changes. In an early stage, the effects are similar to those described for arterial infusion of the drug. In a later stage, after repeated applications of atropine, convulsive discharges become manifest. These discharges are regularly facilitated by reticular stimulation. The spontaneous activity of the cortex contralateral to the atropinized side is characterized by low-voltage fast activity that remains present throughout the period of asymmetrical action of atropine. This finding is interpreted as an indirect consequence of the changes occurring on the atropinized side and not as a direct effect of dtropine. REFERENCES ARDUINI, A,, AND LAIRY-BOUNES, G. C., (1952); Action de la stimulation Blectrique de la formation reticulaire du bulbe et des stimulations sensorielles sur les ondes strychniques corticales chez le chat ‘encephale isol6’. Electroenceph. din. Neurophysiol., 4, 503-512. ARTEMEV, V. V., AND BABSKII, E. B., (1950); Electrophysiological analysis of the action of acetylcholine on nerve centers. 11. Action of eserine, prostigmine and atropine on the electrical activity of the visual lobes of a frog. (Translated from the Russian). Fiziol. Zh., 36, 151-160.

244

F. R I N A L D l

BRADLEY, P. B., AND ELKES,J., (1953); The effect of atropine, hyosciamine, physostigmine and neostigmine on the electrical activity of the brain of the conscious cat. J . Physiol., 120, 13P. N. G.,(1935); On the mechanism of action of atropine on the BRAILOVSKI, V. v., AND PONIROVSKI, nervous system. (Translated from the Russian). Sovetsk. Psichonevrol., 11, 167-171. BREMER, F., A N D STOUPEL, N., (1959); Etude pharmacologique de la facilitation des reponses corticales dans I’eveil reticulaire. Arch. bit. Pharniacodyn., 122, 234-248. J. P., MOREAU, A., BEAULNES, A., AND LAURIN, C., (1962); Topical chemical stimulation CORDEAU, of the brain stem reticular formation in cats. Electroenceph. d i n . Neurophysiol., 14, 583-584. F O R R ~G. R , R., (1951); Atropine toxicity in the treatment of mental disease. Amer. J. Psychial., 108,107-1 12. HERNANDEZ-PEON., R, (1962); Sleep induced by electrical or chemical stimulation of the forebrain. Electroenceph. d i n . Neurophysiol., 14, 423424. HERNANDEZ-PEON, R., CHAVEZ-IBARRA, G., MORGANE, P. J., AND TIMO-IARIA, C., (1963); Limbic cholinergic pathways involved in sleep and behavior. Exp. Neurol., 8, 93-1 11. LOEB,C., MAGNI,F., AND RON, G. F., (1960); Electrophysiological analysis of the action of atropine on the central nervous system. Arch. ital. Biol., 98, 293-307. L.ONGO, V. G . ,(1955); Studio sul meccanismo dell’ azione centrale della scopolamina e dell’atropina. Resoconti Istitulo Supeuiore di Sanita (Roma), 18, 1033-1044. MARTIN, W. R., AND EADES, C. G., (1960); A comparative study of the effect of drugs on activating and vasomotor responses evoked by midbrain stimulation; atropine, pentobarbital, chlorpromazine and chlorpromazine sulfoxide. Psychopharmacol., 1, 303-335. M I L L ~ F. R , R., STAWRAKI, G. W., AND WOONTON, G. A., (1940); Effects ofeserine, acetylcholine and atropine on the electrocorticogram. J. Neurophysiol., 3, 131-138. MINVIELLE, J., CADILHAC, J., AND PASSOUANT, P., (1954); Action of atropine on epileptics. Electroenceph. elin. Neurophysiol., 6, 162. RINALDI, F., (1959); L‘attivita elettrica corticale dell’ emisfero isolato cronico di coniglio e le sue modificazioni da atropina e DFP. Riv. Pat. nerv. ment. (Siena), 80, 429-433. RINALDI, F., AND HIMWICH, H. E., (1955); Alerting responses and actions of atropine and cholinergic drugs. A.M.A. Arch. Neurol. Psychiat., 73, 387-395. RINALDI, F., AND HIMWICH, H. E., (1955b); Cholinergic mechanism involved in function of mesodiencephalic activating system. A.M.A. Arch. Neurol. Psychiat., 73, 396-402. SAWYER, CH., EVERETT, J. W., AND GREEN,J . D., (1954); The rabbit diencephalon in stereotaxic coordinates. J . conip. Neurol., 101, 801-824. VELLUTI, R., AND HERNANDEZ-PEON, R., (1963); Atropine blockade within a cholinergic hypnogenic circuit. Exp. Neurol., 8, 20-29. WIKLER,A., (1952); Pharmacologic dissociation of behavior and EEG ‘sleep patterns’ in dogs: morphine, N-allylmorphine and atropine. Proc. SOC.exp. Biol. ( N . Y.), 79, 261-265.

245

Chicken Brain Amines: Normal Levels and Effect of Reserpine and Monoamine Oxidase Inhibitors G O R D O N R. P S C H E I D T A N D H A R O L D E. H I M W I C H Thudiclzum Psychiafric Research Laboratory, Galesburg State Research Hospital, Galesburg, Ill. (U.S.A.)

Norepinephrine and serotonin are considered important chemical constituents of the brain relating to both normal and abnormal physiological processes and have been ascribed a role in higher mental functions as well. Extensive data on the distribution of these amines in the brain and the effects of monoamine oxidase inhibitors and reserpine are available for many mammalian species (Vogt, 1954; Amin et al., 1954; Costa and Aprison, 1958; Costa and Rinaldj, 1958; Bertler, 1960; Himwich and Costa, 1960; Bernheimer et al., 1961; Bogdanski et al., 1961; Kuntzman et al., 1961;Von Euler, 1961 ; Pscheidt and Himwich, 1963a, b; Pscheidt, 1964). Much of this material has been summarized and discussed by Pscheidt et al. (1964) who have pointed out that the highest concentration of amines is usually found in the hypothalamus and that monoamine oxidase inhibitors elevate both norepinephrine and serotonin in most species. Comparable biochemical data on avian material are lacking. In a previous report we determined the normal distribution of these amines in the chicken brain (Pscheidt and Himwich, 1963a, b) and established that the chicken cerebellum contains relatively large amounts of norepinephrine in contrast to other species. In this communication further determinations on the distribution of serotonin and norepinephrine in various regions of the chicken brain are presented as well as the effects of monoamine oxidase inhibitors and reserpine. METHODS

The chickens (Gallus domesticus) used in this study were 3- to 4-month-old white Leghorn cockerels (weight 1.O-1.6 kg) and 2-year-old white Leghorn hens (weight 2.5-4.0 kg). Each sex was studied separately. Nine animals were assigned to each of the control groups and 3 to 6 to each of the experimental groups. The latter received 3 daily intramuscular injections of nialamide (25 mg/kg in saline), isocarboxazid (15 mg/kg in propylene glycol), reserpine (0.5 mg/kg in water), or combination thereof. In one additional experiment 4 hens were given 3 daily injections of reserpine intravenously. On the 3rd day, 6 h after the last injection, the animals were decapitated, the brain rapidly removed, dissected, frozen on solid carbon dioxide and subsequently analyzed for its content of serotonin and norepinephrine as described by Mead and References p. 249

TABLE I BRAIN AMINES

(in m l g )

Treatment

Control

Dose rnglkg

-

Number of Sex animals

Norepinephrine

Serotonin Hemispheres

Midbrain

Pons-

Cerebeilum

Hernispheres

Midbrain

Ponsmedulla

Cerebellum

9 9

M F

0.88 f 0.08 0.70 & 0.10 0.51 0.06 0.16 & 0.06 0.45 f 0.04 0.82 & 0.08 0.65 f 0.08 0.55 f 0.08 0.98 i0.16 0.83&0.11 0.85f0.19 0.23&0.08 0.49f0.10 0.82&0.08 0 . 6 7 i 0 . 1 1 0.44fO.13

+

Reserpine

0.5

3 6

M F

0.40 & 0.04 0.40 f 0.18 0.25 0.37 i0.16 0.31 f0.14 0.32 f 0.12

0.06 0.20 0.05 0.27 f 0.02 0.23 0.07 f 0.03 0.16 & 0.12 0.16 i 0.07 0.10

Reserpine (i.v.)

0.5

4

F

0.30 f 0.07 0.31 = 0.09 0.40

0.10

Nialamide

25

3 6

M F

1.10 f0.02 1.10 0.67 0.17 0.58 0.05 1.20 0.81 0.56 1.80 & 0.74 1.80 f 0.48 2.30 & 0.45 0.31 f 0.09 0.72 5 0.2: 1.34 f 0.19 1.15 f 0.18 0.53 & 0.04

Nialamide Reserpine

25 0.5

3 6

M F

0.05 0.62 0.16 0.52 0.12 0.46 0.57 f 0.15 0.72*0.38 0 . 6 4 f 0 . 2 6 0.04fO.03

Isocarboxazid

15

3 6

M

F

1.40 i0.26 1.74 & 0.15

3 6

M F

0.70 5 0.12 0.58 & 0.02 0.54 1.00 f 0.45 1.26f0.54 1 . 3 5 f 0 . 1 0

Isocarboxazid Reserpine

15 0.5

-

+

-

-

0.05 L- 0.03 0.06 f 0.05 0.10

+ 0.03

0.25 0.08 i0.05

-

-

0.15

0.31 f 0.05 0.33 f 0.06 0.30 0.29 0.13f0.05 0.20+0.07 0.19&0.04 0.12f0.11

1.40 f 0.22 0.95 0.18 0.59 % 0.12 1.10 & 0.04 0.82 0.61 1.88 & 0.22 2.20 f0.05 0.28 f0.05 0.76 & 0.14 1.45 & 0.43 1.30 5 0.17 0.67 f 0.14 0.07 0.07&0.01

0.29

0.07 0.31 & 0.03 0.38 0.34 0.38h0.30 0 . 3 8 i 0 . 0 4 0.14&0.06

0.20+0.17

RESERPINE A N D

MA01

O N C H I C K E N B R A I N AMINES

247

Finger (1961). The brain was divided into the following regions : hemispheres, midbrain (thalamus, hypothalamus and colliculi), pons-medulla and cerebellum. To provide sufficient material for analysis of the latter two structures it was necessary to pool tissue from 2 or 3 animals. RESULTS

The brain amine levels for both experimental and control animals are summarized in Table I. The results are expressed as ,ug of amine per gram of fresh tissue and where more than two determinations were performed the standard deviation is included. The amine levels in the various brain parts were about the same for both the male and female chickens with the exception of the serotonin content of the pons-medulla which had a higher value in the female chickens. The cerebellar norepinephrine content was 0.55 pg/g in the males and 0.44 pg/g in the females, both values being higher than that found in other species. Response to drugs (hens) : With the exception of the cerebellum both monoamine oxidase inhibitors employed (nialamide and isocarboxazid) elevated serotonin and norepinephrine levels in the brain regions. The magnitudes of the increases were all significant to at least the 5% level as determined by the student ‘t’ test. Reserpine depleted the amines from all brain regions (p < 0.05) whether given intravenously or intramuscularly. Slightly lower values for norepinephrine were found after the intravenous injection. Simultaneous administration of the monoamine oxidase inhibitors and reserpine yielded brain amine levels above that obtained when reserpine was given alone (p < 0.05). This occurred in all brain regions except the cerebellum where the inhibitors failed to counteract the depleting effect of reserpine. Response to drugs (cockerels): In general the same drug effects were seen in the 3- to 4-month-old male chickens as in the 2-year-old hens: the monoamine oxidase inhibitors producing an elevation of brain amines, reserpine depleting the amines, and combined administration yielding intermediate values. There was a tendency for the inhibitors to produce a greater elevation of amines in the females than in the males. The pons-medulla showed the most striking difference; in the females the serotonin levels were three times higher than in the males. DISCUSSION

In all mammalian species investigated the hypothalamus and adjacent structures usually are found to have the highest concentrations of serotonin and norepinephrine in the brain (Pscheidt et al., 1964). In the fish higher concentrations of norepinephrine have been found in the telencephalon (Von Euler, 1961). In the frog diencephalic and mesencephalic structures have the highest concentrations of amines, however, epinephrine is the major catecholamine present (Pscheidt, 1964). The chicken follows the general mammalian pattern for norepinephrine but deviates in the case of serotonin. Both males and females exhibit the highest levels of this amine in the cerebral hemispheres. As pointed out previously (Pscheidt and Himwich, 1963a,b), this may be due to the fact that the avian telencephalon is comprised mainly of pallial structures to the relaReferences p . 249

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G. R. P S C H E I D T A N D H. E. H I M W I C H

tive exclusion of neocortex, a structure which is known to contain relatively small amounts of serotonin in mammals. With only three exceptions elevated levels of serotonin and norepinephrine are found in the brain of all species investigated after administration of monoamine oxidase inhibitors. The three exceptions are the frog (Pscheidt, 1964) the cat (Brodie et al., 1959a, b; Funderburk et al., 1962; Pscheidt et al., 1963) and the dog (Brodie et al., 1959a; Pscheidt et al., 1964). In these species norepinephrine levels (or epinephrine in the case of the frog) are not increased after administration of amounts of monoamine oxidase inhibitors sufficient to elevate serotonin concentrations appreciably. The underlying biochemical reasons for these differences are not understood. The results of this investigation clearly demonstrate that the chicken brain, except for the cerebellum as discussed below, is no exception to the general mammalian pattern inasmuch as the inhibitors elevated both amines in the various brain regions. The differences between the male and female chickens with regard to amine content and metabolism are appreciable in some instances, particularly the serotonin content of the pons-medulla. We are inclined to view these as true sex differences despite the disparity in ages between the two groups of animals inasmuch as there was only a small difference in the average brain weights, and the development of the brain probably was largely completed in the cockerels. The chicken cerebellum did not give the same biochemical response as the other brain regions. Its content of serotonin and norepinephrine was depleted by reserpine as were the other brain regions but normal levels were not restored by simultaneous administrations of monoamine oxidase inhibitors sufficient to restore amine levels in the rest of the brain. Similarly the inhibitors when given alone did not produce any significant elevation of amines in this structure. This suggests that in the chicken the cerebellum has a slower rate of synthesis for these compounds than other parts of the brain. SUMMARY

Norepinephrine and serotonin concentrations were determined in the major brain regions of 3- to 4-month-old male and 2-year-old female white Leghorn chickens (Gallus domesticus) which had received intramuscular injections of nialamide, isocarboxazid, reserpine, or combinations thereof. The control values for serotonin in pg/g, of fresh tissue for males and females respectively were: hemispheres, 0.88, 0.98; midbrain, 0.70, 0.83 ; pons-medulla, 0.51,0.85; cerebellumo. 16,0.23. The corresponding values for norepinephrine were: hemispheres, 0.45, 0.49; midbrain, 0.82, 0.82; pons-medulla 0.65,0.67 ; cerebellum, 0.55,0.44. Relatively high amounts of norepinephrine were found in the cerebellum compared to other species. lsocarboxazid and nialamide, monoamine oxidase inhibitors, elevated both norepinephrine and serotonin in the brain, whereas reserpine lowered the brain biogenic amines. Combined administration of reserpine and a monoamine oxidase inhibitor gave brain amine levels between those found with either drug alone. Thus with the exception of the cerebellum, in chickens as well as in mammals, reserpine and monoamine oxidase inhibitors change brain amine levels in opposite directions.

RESERPINE AND

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ON C H I C K E N BRAIN AMINES

249

ACKNOWLEDGEMENTS

We wish to thank the following organizations for their generous supplies of drugs: Chas. Pfizer and Company for nialamide, Hoffmann-LaRoche, Inc. for isocarboxazid, and Ciba Pharmaceutical Products, for reserpine. REFERENCES AMIN,A.H., CRAWFORD, T. B. B., AND GADDUM, J. H., (1954);Thedistributionof serotonin 5-hydroxytryptophan decarboxylase, and monoamine oxidase in brain. J. Neurochem., 126, 596-61 8 . BERNHEIMER, H., BIRKMAYER, W., AND HORNYKIEWICZ, O., (1961); Verteilung des 5-Hydroxytryptamins im Gehirn des Menschen und sein Verhalten bei Patienten mit Parkinson-Syndrom. Klin. Wschr., 39, 1056-1059. BERTLER, A., (I 960); Occurrence and localization of catecholamines in the human brain. Acta physiol. scand., 50, 1-1 1 . BOGDANSKI, D. F., WEISSBACH, A., AND UDENFRIEND, S., (1961); The distribution of serotonin, 5-hydroxytryptophan decarboxylase, and monoamine oxidase in brain. J. Neurochem., 1,272-278. BRODIE, B. B., SPECTOR, S., AND SHORE, P. A., (1959a); Interaction of monoamine oxidase inhibitors with physiological and biochemical mechanisms in brain. Ann. N . Y. Acad. Sci., 80, 609-614. S., AND SHORE, P. A., (1959b); Interaction of drugs with norepinephrine in BRODIE, B. B., SPECTOR, the brain. Symposium on Cutecholamines. 0. Krayer, Editor. Baltimore, Williams and Wilkins (p. 548-564). COSTA,E., AND APRISON, M. H., (1958); Studies on the 5-hydroxytryptamine (serotonin) content in human brain. J . new. nient. Dis., 126, 289-293. F., (1958); Biochemical and electroencephalographic changes in the brain COSTA,E., AND RINALDI, of rabbits injected with 5-hydroxytryptophan. Am. J . Physiol., 194, 214-220. FUNDERBURK, W. H., FINGER,K. F., DRAKONTIDES, A. B., AND SCHREIDER, J. A,, (1962); EEG and biochemical findings with M A 0 inhibitors. Ann. N . Y. Acad. Sci., 96, 289-302. HIMWICH, W. A., AND COSTA,E., (1960); Behavioral changes associated with changes in concentrations of brain serotonin. Fed. Proc., 19, 838-845. R., SHORE, P. A,, BOGDANSKI, D., AND BRODIE, S. B., (1961); Microanalytical procedures KUNTZMAN, for fluorometric assay of brain DOPA-5HTP decarboxylase, norepinephrine, and serotonin, and a detailed mapping of decarboxylase activity in brain. J . Neurochem., 6 , 226-232. MEAD,J. A. R., AND FINGER, K. F., (1961); A single extraction method for the determination of both norepinephrine and serotonin in brain. Biochem. Pharmacol., 6, 52-53. PSCHEIDT, G. R., (1964); Effects of reserpine and isocarboxazid in the frog. Developing Brain, Progress in Brain Research, Vol. 9. H. E. Himwich and w. A. Himwich, Editors. New York-Amsterdam, Elsevier (p. 213-21 6). G. R., AND HIMWICH, H. E., (1963a); Reserpine, monoamine oxidase inhibitors, and distriPSCHEIDT, bution of biogenic amines in monkey brain. Biochem. Pharmacol., 12, 65-71. 0. R., AND HIMWICH, H. E., (1963b); Chicken brain amines, with special reference to cerePSCHEIDT, bellar norepinephrine. Life Sciences, 8, 524-526. G. R., MORPURGO, C., AND HIMWICH, H. E., (1964); Studies on norepinephrine and seroPSCHEIDT, tonin in various species. Comparative Neurochemistry. D. Richter, Editor. 5th International Neurochemical Symposium. Oxford, Pergamon Press (pp. 401-412). VOGT,M., (1954); The concentration of sympathin in different parts of the central nervous system under normal conditions and after the administration of drugs. J . Physiol., 123, 451481. VON EULER,U. S . , (1961); Occurrence and distribution of catecholamines in the fish brain. Acta physiol. scand., 52, 62-64.

Spinal Input to the Midbrain Reticular Formation : Pharmacological Investigation A. MORILLO*, A. R. D R A V I D A N D R.D I P E R R I Thutlichuni Psychiatric Research Lahouafory, GaleJburg State Research Hospital, Gatesburg, Ill. ( U.S.A.)

INTRODUCTION

Studies on the D-lysergic acid diethylamide (LSD-25) (Bradley and Elkes, 1953, 1957) indicate that this substance produces electrocortical arousal in the relaxed, conscious cat. and that such an effect is not observed in the ‘enckphale isolC’ or in the ‘cerveau isolk’ preparations. These and other results have led Bradley (1958) to postulate that LSD-25 ‘exerts an action at the brain stem level’. Experimental data in the literature also indicate that the action of LSD-25 on the electrocortical activity of the cat is somehow related to the availability of sensory input (Adey et al., 1962; Bradley and Elkes, 1953). A review of the neurophysiological effects of LSD-25 upon the sensory (visual) system has been made by Evarts (1957). In an attempt to determine to what extent the observed effect of LSD-25 on the midbrain may have been partly, at least, due to an action upon the spinal afferents we have conducted the experiments reported here in which we investigated the action of LSD-25 upon evoked potentials simultaneously recorded from the lateral funiculus of the spinal cord at upper cervical level and from the brain stem reticular formation at intercollicular level. For the purpose of comparison, the effect of pentothal was investigated by the same method. METHODS

Acute experiments were carried out in 18 intact adult cats. Under ether anesthesia the animals were tracheotomized, one of their femoral veins was cannulated and a sciatic nerve was dissected and cut. The animals were placed in a stereotaxic apparatus and nichrome wire electrodes were stereotaxically placed through a burr hole into the mesencephalic reticular formation at intercollicular level. The electrodes in the midbrain were located contralaterally to the dissected sciatic nerve. Under visual inspection electrodes were inserted into the lateral funiculus of the spinal cord at the level of the second cervical segment, ipsilateral to the-sciatic stimulated. -

* Present address : Departamento de Fisiologia, Facultad de Medicina, Univcrsidad Javeriana, BogotZI (Colombia).

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Fig. 1 . Evoked responses recorded monopolarly from different sites of the lateral funiculus of the spinal cord at the level of C2. The ipsilateral sciatic nerve was stimulated with singlerectangular shocks every 2 sec; other parameters were 0.1 msec and 7 V. The hexagons indicate locus of the electrode tip. The number refer to each experiment. In all but in experiment No. 12 5 consecutive responses were superposed. Negativity is indicated by an upward deflection. Calibration is 100 pV but for experiment No. 10 in which it is 50 pV.

The dorsal columns were severed one segment below the recording electrode in the spinal cord to rule out the possibility of their contribution to the evoked potentials recorded from the most dorsal part of the lateral funiculus. When the responses had attained their control values after recovery from the action of the drug, the spinal cord was carefully sectioned at the level of C1 as a control measure to discard ‘centrifugal’ components of the responses in the lateral funiculus. This was the last step in each References p . 2541255

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experiment since afterward no more responses in the midbrain were available for study. The stimulating and recording rechniques were the conventionally used in this type of experiments and similar to those described in detail in a previous paper in which we also described the method used for the histological identification of the electrodes (Morillo and Baylor, 1963). The drugs (LSD-25 and pentothal) were injected through a cannula in the femoral vein. The pH of the solutions of LSD-25 was adjusted to 7 in all the experiments. RESULTS

Fig. 1 illustrates the evoked potentials that are recorded from the lateral funiculus of the second cervical segment in response to a single rectangular pulse applied to the central end of the ipsilateral sciatic nerve. The results from 1 1 experiments are illustrated; each point indicates the site of the electrode tip as demonstrated by histological identification of the coagulating lesion made through the electrode tip at the end of each experiment. Under each point is a picture of a single or 5 superposed traces of the response recorded during the experiment identified by the number beside each point. These records were taken from intact, curarized animals in experiments in which the response in the midbrain reticular formation was simultaneously recorded. 1

2

39 A A M -66 Fig. 2. In each picture the upper trace reproduces the evoked potential recorded from the midbrain reticular formation; the lower trace the evoked potential recorded simultaneously from the lateral 5 min funiculus of the spinal cord a t C2. The sciatic nerve was stimulated. A I = control; A 2 after i.v. injection of 5 mg/kg of pentothal; B 1 - control; B 2 = 20 min after i.v. injection of 25 pg of LSD-25 per kg. Monopolar records. Five superposed responses in each picture. Negativity is indicated by an upward deflection. Calibration signals: 20 pV for the upper traces; 100 ,uV for the lower ones. Time signal is 20 msec.

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The fact that these responses were little affected by careful spinal transection at the level of C1 demonstrates that they represent ascending volleys. Fig. 1 also shows that the features of the responses, i.e. latency, wave configuration, amplitude and duration vary as a function of the electrode placement. It is most likely that these changes are related to the various spinal tracts in the vicinity of the recording area of each electrode. The results illustrated in Fig. 2A show the well-known action of a barbiturate, pentothal at a dose of 5 mg/kg intravenously upon the evoked response in the midbrain reticular formation (refer to Fig. 1, experiment No. 9 for identification of the electrode position in the spinal cord). It is clearly demonstrated that 5 min after the injection the reticular response is completely abolished while the spinal evoked potential is only slightly depressed (negative phase). A similar experiment investigating the action of LSD-25 in the same animal 1 h after it had recovered from the pentothal is illustrated in Fig. 2B. The intravenous injection of 25 pg of LSD-25 produced in 10 min moderated depression of the reticular response and practically no effect upon the potential in the spinal cord. Higher doses of LSD-25 produced a more intense depressant effect upon the reticular potentials. The results are representative of the findings in our series of experiments, namely the high resistance of the spinal cord potentials to the action of LSD-25. This holds true for all the responses recorded from the lateral funiculus, irrespective of the position of the electrode. DISCUSSION

The variability of the configuration of the responses recorded from the spinal cord as a function of the position of the recording electrode, suggests that the different ascending paths topographically distributed in the lateral funiculus contribute to the various characteristics of the responses. At the present stage of our investigation we are in no position to ascribe with certainty these responses to any particular tract, but to infer from the location of the electrode which paths in its vicinity may have contributed to the recorded evoked potential. Irrespective of any further anatomical identification it is interesting to note that the electrodes were placed in most of the cases in an area of the lateral funiculus that has been previously identified as being one along which travels part of the ascending spinal volleys contributing the potentials recorded from the midbrain reticular formation (Morillo and Baylor, 1963). The lack of significant action of LSD-25 and of pentothal upon the paralemniscal paths, suggests that the effect of these drugs upon the reticular evoked potential is the result of their action at an upper most level. Although the depressant action of the barbiturates upon the reticular formation has been recognized (Arduini and Arduini, 1954; French et al., 1953; Loeb et al., 1960), the possibility that such an action was the result of an effect at spinal level had to be tested in our experimental conditions, in which the impulses generating the response in the midbrain had to be conveyed by ascending spinal tracts known to provide input to the reticular formation. Similar considerations are relevant for our experiments with LSD-25. Our results with LSD-25 lend support to Bradley’s hypothesis according to which the action of LSD-25 would be at midbrain level (BradRcfercnres p . 254jZ55

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ley and Key, 1958). Whether or not this effect is the result of an indirect action, was not determined. The effect on corticifugal influences (Adey et al., 1957) were not investigated, though there is evidence indicating their contribution to the responses in the midbrain (Morillo and Baylor, 1963). It is interesting to note, how these two drugs having a similar action upon the responses in the reticular formation, have completely opposite effects upon the electrocortical activity at the doses that were used in our experiments. While the barbiturates induce electrocortical synchronization and sleep patterns, LSD-25 produces desynchronization of the cortical rhythms and electrical activity similar to that of arousal. Such contrasting actions are also apparent in their behavioral effects, for barbiturates induces sleep, and LSD-25 produces 'excitation' (Bradley, 1958). Furthermore, it is known that LSD-25 does not affect the threshold to arousal by direct electrical stimulation of the reticular formation, but the barbiturates markedly raise it (Bradley and Key, 1958). All these observations and our results would suggest a plurality of mechanisms responsible for arousal, both electrographical and behaviorally, but the limited number of our observations and the fact that our recording electrodes in the reticular formation were placed in a restricted area does not warrant further elaboration on these matters. SUMMARY

In 18 intact adult cats, records of evoked potentials were taken simultaneously from the lateral funiculus of the spinal cord at upper cervical level and from the midbrain reticular formation. LSD-25 had no effect upon the evoked potentials in the lateral funiculus of the spinal cord, but depressed the response in the midbrain reticular formation. The results support the hypothesis according to which the effect of LSD-25 is exerted at midbrain level. Contrasting properties of LSD-25 and penthothal are discussed. REFERENCES ADEY,W. R., BELL,F. R., AND DENNIS, B. J., (1962); Effects of LSD-25, psilocybin and psilocin on temporal lobe EEG patterns and learned behavior in the cat. Neurology, 12, 591-602. ADEY,W. R., SEGUNDO, J. P., AND LIVINGSTON, R. B., (1957); Corticifugal influences on intrinsic brain stem conduction in cat and monkey. J . Neurophysiol., 20, 1-16. ARDUINI, A., AND ARDUINI,M. G., (1954); Effects of drugs and metabolic alterations on brain stem arousal mechanisms. J. Pharmacol. exp. Ther., 110, 76-85. BRADLEY, P. B., (1958); The central action of certain drugs in relation to the reticular formation of the brain. Rericular Forniation of flze Brain. H. H. Jasper, L. D. Proctor, R. S. Knighton, W. C . Noshay and R. T. Costello, Editors. Boston, Little, Brown (pp. 123-149). BRADLEY, P. B., AND ELKES, J., (1953); The effect of amphetamine and D-lysergic acid diethylamide (LSD-25) on the electrical activity of the brain of conscious cat. J. Physiol., 120, 13P-14P. BRADLEY, P. B., AND E L K ~J., S , (1957); The effects of some drugs on the electrical activity of the brain. Brain, 80, 77-1 17. BRADLEY, P. B., A N D KEY,B. J., (1958); The effects of drugs on arousal responses produced by electrical stimulation of the reticular formation of the brain, Eledroenceph. din. Neurophysiol., 10, 97-1 10.

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EVARTS, E. V., (1957); A review of the neurophysiological effects of lysergic acid diethylamide (LSD) and other psychotomimetic agents. Ann. N . Y . Acad. Sci., 66, 479495. FRENCH, J. D., VERZEANO, M., A N D MAGOUN, H. W., (1953); A neural basis of the anesthetic state. Arch. Neurol. Psychiat., 69, 519-529. LOEB,C . , MAGNI,F., AND Rossr, G. F., (1960); Electrophysiological analysis of the action of atropine on the central nervous system. Arch. ital. Bioi., 98, 293-307. MORILLO, A,, AND BAYLOR, D., (1963); Electrophysiological investigation of lemniscal and paralemniscal input to the midbrain reticular formation. Electroenceph. clin. Neurophysiol., 15, 455-464

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Bacterial Neurotoxins D.A. B O R O F F Laboratory o f Immunology, Albert Einstein Medical Center, Philadelphia 41, Pa. (U.S.A.)

Much has been written about the toxin of C. botulinum. There is still, however, only scant knowledge of its chemistry, mode of action or the site of its action. The toxin is elaborated by Clostridium botulinum of which six serologic types are known. The organism is an anaerobe, widely spread in the soil, which becomes toxigenic only under certain special conditions. The disease is a result of the ingestion of the preformed toxin. Once a lethal dose has been ingested, very little can be done for the affected animal or man. The antitoxin, unless administered very early after intoxication, is not very effective and there are no other substances or drugs known that will counteract the lethal action of the toxin. Even if the animal survives, convalescence is prolonged and difficult. The only sure protection against this disease is prophylactic immunization. Presently available vaccines, however, are still in developmental stages. Neurological symptoms invariably observed in botulinum intoxication directed attention to various functional disturbances that could be demonstrated in the peripheral nervous system (Schubel, 1921). The particular sites of action were, however, described by Dickson and Shevky (1923) who pointed out the similarity between the sites of action of the botulinum toxin and those of acetylcholine. They further showed that motor paralysis was not due to blocking impulses along the nerve trunk but was dependent upon the intoxication of some endorgan (myoneural junction). This was later confirmed by Guyton and MacDonald (1947) and Ambache (1 949). However, the manner in which botulinum toxin interferes with acetylcholine release at the neuro-muscular junction still remains obscure since Brooks (1954) observed that the acetylcholine release mechanism is not in itself damaged. Nonetheless, all evidences so far accumulated show that botulinum toxin acts on those portions of the peripheral nervous system that are cholinergic, whether they are pre- or postganglionic components of the nervous system. Neither in clinical no laboratory botulism is there an indication that the central nervous system is involved. However, Matveev (1959) maintains that the central nervous system is also involved. Tn 1959 Winbury endeavored to show that botulinum toxin, besides affecting the cells of the striated muscles, also had an effect upon the smooth muscles of the blood vessels. The exceptional lethality of this toxin places it in an unique position among bacterial and other poisons. About 10-4 pg is sufficient to kill a 20-g mouse. Tt has been

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established that the toxin is a protein of no unusual amino acid composition (Buehler et al., 1947). This protein has been crystallized by Lamanna et al. (1946) and also by Abrams et al. (1946). When examined in the ultracentrifuge or electrophoretically, the crystalline toxin appeared to be homogenous with an estimated molecular weight of about 900,000 (Putnam et al., 1948). Due to its high toxicity and its ability to induce the formation of antitoxin, it was thought to be an exotoxin, that is, a substance secreted by the clostridia as a product of their metabolism. This hypothesis was disputed by Raynaud and Second (1949) and later by Boroff and Raynaud (1952), who succeeded in obtaining a toxin as powerful as that found in the culture filtrates by the extraction of washed organisms. It was further established that a relationship existed between the rate of autolysis of the clostridia and the accumulation of toxin in the culture, which suggested the probability that the toxin is a part of the bacterial soma (Boroff, 1955; Gendon, 1957; Bouisset et al., 1957). The homogeneity of the crystalline toxin was also questioned by Lamanna and Lowenthal(1951), who reported that the toxin contained a separable component responsible for hemagglutinating properties previously thought to be property of the pure toxin. Our first concern was to obtain toxin in pure form. The organism used was Clostridium botulinum type C isolated from an outbreak of botulism among horses in France by Prtvot and Brygoo (1949). The organism produced toxin containing 1.8 x 106 MLD per ml for mice of 20 g. For media we employed cornsteep liquor according to formula described by Sterne (Sterne and Wentzel, 1950). These authors also suggested a method for obtaining the toxin free from contaminating substances of the medium which we adopted and modified for small amounts of culture. The method consisted of growing the cultures in sacks made of dialysis casing immersed in the media. Since only dialysable nutrient can penetrate the casing, all proteins found within had to be of bacterial origin. These cultures were incubated at 37" for 6 to 8 days. The toxin found in the supernate was freed from the organisms by centrifugation and filtration, and finally separated from all small molecular weight substances by dialysis against saline. Further purification was accomplished by dialysis against distilled water which precipitated the toxin within the cellophane sack. The precipitate dissolved in 0.1 M buffer pH 6.8 and again by acidification with dilute HCl at pH 3.5-4. This procedure yielded a preparation containing about 107 MLDs for mice of20 g per mg of nitrogen. At the same time a method was developed which permitted a very rapid and fairly accurate estimation of the toxin concentration. The usual method is injecting aliquots of serially diluted toxin intraperitoneally into groups of mice and noting the least amount of material which will still kill 50% of the mice in the group. This established the LD50 but took 4 days for each determination. By injecting the mice intravenously, with 0.1 ml of concentrated toxin, we observed that the time of survival of these mice in minutes was inversely proportional to the logarithm of concentration of the toxin. Thus in a few minutes we could estimate the potency of the preparation on hand. In the course of this work we noted that the maximal amount of toxin appeared in the culture medium on about the 8th day. This observation is not consistent with the theory that this toxin is an exotoxin, i.e. a product of bacterial metabolism, and is References p. 2611262

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excreted by the organism into the surrounding medium during the life of the clostridia. Ordinarily, a bacterial culture, especially fast-growing clostridia, is by the 8th day long past its logarithmic phase and in either the plateau or declining stage. If C. botulinum produces an exotoxin, we should expect the toxin to manifest itself at the maximum when the organisms grow and metabolize at the maximum rate, that is, at the logarithmic stage. As we stated above, the contrary is, however, true. Raynaud and Second (1949) and Boroff and Raynaud (1952) were able to demonstrate that while there is still little toxin in the supernate, a considerable amount of toxin is obtained by extracting the washed organisms of young cultures. We observed that C. botulinum autolyzes very rapidly. This observation, combined with the observation of the lateness of the appearance of the maximal amount of toxin in the culture supernate, prompted us to investigate the events surrounding both the toxin production and its relation to the autolysis of the organisms. We realized that linking of autolysis to toxin production would depend upon our interpretation of what we observed of the appearance of the organisms in an aging culture and our attempts to correlate these observations with the appearance and augmentation of the titer of the toxin. We were, however, able to relate the increase of protein in culture fiitrates with apparent autolysis of the cells and thus with toxin production. In another set ofexperiments we succeeded in inducing early autolysis in ayoungculture by the additionofcell-freefiltrate from an8-daygrowth. Therapidlysiswhichensued was followed by a steep increase in titer of toxin found in the filtrate of these organisms. A striking morphological change which can be observed even in a fairly young culture, is the loss of its ability to stain with Gram’s stain. After this, the organisms become progressively thinner until they are only a thread which final1y.disintegrates. Another observation which was of interest was that in all our attempts to purify the toxin for the use in antibody production in rabbits we never succeeded in obtaining an antigen which would not, besides inducing neutralizing and flocculating antibody, induce the formation of agglutinins at a high titer. Agglutination is a surface phenomenon so that the antigens producing agglutinins should be situated on the surface. Extraction of organisms with 1 M NaCl left the organisms retaining their shape but becoming Gram negative. Finally, a suspension of washed organisms, when injected intravenously into mice, killed almost as fast as the soluble toxin. Since death occurred in the course of 30 to 40 min it is not likely that the injected organisms were lysed in this period. Although these observations were not intended to serve as prima facie evidence for our hypothesis, they were not in contradiction with our contention that the toxin of Cfostridium botulinum is a part of the soma and is released into the culture medium as a product of autolysis. Thus, toxin does not belong to the class of exotoxins as defined in Zinsser and Bayne-Jones (1939). The possibility that the bacterial soma might comprise the toxin raised the question whether protection against the toxin could not be achieved by the injection of bacterial vaccines*. I n the course of this work we observed that under certain cultural

* The usual mode of antiserum production is by the injections of formalin inactivated toxin.

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conditions our toxic strain of Clostridium botulinum type C gave rise to a variant which when injected intraperitoneally or intravenously into mice in relatively large amounts failed to affect the animals noticeably. We thought it of interest to investigate whether the antibodies elicited by a nontoxic variant would protect against soluble toxin and react serologically in tests with soluble toxin. By repeatedly subculturing the organism in beef heart infusion broth (Difco) we obtained a variant which on microscopic examination resembled in all respects the parent strain but which was devoid of its toxigenic properties. Suspensions of such organisms agglutinated readily with serum against the toxin. These organisms injected in rabbits resulted in the formation, in the blood of these animals, of antibodies which were fully capable of reacting in serological tests with toxic parent strain and precipitate the toxin from solution. Immunized rabbits could withstand 20,000 mouse MLDs and the sera in 0.5 ml amount injected intraperitoneally protected mice against 1000 MLDs either of toxic organisms or type C toxin. These results show that an atoxic variant of Clostridium botulinum was capable of inducing formation of circulating protective and flocculating antibodies. This evidence is not in accord with the commonly accepted view that formation of flocculating antibodies is an exclusive property of exotoxins. Nor are these observations solely confined to Clostridium botulinum. Boroff and Macri (1949) were able to show that it is possible to induce formation of these antibodies with a rough strain of Shigellu dysenteriu devoid of exotoxin. It is, therefore, obvious that the exotoxin theory will not suffice to explain this phenomenon. If the hypothesis discussed above is to be considered and we are allowed to conceive the toxic principle as being an active grouping or configuration of groupings on the molecule of bacterial protein, while the whole molecule of the protein is responsible for the antigenic properties, then the formation of an antigenically active toxoid with the aid of reagents which act upon specific chemical groupings would be easily understood and the phenomenon observed with atoxic Clostridium botulinum readily explained. To forestall the objection that we might have been dealing with an organism of very low toxicity, but nonetheless containing enough toxin to be antigenic, our experience with our attempt of immunized rabbits with sublethal doses of the toxin point to the contrary. Injection of sublethal amounts of toxin did not result in antibody formation and although the injections were given at long intervals all rabbits died after the 5th or 6th injection. Apparently the toxin was not eliminated from the body, and since no immunity was established, as soon as sufficient toxin accumulated to amount to a lethal dose the rabbits perished. These observations laid the basis for the study of bacterial proteins with the hope that this might lead to a better understanding of the chemical structure of substances showing this remarkable biological activity. This work began chiefly at the Thudichum Psychiatric Research Laboratory and has been continued to date. However, while still at the Galesburg State Research Hospital our interest in bacterial toxins led us to the investigation of toxins of pathogenic Escherichia coli. These common intestinal parasites are also common pathogens of the genito-urinary tract and are often the cause of a prolonged chronic disease. Pathogenic coli, accorReferences p . 2611262

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ding to Vincent (1933) elaborate two toxins: one, thermostable enterotoxin, and the other, a thermolabile neurotoxin. The latter is found in the culture media afterbabout 5 days incubation. Baruk (1949) is of the opinion that the neurotoxin affects the central nervous system and is responsible'for certain mental disorders. The idea of bacterial etiology of mental diseases is a very intriguing one and it prompted us to search for a pathogenic E. coli from which we could obtain this neurotoxin. Many strains were examined until one was found which had this property. This strain was isolated from a parturating woman suffering from a coli pyelonephritis. The organism was grown on a beef heart infusion broth (Difco) for 5 days. The media was freed from the organisms by filtration and the clear sterile culture filtrate injected in various amounts into mice, guinea-pigs and rabbits. Only a few of each species of animals were affected by the filtrates. The effect upon the susceptible animals was dramatic. Within 30 to 120 min after injection the animals became passive and lost interest in their surroundings. The animals could be placed in various unnatural positions. However, when there was danger of an imminent fall they moved to regain balance. The animals also moved when some external force was applied but as soon as the force was withdrawn at once relapsed into passivity. Muscle tones and the righting reflex were always present. Breathing and temperature remained normal. This state resembled in many respects one described by De Jong (1 93 1) in his study of bulbocapnine induced catatonia. Rabbits, guinea-pigs and mice were susceptible to the E. coli toxin. However, not every strain of pathogenic coli elaborated this toxin, nor were all animals susceptible to it. Of the 17 strains of E. coli from various human infections only 1 produced this toxin and only about 10% of all the animals tested exhibited these symptoms. All animals recovered within 24 h with no apparent ill effects. These observations corroborate Baruk's findings. He, however, contends that in humans, chronic genito-urinary infections due to toxigenic strains of E. coli are responsible for schizophrenia and the curing of such patients of the bacterial infection is followed by the cure of mental disease as well. Unfortunately there is little in the literature so far to substantiate such claims. These considerations notwithstanding, the possibility of the existence of bacterial products which are in some way responsible for controlling a mental function is intriguing and warrants further investigation. The work described was either accomplished or initiated at the Thudichum Psychiatric Research Laboratories at the Galesburg State Research Hospital to which I came in late 1950 before Dr. Harold Himwich joined us as the director of the laboratories and before the laboratories were built and named. It was my privilege to welcome Dr. Himwich to Galesburg as well as participate in creating the research laboratory facilities. SUMMARY

The most powerful neurotoxin is produced by several serological types of soil anaerobes of Clostridium botulinum. It has been shown that the toxin is a part of the bacterial soma and is released into the culture medium only upon the death and autolysis of the

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organism. This makes the toxin more akin to an endotoxin than the exotoxin which it was previously believed to be. A method for growth of C. botulinum type C within dialysing casing was adopted, which permitted an easy separation of large molecular weight substances of bacterial origin from substances of the media. This procedure permitted a simple and more rapid purification method which yielded toxin of high purity and specific activity. Furthermore, a rapid method for assaying the potency of the toxin was developed, which permitted the determination of the concentration of the toxin in mouse MLD units within minutes. Another study of a bacterial neurotoxin was that obtained from Escherichia coli. The substance found in the culture supernate has the power, when injected into animals, to produce a state akin to human catatonia. The animals lost the power of voluntary movement which, however, was overcome if the animal was later placed into an unbalanced position or was in danger of falling. All affected animals regained normal state 24 h after the onset of symptoms.

ACKNOWLEDGEMENTS

This investigation was supported by the U.S. Public Health Grant Nr. E4180, and National Science Foundation Grant Nr. G17286. REFERENCES ABRAMS, A., KEGELES, G., AND HOTTLE, G. A., (1946); The purification of toxin from Clostridium botulinuin Type A. J . biol. Chem., 164, 63-79. AMBACHE, N., (1949); Peripheral action of botulinum toxin. J. Physiol., 108, 127--141. BARUK,H., (1949); Experimental catatonia and the problem of will and personality. J . new. menf. Dis., 110, 218-234. BOROPF, D. A., (1955); Study of toxins of Clostridium botulinum. 111. Relation of autolysis to toxin production. J . Bacreriol., 10, 363-367. BOROFF, D. A., AND MACRI,B. P., (1949); Studies of toxins and antigens of S. dysenteriuein activeprotection of rabbits with whole organisms and various fractions of S. dysenteriae. J . Bacteriol., 58, 3 87-394. BOROFF,D. A., AND RAYNAUD, M., (1952); Studies of toxin of Clostridium botulinum, Type D. J. Immunol., 68,503-5 1 1 . BOUISSET, L., BREUILLAND, J., AND GRIZOV, V., (1957); Croissance et toxinogenkse chez Clostridium botulinum, Type A. C . R. Soc. Biol. (Paris), 151, 387-390. BROOKS, V. B., (1954); The action of botulinum toxin on motor nerve filaments. J . Physiol., 123, 501-5 15. BUEHLER, H . , SCHANTZ, E., AND LAMANNA, C., (1947); Elemental and amino acid composition of crystalline Clostridium botulinum, Type A toxin. J . biol. Chem., 169, 295-302. DEJONG, H., (1931); Uber Katatonie-Erzeugende Stoffe. De Physiol. Pharmacol. Microbiol. E.A., 1,4-6.

DICKSON, E. C., AND SHEVKY, E., (1923); Botulism studies on the manner in which the toxin of Clostridium botulinuin acts on the body. I. The effects on the autonomic nervous system. J. exp. Med., 37,711-731. GENDON,U. Z., (1957); Growth and toxigenicity of B. botulinum in cellophane sack cultures. J. Microbiol. Immunol. (Russian), 3, 67-70. GUYTON, A. C., AND MACDONALD, M. A., (1947); Physiology of botulinum toxin. Arch. neurol. Psychiat. (Chic.), 51, 579-592. LAMANNA, C., AND LOWENTHAL, J. P., (1951); The lack of identity between hemagglutinin and the toxin of Type A botulinal organism. J. Bacteriol., 61, 751-752.

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LAMANNA, C., MCELROY, 0. E., AND EULAND, H. W., (1946); The purification and crystallization of Clostridium botulinum, Type A toxin. Science, 103, 613-614. MATV~LV, K. l., (1959); Botulim. Moscow, State publishers of Medical Literature. PR~VOT, A. R., AND BRYGOO, E. R., (1949); Etude de la premibe souche FranCaise de C. botulinum. Bull. SOC.vPt. Pratique de France, 33, 1-1 1 . PUTNAM, F. W., LAMANNA, C., AND SHARP,D. G., (1948); Physicochemical properties of crystalline Chtridium botulinum Type A toxin. J. biol. Chem., 176, 401-412. RAYNAUD, M., AND SECOND,L., (1949); Extraction des toxines botuliniques a partir des corps microbiens. Ann. Inst. Pasteur, 77, 316-319. SCHUBEL, K., (1921); Uber das Botulinus Toxin. Dtsch. med. Wxhr., 47, 1047. STERNC, M., AND WENTZEL,L. M., (1950); New method for large-scale production of high-titre botulinum formol-toxoid Types C and D. J . ImmunoZ., 65, 175-183. VINCENT,H., (1933); R81e de I’intoxication colibacillaire dans la genese de certains troubles mentaux. Leur gukrison par la serotherapie anticolibacillaire. Ann. Acad. Sci., 28 aofit, Paris. WINBURY, M. M., (1959); Mechanism of the local vascular actions. J. Physiol., 147,l-I3 ZINSSER,H., AND BAYNE-JONES, S., (1939); A Textbook of Bacteriology. 8th Ed. New York, D. Appleton-Century Company (p. 153).

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Variations in Water Ingestion: The Response to Barbiturates H. SCHMIDT, JR. Department of Psychiatry, Washington University, School of Medicine, St. Louis, Mo. (U.S.A.)

Despite the present recognition oftherole of thecentral nervous system, more particularly the brain, and perhaps most particularly the hypothalamus in water ingestion, study of drug effects upon water intake, evaluation of neurotropic drugs with regard to water ingestion, is only poorly studied. In a recent review I cited perhaps 25 papers particularly germane to this topic and virtually exhausted it (Schmidt, 1963a). On the other hand, that other great regulator of water, the kidney, is well-represented in the pharmacological literature. Perhaps the reasons for this disparity are not hard to seek. The kidney controls what has always been regarded as a more or less automatic activly and impairment of the organ is not without dire consequences for drug activity and fate, therapeutics, and general health. On the other hand, the ingestion of water, in man at any rate, is believed to be vo1it;onal and not so subject to impairment as long as the drug recipient is conscious. To see the flaw in this argument all we need do is ask ourselves: How volitional is the hyperdipsia of diabetes insipidus whether hypothalam'c or nephrogenic? Having established that at least under some circumstances, water ingestion can be regarded as an automatic, essentially non-volitional activity, the mode of approach to the whole problem may be considered. First of all, I would like to avoid consideration of thirst as a sensory condition. My reasons for this evasion are centered upon the difficulty of defining thirst. The most obvious definition is lhat which instigates water ingestion fails for reasons of subjective experience described most eloquently by Wolf (1958). To add such a proviso that thirst is the central state which instigates drinking while quite in keeping with contemporaneous thought implies a unitary state which remains yet to be elucidated. A second fiat will be the consideration of water ingestion as an automatic activity or perhaps more significantly as an outcome of a number of automatic activities. Within the confines of this latter notion would fall the experiment of Teitlebaum in which the rat laps at a tube of water (ingesting the water) in order to avoid a shock (Williams and Teitlebaum, 1956). While that latter experiment is not precisely relevant to the present paper, it leads to the third consideration to be made at this point. Water ingestion need not be regulative in the sense of being simply and only a means of maintaining the internal environment a t a given References p. 2831284

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level. This role of water ingestion is not denied but need not be of concern. (For a more extended discussion of the regulative and non-regulative facets of drinking, see Falk, 1961). The barbiturates comprise an extensive family of compounds. Relatively few of them have found therapeutic application. All classes of nervous system activity are to be found within this family, namely, stimulants, hypnotics, and agents with no action at all. The present report is concerned with the activity of only a limited sample of even the drugs which are presently therapeutically significant. The underlying assumption is that the sampling given is sufficient to describe the activity of all drugs within a subclass adequately in one way or another. However, development of the topic will provide someinformation that should lead to healthy doubt ofthis sampling assumption. THE B E G I N N I N G S : T H E O N E - D R U G M O D E L

The earliest literature concerning the effect of barbiturates upon water ingestion were the studies of Jones (1943), Alexander and Siegel (1947), and O’Kelly and Weiss (1 955). Both Jones and O’Kelly and Weiss found marked increasesi n drinking associated with particular dosages of phenobarbital and Dial, respectively. Both of these reports concern water deprived rats. Alexander and Siegel report negative findings, even some depression of drinking, following pentobarbital treatment in the rat. The procedure of this latter study was to inject intraperitoneal isotonic saline to induce drinking and is unique not only for its method of initiating drinking response but also for the reduction of drinking associated with a hypnotic barbiturate. In no case have I found that small to moderate doses of hypnotic barbiturates reduce drinking. However, I have not employed the method of Alexander and Siegel (1947) to induce drinking. At the time I began research in this area the findings were as described in the paragraph immediately preceding. Assuming that there was anything to the reports of Jones (1943), and O’Kelly and Weiss (1955), it was easy to construct a prediction based upon a few simple considerations. First, water ingestion rose at a dosage less than that producing hypnosis in the findings reported. Second, increments in dosage, if sufficient in quantity, resulted in hypnosis at which point no drinking will occur. Finally, it was reasonable to believe that there were degrees of depression at dosages lower than that which would produce hypnosis, i.e. a n assumption of continuity. These considerations led to a concrete prediction of a double action - a rise in drinking response at low dosages to a limit with a subsequent fall at higher dosages. The initial tests of this idea were consistently disappointing. The data obtained all pointed to a rise to a maximal response with a levelling off at all higher dosages with no sign of a relative decline in the amount of water the rat would ingest. The drug in this case was pentobarbital with a 15-min delay interval between drug administration and access to water in the 23+ h water deprived rat (Schmidt, 1958). Dr. Erminio Costa suggested that the administration time might be too short for drug absorption from the injection site to suffice to produce a depressant response. Application of this idea to the problem by increasing the time interval between drug ad-

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ministration and drinking to 1 h resulted in good confirmation of the prediction as laid out above (Schmidt, 1958). The kind of conceptual scheme which might be elaborated from these data is very similar to that of barbiturate hypnosis. Essentially all of the hypnotics are believed to have similar actions which produce hypnosis varying in potency (a function of the reciprocal of dosage) and duration of action. The initial experiments in the series of studies done after the confirmation of the notion of double action were concerned with simple dosage problems. At this time a concept of a family of barbiturates varying in effect upon water ingestion was entertained. It was felt that the hypnotics would have facilitating (incremental) effects upon ingestion in suitable dosages, while the inert products would have no effect, and the stimulants would have depressant (decremental) effects upon drinking. The other variation would be in terms of dosage, e.g. the same small dose of phenobarbital would increase water intake to a lesser extent than the same dose of pentobarbital as long as the dose of the latter drug does not exceed that producing maximum drinking. The data obtained definitely confirmed the notion of a family of barbiturate actions varying in hypnotic activity (Schmidt and Moak, 1957). A disquieting finding was that chlorpromazine, which by itself has some depressant effect upon drinking, totally blocks the facilitating action of pentobarbital upon drinking (Schmidt and Moak, 1959). While this indicated that the pharmacology of water ingestion involves a number of interactions, the notion of a unidimensional barbiturate action upon drinking was not materially affected. Becoming more detailed, the notion of a one-drug model can be more closely examined. In the model, small dosages of hypnotic barbiturates increase drinking to a maximum,rthe same maximum for all barbiturates. Two hypotheses might be readily advanced. The first is the osmomimetic hypothesis advanced by O’Kelly and Weiss (1955) in which it is postulated that the hypothalamic vasodilation associated with barbiturate anesthesia (Laidlaw and Kennard, 1940) occurs at definitely subhypnotic doses and this vasodilation irritates osmoreceptive elements. Another theory of the action of hypnotic barbiturates producing its facilitating effect upon drinking is by obtunding other undefined but competing responses. Further increments of dosage result in a falling water intake below the maximum and then terminate in zero response in the vicinity of the hypnotic dose. Ostensibly, the decrease in drinking below the maximum response is due to progressive sedation. Unfortunately, there is one thing to be said for the one-drug model, it is false! Systematic examination of the effects of other drugs upon drinking yields results in contradiction to the assumptions made. THE NECESSITY O F TWO D I M E N S I O N S : THE T W O - D R U G MODEL

Careful comparison of dose-drinking response curves obtained with various barbiturates reveals a striking finding in addition to the expected dosimetric variation attributable to variations in hypnotic activity: the magnitude of the maximum response varies (Schmidt and Moak, 1959). Phenobarbital is the most active barbiturate in the References p . 283!284

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facilitation of water intake at a maximum of all those presently studied while pentobarbital is appreciably less active. Several drugs seem to be about as active as pentobarbital, e.g. secobarbital or amobarbital (Schmidt and Dry, 1963b). On the other hand, no other barbiturate is known to be even close to phenobarbital in incremental activity upon drinking. Barbital has been found to be intermediate in its facilitating effect upon the measure of water ingestion (Schmidt and Moak, 1959). As a consequence of these findings, it was concluded that pentobarbital and phenobarbital did not facilitate drinking by the same action or at least that pentobarbital was quite low in an action strongly represented by phenobarbital facilitation of drinking. The only possible conclusion was that in addition to the hypnotic action of barbiturates some other undefined action affects the drinking response (Schmidt and Moak, 1959). One linc of investigation undertaken in this area was to induce drinking with hypertonic saline solution in the rat in water balance. Two measures of response were generally employed: (1) response latency, i.e. time to initiate drinking, and (2) 3 h volume of water ingested. Shirley Moak and I found that pentobarbital had an unreliable effect upon the volume of water ingested under these conditions, apparently showing an increase in ‘naive’ animals and not in previously saline treated animals (Schmidt and Moak, 1959). In a later series of studies, even this latter possibility was not borne out; there was no effect if there was no delay between injection of the hypertonic saline and access to water if pentobarbital was administered as compared with placebo controls. Neither latency decreased nor volume ingested increased after pentobarbital treatment as compared with control (Schmidt and Dry, 1963a). Phenobarbital both decreased latency and increased 3 h intake when access to water immediately followed injection (Schmidt and Dry, I963a). Curiously, if a 53-h interval was interposed between saline injection and access to water, pentobarbital markedly increased drinking over control values (Schmidt and Dry, 1963a). Regrettably, latency measures are of no value in this delay experiment. It is of some note that this kind of delay is believed to have properties like water deprivation despite no increment in drinking or even a decrement with small amounts of saline as compared with immediate access to water after saline ingestion (O’Kelly and Heyer, 1949; Miller, 1956; Wayner and Emmers, 1959). At this point the O’Kelly and Weiss (1955) theory of hypothalamic irritation by vasodilation induced by hypnotics can be evaluated. If one can consider the response latency to be related to the time needed for serum saline to reach the threshold of the drinking response, it may fairly be said that phenobarbital lowers the threshold while pentobarbital has no such effect. Furthermore, if irritation of the osmoreceptors by vasodilation occurs, one would expect the threshold to be lowered by the agent producing vasodilation. Following this reasoning, phenobarbital might have the necessary irritative property while pentobarbital clearly does not. Thus again two factors must be invoked, one factor does not suffice to explain the available data. These considerations do not necessarily preclude other explanations of the phenobarbital action upon drinking latency. In order to demonstrate the insufficiency of the O’Kelly-Weiss notion of threshold

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alteration, it is necessary to present evidence that response latency is correlated with the drinking threshold as postulated and also to demonstrate that the stimulus itself is not altered. Wayner et al. (1962) have shown that with 3.2 mequiv NaCl there is a rise in serum sodium over a period of about half an hour after which there is a slow steady decline over 5 or perhaps more hours. This value of the amount of salt is quite close to that used in this laboratory (3 mequiv) and the latency of the untrained rat, or at least a rat with no history of adaptation to a deprivation schedule, is approximately 73 to 8 min as a median estimate (Wayner and Reimanis, 1958; Schmidt and Dry, 1963a). This small amount of evidence favors the use of latency measures as an index of threshold though more precise specification can be gotten only through further work in this area. Precise quantitative correlations remain to be obtained between latency and value of serum sodium concentration especially for short times so as to specify more exactly the function followed. The possibility of alteration of the stimulus itself by inhibiting diffusion of sodium from the injection site by pentobarbital or promotion of such diffusion by phenobarbital must be considered. There is no evidence for such an alteration of diffusion rates by phenobarbital, at least. Serum sodium of male and female rats after phenobarbital or placebo treatments and 3 mequiv NaCl 3 to 5 min after saline injections have been examined and no systematic differences attributable to drug treatment have been found. Thus, I believe that the barbiturate does not alter the diffusion of saline from an injection site and coordinately does not affect the adequate stimulus for drinking itself. These data tend to exclude the periphery from consideration as a significant site of barbiturate action upon drinking. On the contrary, the implication seems to be that an actual alteration of the ‘thirst receptor’ itself is involved, at least in the case of phenobarbital. In this, the O’Kelly-Weiss notion (1955) and the present available data are in agreement even though the mechanism involved may well be different than that suggested by those earlier workers. Further evidences of differences between phenobarbital and pentobarbital were obtained. Previous data had shown that chlorpromazine totally blocks the facilitating action of pentobarbital upon drinking (Schmidt and Moak, 1959). It was speculated that phenobarbital would be only partially blocked by chlorpromazine. The basis of reasoning underlying this speculation was that phenobarbital simulated the ‘adequate stimulus’ for drinking while chlorpromazine has only a subtractive effect in a sufficiently large fixed dose. If 2 3 t h water deprived animals are used with 40 mg/kg phenobarbital and 1.5 mg/kg chlorpromazine, the experiment fits expectations as stated above. However, if the same dosages of drugs are used in animals given 3 mequiv NaCl to induce drinking, the phenobarbital-chlorpromazine combination does not significantly differ in effect upon drinking from that obtained with chlorpromazine alone. Moreover, the drinking latencies of the chlorpromazine-phenobarbital combination are not appreciably different from that obtained with chlorpromazine alone. These latter findings tend to undercut the threshold alteration hypothesis proposed above. There are some considerations which may save that notion if certain other regularities can be found. A gross observation made in this [investigation was the very profound sedation of animals when given the chlorpromazineReferences p . 2831284

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phenobarbital combination, particularly when given saline solution. The observations of the sedative effect of this combination of drugs under deprivation conditions are not remarkable. However, these observations do not suffice for a sound judgment of relative hypnotic activity. Recent studies of hexobarbital sleeping times in the mouse have shown water balance to be a critical variable with regard tosleeping time and as a corollary, hypnotic activity itself. It has been shown that overhydration lengthens sleeping time (Borzelleca and Manthei, 1957; Bhide, 1960) while water deprivation as short as 24 h appreciably diminishes sleeping time (Ramwell and Lester, 1961). A comparable experiment with hypertonic saline solutions has been done. If the length of time the saline acts is not so long as to cause appreciable diuresis, sleep time should serve as an index of sedation of hypnosis in the relative dehydration state. Relatively large amounts of salt (2 g/kg) definitely increase sleep time following pentobarbital hypnosis (Borzelleca and Manthei, 1957). Ostensibly, a smaller dose of phenobarbital, sufficient to be quite active in increasing drinking in the deprived animal, should serve to decrease response latency and increase drinking either singly, or when combined with chlorpromazine in the hypertonic saline treated animal. Stress markedly affects barbiturate action upon the drinking response of the rat. If an animal is'immobilized for 2 h and then removed from immobilization 1 h before the half hour drinking period, no effect upon volume ingested is obtained in rats either placebo treated or not otherwise treated. If, on the other hand, the animals are injected with doses of pentobarbital (9 mg/kg) or phenobarbital (40 mg/kg) which give a near maximal drinking response and are immobilized as above, drinking is reduced below that found in non-immobilized rats treated similarly in terms of drugs. It is a fact of some importance for what follows to note that stress reduces drinking by the same amount for both drugs. Rupe et al. (1963) have described what may be a similar phenomenon in terms of sleeping time in the rat. The stressor was ligation of a posterior leg of the animal. All barbiturates except barbital showed a significant reduction in sleeping time as compared with non-stressed controls. Considering these facts and others detailed in the study, it was thought that ACTH or a consequence or its release resulted in increased drug metaholisrn so as to reduce sleeping time. This interpretation states that barbital is not affected because it is normally excreted without metabolic alteration. Could a similar interpretation be made of the effect of stress upon barbiturate facilitation of drinking? Of course, one can conceive of this possibility being true. There is one basis of doubt however. It is hard to believe that an increase in metabolism produced by stress would produce just that reduction of phenobarbital activity upon drinking to equal the reduction of pentobarbital activity in the face of widely differing usual ratec of metabolic degradation. This raises only an implausibility, not an impossibility. Considering that barbital is excreted unaltered, this latter drug should be useful in answering the question raised by these findings. There may well be only one mechanism of stress reduction of barbiturate activity but present evidence does not preclude the possibility of two separate phenomena. If one considers the variety of phenomena demonstrating differences between phenobarbital and pentobarbital facilitation of drinking, the relevant question is the

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number of dimensions of action revealed by the drinking measure. Tentatively, two dimensions have been identified: ( I ) an anti-satiating activity which is to be found associated with the action of both pentobarbital and phenobarbital, and (2) a threshold reducing action which is only negligibly, if at all, present in pentobarbital facilitation of drinking. Current thinking in this area does not eliminate the possibility of other dimensions but neither is our present conceptualization so advanced as to foresee the consequences of introducing one or more dimensions or conversely not doing so. Ockham’s razor is perhaps the heuristic principle to be adhered to but that may reflect ignorance rather than satisfactory explication. T I M E O F A D M I N I S T R A T I O N AS A C O M P L I C A T I N G F A C T O R

One variable of drug action of considerable importance is duration of action. After some dosirnetric considerations, no other variable can be of greater importance. My colleagues and I have largely confined ourselves to examining barbiturate effects upon drinking at about 1 h after drug administration. More recently we have employed 45 min as the time after drug administration at which access to water is allowed. This latter change seems to have negligible effects upon our results but might be of some moment if very short acting agents were to be examined. This minor temporal change does not serve to define duration of action of a drug in the drinking situation. The assumption upon which the procedure of these drug studies was postulated was that the response would be proportional to the amount of active drug in tissue. In its simplest form one can imagine a situation where the drug is metabolized into inert products or excreted unchanged. Assuming this to be the case, it can be expected that the half life of the drug is a significant parameter which will sufficiently define sequential changes in response t o be observed. The examination of barbital should prove of value in this regard since it is largely excreted unchanged. If the response is proportional to the amount of active drug remaining in tissue at any time, the curve should follow the dosimetric curve more or less as a function of time, only the curve will be reversed. No values below the control level would be expected. However, we have today some definite evidence which elicits serious doubts at the validity of this simple scheme. The usual procedure in our studies has been to adapt our rats for a period of lodays. On the I I th day, and every 3rd day thereafter the animals are treated until all animals receive all treatments. At this time, if sufficient time elapses, the characteristic rising and falling barbiturate dose-drinking response curves are obtained. If one looks at what happens upon the following 2 days (24 and 48 h after drug administration), there are other findings. If short acting drugs such as secobarbital or amobarbital are given, the curves are deflected downward so that there is a falling and then rising curve as a function of dosage 1 and 2 days after drug administration (Schmidt and Dry, 1963b). Approximately 2 days after drug administration these curves suggest water balance since the departures are small though significant. With phenobarbital, on the other hand, there is the falling then rising function 24 h after drug administration which at the highest dosages is above the control value at that time. The curve essentially only falls, 48 h after drug treatment, References p . 2831284

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as a function of the dosage (Schmidt and Dry, 1963b). At first it was thought that these late curves were reflections of the overhydration of animals somewhat contaminated with residual drug actions in the case of phenobarbital. Note the acceptance of a simple time of action theory above implied by this explanation. Experimental test of this interpretation totally refuted the notion that regulation of overhydration was involved, at least 48 h after phenobarbital treatment. Two groups of rats were adapted to a 23%h water deprivation schedule. On the 1 1 th day and every 3rd day thereafter one group of animals was treated as usual in our experiments. No restriction of water in the half hour drinking period was made at any time. The other group was allowed to drink only 17 ml, 45 min and 24 h after drug administration, in the half hour drinking period. Two days after drug administration, ad lib. drinking by this group was allowed. Whileit istrue that this latter group drank more than the former on the test trials, the curves for both groups sloped down as a function of the increasing dosage of phenobarbital administered 2 days before (Schmidt and Dry, 1963b). Thus there is a dosage effect at this time which is not a product of water regulation per se. The test is not an overwhelming refutation of the notion that the response is proportional to the amount of active drug in tissue since there are phenobarbital metabolites and they might be the active drugs sought for. Barbital should obviate the restrictions upon the conclusion since it is largely, if not entirely, excreted i n an unaltered form. Tf I may hazard a prophecy, and in an area as complex as this prophecy is hazardous, barbital will also show an effect which cannot be regarded as related to the amount of active drug remaining. EFFECTS U P O N S A L I N E A C C E P T A B I L I T Y

An area of drug investigation which in times yet to come should yield new vistas, is that of acceptability and preference. The major distinction between these two concepts is operational: Acceptability refers to a measure, usually volume ingested, in a one bottle test, i.e. one bottle at a time, while preference refers to a choice between two or more solutions or substances at a time. Both acceptability and preference tests generally give rise to similar findings but there are puzzling exceptions which are not explicable within any current conceptual framework. Whether preference and acceptability refer to processes which must be regarded as fundamentally different remains to be seen both experimentally and theoretically. At the time the work was undertaken in this limited area, I had expected to find a shift in maximum acceptability of saline solutions from approximately 0.9 % NaCl to a lower value. In fact, the only data bearing on the issue were obtained in a pilot preference study undertaken to get a preliminary estimate. These latter data indicated that rats given 40 mg/kg phenobarbital daily drank very little 0.9 % saline solution while control rats drank appreciable quantities of saline. A formal test was proposed in which a variety of solutions would be tested for acceptability. These solutions ranged from 0.0 to 3.0% NaCl with the intervals between solutions unequal. Rats were adapted to a split drinking schedule so that a failure to drink during the first period because of very low solution acceptability could be overcome during the

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second drinking period I + h later. The animals were divided into 2 groups, one of which was given 40 mg/kg phenobarbital while the other received physiological saline in the same relative volume. The phenobarbital increased the intake of all solutions markedly. The greatest increase was in the vicinity of 0.9 %, maximally accepted by both groups (Schmidt, 1963b). Some phenobarbital treated rats drank so much at that concentration so as to overload the absorptive mechanisms of the gut and expel water through the anus. The experiment just cited was done with albino rats about 120 days of age. A second experiment of similar design was done with 200-day-old hooded rats. The results in this study differed somewhat in that only at higher concentrations did phenobarbital give rise to greater solution acceptability. Pentobarbital had even more striking effects in that only at the maximally acceptable concentration of saline did the pentobarbital treated animals drink more than the controls. Usually the same amount was ingested but on the rejection limb of the curve the pentobarbital treated hooded rats drank less than controls. Replication of this experiment with 200-dayold albino rats gave no evidence that pentobarbital gave rise to a more aversive response to salt on the rejection limb of the curve than did placebo treatment. Perhaps there is a strain difference which accounts for this disparity. However, the groups of hooded rats were so small that chance factors may have determined the results. The splitting of the drinking period involves problems of drug action as well as problems of response to various concentrations of saline. The split drinking schedule employed in this laboratory consists of 21+ h of water deprivation, 3 hour drinking, 13 h in the home cage or, on test days, in other cages without food or water, and finally a second half hour drinking period. During the first half hour drinking period, phenobarbital treated rats (albino animals as above) drink appreciably more of all test saline solutions than do the comparable controls. Also, they ingest an appreciably greater quantity of salt, reaching a maximum of approximately 450 mg salt on the average at a 1.5 % saline solution; the controls ingest a maximum of approximately 250 mg salt at that same concentration, which also appears to be the upper limit in that case. While it is true that the phenobarbital treated animals drink more tap water during the drinking period following the saline test period ( I + h later) and the curves are linear, increasing functions of saline concentration up to 1.5 % saline, the curves for placebo and phenobarbital treated animals are parallel throughout this range. The finding of linearity may or may not be surprising but the parallelism between functions is quite puzzling. Presumably, the increase in drinking as a function of the earlier saline concentration is a result of increasing salt ingestion. Certainly there is nothing in my experience to suggest that phenobarbital blocks salt activity upon drinking. Why do not the two curves diverge as a function of increasing saline concentration? A careful review of the data resulted in the extraction of another variable. If one examines the correlation of amount drunk during the earlier, with amount drunk during the later, drinking periods, within any concentration, a negative correlation is observed. Tap water ingestion 1%h after the saline test is a multiple function of both amount of salt ingested and volume of water ingested. Increasing salt ingested increases drinking while increasing water volume decreases drinking. This essentially References p . 2831284

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trivial relationship describes almost totally the behavior of placebo treated animals. However, a significant residual variance remains after these variables are extracted with phenobarbital treated animals. Thus, other variables than those noted immediately above must contribute to the phenobarbital effect. The most obvious explanation of the failure of the functions to diverge is that there is sufficient volumetric inhibition of drinking to balance the salt enhancement of drinking in the case of the phenobarbital treated animals. This explanation requires a discontinuity at the maximally accepted concentration with an increase of slope as a function of concentrations above the point of maximum acceptability when drinking tap water during a later period. The finding of linearity to 1.5% tends to discount this possibility though the number of points does not suffice to make this reason overwhelming. However, if the effect of saline solutions above 1.5 % is considered,there is indeed a change in slope,but downward in a direction ofrelativedecrease rather than increase. This latter point excludes the possibility that volumetric inhibition is at work to prevent the phenobarbital curve from diverging from the placebo curve. Another consideration which seemed important was the possibility that more sodium was excreted by the phenobarbital treated animals than the controls. As a consequence, the effective sodium at the time of the later drinking period would be equal in both groups. Examination of this possibility in terms of urinary sodium indicated nothing to support this. While it was true that the phenobarbital treated animals excreted a somewhat more sodium concentrated urine than the placebo controls, insufficient amounts were excreted to even grossly equate both groups. C H R O N I C R E P E A T E D B A R B I T U R A T E EFFECTS

Most of the previous description is in large part based upon single treatment instances with fairly large numbers of animals. There were noted a few exceptions of little definitive value in describing any effects of repeated barbiturate treatments. One experiment described heretofore of some interest with regard to long term effects of barbiturates and the consequences of repeated treatment involved the finding that there are long term barbiturate effects which can be related to dosage of the drug administered. One can see from that result, there are some persistent effects of at least phenobarbital treatment over what in these studies is a long period. Presumably, shorter tempera1 intervals might demonstrate similar phenomena for other shorter acting barbiturates. Repeated treatments should give answers to the problems of tolerance to the drugs, changes in sensitivity, withdrawal symptoms, and changes which can be described as ‘psychic craving’. Tolerance can, i n principle, be described as of two possible kinds: (1) tolerance which affects the facilitation of drinking so that it is necessary to raise the dose in order to produce a maximum drinking response, and (2) tolerance to the depression of drinking. Sensitivity would be the opposite to tolerance, namely, enhanced response to specified doses of a barbiturate in this case. A withdrawal response might be considered to be any reduction in behavior not a physiological regulation per se at the time a chronic course of medication is terminated or shortly thereafter.

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Finally, ‘psychic craving’ might be defined as the performance of some work or behavior in order to receive a continuation of the drug. These areas of barbiturate pharmacology are a mare’s nest of contradictions and refutations. Several studies have shown tolerance to barbiturates in rats (Fitch, 1930; Nicholas and Barron, 1932; Moir, 1937; Gruber and Keyser, 1946). Moreover, tolerance development appears less persistent in females than males (Moir, 1937). There are, however, contrary data to both the development of tolerance and sex differences (Ravdin et al., 1930; Stanton, 1936a). The only study which might be considered to demonstrate sensitization to barbiturates showed that after a period of repeated treatment and then withdrawal female rats, though not males, were hypnotized with lower doses of these drugs than was typical of animals of the same age and sex without that course of treatment (Moir, 1937). Stanton (1936b) found no increase of struggling response after withdrawal from a prolonged course of pentobarbital or phenobarbital treatment as he had found after morphine withdrawal (1936a). From this he concluded that the rat showed no physical dependence upon barbiturates. Jones (1943) reported some depression of eating after withdrawal from phenobarbital. However, these data were not sufficiently controlled to be so sure that this was truly a withdrawal effect. This finding has had no substantial effect upon thinking with regard to withdrawal effects of barbiturates in the rat. Jones reported no change in water intake except a return to its normal level after withdrawal from phenobarbital. Finally, I have not been able to find any reports of attempts to ascertain the existence of ‘psychic craving’ for barbiturates in the rat. A couple of years ago my wife and I performed an experiment in which 70 mg/kg phenobarbital was given to a group of rats each day. A second group received only 40 mg/kg daily, while a control group was injected with physiological saline. This schedule of treatments was maintained for 17 days during which the animals were housed singly in cages. All eating and drinking were ad lib. During the early part of the treatment phase of this study both barbiturate treated groups drank appreciably more than the controls but not much differently from each other. As treatments progressed, drinking fell off progressively to only slightly and probably insignificantly above the control group. Weight and food intake did not appear to be greatly affected at this time. When treatments were withdrawn the animals given 70 mg/kg phenobarbital drank significantly less than did the controls. On the other hand, those animals given only 40 mg/kg phenobarbital showed only a slight insignificant decline in drinking at this time. Again weight and food intake appeared unremarkable. This latter study was the basis upon which we have continued work in this area. It differs from what have been essentially replications of it only in two significant respects. First, food intake and weight have been affected in all later studies in which they have been measured. Both food intake and body weight fell off after withdrawal from a chronic course of phenobarbital treatment. Second, all later ad lib studies of the changes during the treatment phase of chronic phenobarbital treatment have shown a less gradual reduction to the control level than was originally found when daily water ingestion is measured. In effect there is a sharp rise to a maximum with a very rapid return to the baseline. Pentobarbital has not been found to have an effect References p . 2831284

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when drinking is ad lib. Chlordiazepoxide has an effect much like phenobarbital when administered chronically. A repetition of the phenobarbital treatment after a course of 30 days of phenobarbital treatments and a short period without treatment result in a marked enhancement in the drinking response as compared with the first treatment although the enhanced response is no more persistent. In this respect, chlordiazepoxide is not similar in its effects upon water intake to phenobarbital. Three further points deserve mention. First, despite the fact that the rat at the time of phenobarbital withdrawal seems grossly more tense and presumably more stressed than their counterpart controls, he is no more susceptible to gastric lesions from immobilization. Second, activity in an open field test is below the control level after phenobarbital withdrawal rather than above that level. Third, the weight loss of rats after phenobarbital withdrawal is not simply a loss of water nor a reduction of food ingestion. These few preliminary remarks answer a number of questions raised above. There is evidence here of tolerance to the drug, at least in the sense that the facilitating effect of phenobarbital diminishes after repeated injections. Evidence of sensitization is also found in that the facilitation of the drinking response is much greater during a second course of treatment than it is during the first. Finally, a number of different measures indicate the presence of a withdrawal syndrome. The specific picture of physical dependence revealed is a pattern of what have been known as secondary symptoms of withdrawal rather than the so-called primary symptoms which are life and death matters. The data available to us suggest that the secondary symptoms may have a much greater species generality than do the primary symptoms. If similar procedures are employed in deprived rats, the overall picture of the effects of repeated barbiturate treatments changes somewhat. N o evidence of a clearcut reduction of facilitation of drinking is found during a prolonged course of phenobarbital or pentobarbital treatments. If there is such an effect, i.e. tolerance to the facilitating action, it does not make an appearance within 30 days. Perhaps a much longer course of repeated treatments would show some tolerance of this kind. The general course followed by the barbiturate treated rat on 233 h water deprivation with daily injection is fairly steady depending upon (1) doses, (2) drug, and (3) changes in dose. Dose effects under conditions of chronic treatment are related to acute effects. Those doses giving the greatest effect upon single injection give the greatest effect upon occasions of multiple daily injections. A similar statement applies to the effect of the barbiturate selected. Changes in dosage can result in a rise in drinking, a fall in drinking, or even only a transitory fall in drinking. These variations in effect of change of dosage depend upon the initial dosage and the next dosage selected. Barbiturate tolerance can be found in the water deprived rat when dosages above that producing maximum drinking are employed and dosage is raised. However, this is tolerance to the depressant effect of the barbiturate and not the facilitating effect that occurs in rats with ad lib. water. At present, the possibility that tolerance to the depressant actions of barbiturates in the drinking situation with ad lib. water intake occurs has not been fully investigated. It is not particularly difficult to imagine another experiment which might yield

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additional information about the effects of chronic repeated barbiturate treatment. The paradigm would basically consist of a determination of a dose-response curve, the application of some treatment, and finally a repetition of the dose-response curve. An initial phenobarbital dose-drinking response curve was obtained for 2 different groups. These curves departed from each other by no more than would be expected by chance about half of the time. One group was given 70 mg/kg phenobarbital for 17 days while the other was given a physiological saline placebo for the same length of time. At that time the treatments ceased for 15 days with a precipitous decline in drinking and body weight in animals previously given daily phenobarbital. The decline below control level in drinking persisted for 2 days. Body weight loss was sharpest for this period as well, but continued at a slow rate for several more days, then rose back to the control level just before the beginning of the test dose-response curves. The test curve obtained with placebo treated animals was not significantly different from the original curves. The curve for the animals treated with daily phenobarbital did not differ from the control curve at low dosages of phenobarbital but continued to rise to a maximum response at 53 mg/kg at which dose the control curve was already falling. (The usual dosage of phenobarbital producing a maximum drinking response is in the vicinity of 40 mg/kg. The control curve gave a maximum response at about 36 mg/kg phenobarbital.) Another feature which was quite remarkable was that the maximum response was raised above that found for phenobarbital when given acutely without previous treatment. This experiment provides definite evidence for tolerance to the depressant action of phenobarbital upon drinking in higher dosages and also gives evidence of some response which might be considered to be sensitization to the facilitative action. Perhaps a more parsimonious view of this so-called sensitization would be an unmasking of additional facilitative activity of phenobarbital by reducing the depressant effect. However, further studies have not found anything which can be clearly identified as tolerance while sensitization or at least an increment in the response to phenobarbital or even pentobarbital has been demonstrated after a chronic course of medication has been followed. Considering the kinds of results obtained in the immediately previous study and the possible significance and interpretation of the results several questions need consideration. What would dosage variations of a chronic course of medication do to a later dose-response curve? What about variations in the duration of treatment? Is the drug chosen a critical variable? No doubt there are numerous other questions that are relevant and material to an understanding of even the few phenomena already demonstrated. Moreover, even within the confines of the few questions raised, more might be done in terms of extending the data to cover far broader areas of quantitative and drug variations. A beginning was made, however, in a study conducted by Ken Kleinman and me. Some of this investigation was reported above in terms of the effects of the variables of dose, drug, and duration upon a chronic course of medication itself. The same kind of procedure was followed as in the previous experiment. Essentially this consisted of obtaining dose-response curves, applying various treatment conditions, withdrawal of the treatment conditions, recovery, and finally another set References p. 283!284

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of dose-response curves. Phenobarbital dose-drinking response curves were obtained. The treatments:consisted of 0,40 and 70 mg/kg daily phenobarbital and 15 mg/kg daily pentobarbital for 30 days; 70 mg/kg daily phenobarbital for 10 days, then 85 mg/kgIdaily phenobarbital for 10 days, and then 100 mg/kg daily phenobarbital for the final 10 days; placebo treatment for 15 days followed by 70 mg/kg daily phenobarbital for the final 15 days. Withdrawal and recovery occupied 1.5 days between the application of treatments and the determination of the second dose-response curves. The effects of these variations during the application of the treatments have been described above and need not be repeated here. Withdrawal effects were obtained with all groups. The magnitude of depression of drinking below the control level was not different for the various treatment conditions. However, the duration of the depression varied. Increasing the duration of 70 mg/kg phenobarbital treatment from 15 t o 30 days resulted in an increase in duration of depressed drinking from 2 to 3 days. Dosage increments had an effect upon length of withdrawal hypodipsia also: 40 mg/kg daily phenobarbital resulted in only 1 day of depressed water ingestion while 70 mg/kg phenobarbital, or 70 mg/kg increased to 100 mg/kg phenobarbital resulted in 3 days of significant decrement in water intake. Finally, 70 mg/kg phenobarbital produced a decrement in drinking below the control level for a longer period than did 15 mg/kg pentobarbital. However, withdrawal effcct of 40 mg/kg phenobarbital was of no greater duration than that produced by pentobarbital withdrawal. Certainly in the case of the duration of withdrawal action of pentobarbital it may be suspected that repeated dosages of 15 mg/kg on the same day so that 45 or 60 mg/kg pentobarbital would be given daily for the 30-day period might promote a much more prolonged hypodipsia. It might seem that the supposed withdrawal symptoms in this experiment are a regulation of long persisting overhydration. However, there is no correlation between the duration of withdrawal hypodipsia and amount of drinking during the treatment phase. Moreover, the magnitude of depression of drinking when it occurs is not differentially affected by the treatments within the confines of this experiment. On the other hand, it is evident that there are definite limits to this conclusion since single doses of phenobarbital, if large enough in amount (60 or 70 mg/kg), exhibit residual hyperdipsia a day after treatment. Only two days after treatment do single doses of phenobarbital of those large dosages have a hypodipsic effect while the depression of drinking after several daily repeated treatments with 60 or 70 mg/kg phenobarbital results in prompt hypodipsia after withdrawal. Perhaps this reveals the existence of some increase in rate of metabolic alteration or rate of excretion of phenobarbital after a chronic treatment course. Another facet of these results is the recovery from withdrawal hypodipsia. It is possible that termination of the decreased drinking is in part determined by the progressive dehydration of the animals resulting in a strengthening drive state. At present there are sufficient data to suggest that regulation of water balance by ingestion is asymmetrical with regard to under- and overhydration. Underhydration can only be rectified by increased ingestion in all mammals except those that can effectively conserve metabolic water. On the other hand, overhydration usually, if not invariably, is

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regulated by increases in urine excretion. Returning to theoriginal point, it is not obvious how to investigate the termination of withdrawal hypodipsia in the context of controlling underhydration at various levels. The second dose-response curves found in this series of experiments puzzled us. There were no signs of tolerance to be found. The curves differed from one another in part reflecting some initial differences, though such variations were not of the kind that a shift in the dosage producing maximum drinking was to be found after any of the treatments investigated. On the other hand, sensitization to the barbiturates was much in evidence. The enhancement of response was in proportion to the effect of the treatment during the chronic phase. Thus the greatest sensitization was found when 40 mg/kg phenobarbital had been previously given while a chronic course of 15 mg/kg pentobarbital had the least enhancing effect upon the later dose-response curve. There was some slight increase in sensitization to phenobarbital when the phenobarbital was administered for 30 days as compared with only 15 days of drug treatment. In order to attempt to further examine tolerance to phenobarbital, another study was done. The earlier dose-response curves were not obtained. Moreover, the differential treatments were administered while the animals were maintained upon ad lib. food and water in contrast to the water deprivation schedules used in the earlier studies. Phenobarbital was administered for 15 days in a dosage of 70 mg/kg to one group while another was given a physiological saline placebo. Withdrawal and recovery occupied 15 days during which time the rats were adapted to a water deprivation schedule. The usual phenobarbital dose-response curve was then obtained. No evidence of either tolerance or sensitization was to be found in these data. At present there is nothing definite to suggest the explanation for this unexpected finding. Presumably, the conditions under which the chronic treatment was administered were critical but the significance of such variations remains to be elucidated. Summing up the experience with dose-response curves after repeated phenobarbital treatment one may say that tolerance was proven elusive. Intuitively, there may well remain some expectation of reliably producing tolerance to the hypnotic action of phenobarbital in the face of the several findings of that kind. Sensitization is somewhat better represented by the data heretofore collected being demonstrated upon 6 of the 7 instances where the conditions of its production might be presumed to exist. Even the negative instance may not prove to be a compelling exception if it can be shown that the conditions set up in the ‘exceptional’ case preclude the enhancing effect called sensitization. These studies have brought no nearer any understanding of the ‘nature’ of tolerance or sensitization nor have they indicated what, if any, relationship they bear to each other. Having demonstrated withdrawal symptoms to barbiturates in the rat unequivocally and tolerance with a large surrounding penumbra of doubt, the question of ‘psychic craving’ for barbiturates needs consideration. No previous work in this area is available, none has been reported, and quite possibly none ever done with the rat. However, Nichols et al. (1956) has developed a preference procedure which he used with morphine with great success. Ken Kleinman of this laboratory adapted this procedure to barbiturates. Two groups of rats were placed on a drinking schedule References p. 2S3f284

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and after adaptation to the schedule given 70 mg/kg phenobarbital daily for 30 days. Phenobarbital was then withdrawn for both groups. The animals had no water for 48 h at which time they were given two drinking tubes, one containing tap water, the other containing 1 mg/ml phenobarbital. At this time no rats drank much of the phenobarbital solution. One group was put on a schedule of deprivation of water for 48 h, drink 1 mg/ml phenobarbital solution, 24 h deprivation drink tap water, 48 h deprivation, etc. while the other groups followed the same schedule except those latter animals had tap water at all times. Each animal in this latter group was matched by weight with an animal in the former group and was injected with the same amount of phenobarbital as its matched counterpart received in the drinking solution. On the 5th and 10th occasions of phenobarbital solution drinking or injection, a preference test was given to all animals to determine relative ingestion of tap water or phenobarbital solution. The preference tests were negative with regard to differential results in the drinking of phenobarbital solution. There was no increment in ingestion of the phenobarbital solution when tap water was also available from one occasion to the next. Thus, no ‘psychic craving’ was demonstrated by this experiment in any meaningful sense. However, the single bottle test of phenobarbital ingestion showed some sequential incremental change. The phenobarbital solution offered, 1 mg/ml, is less acceptable than tap water to 233 h water deprived rats. Even the first occasion of 48 h water deprivation finds the phenobarbital solution less acceptable than tap water. But successive presentations resulted in an appreciable rise in phenobarbital ingestion to well above the amount of Auid consumed by the controls though there was a later fall to the control level of fiuid ingested. Consequently, the results of this study are not entirely conclusive but need supplementation of other data. At this point it is quite material to ask whether the rat is subject to barbiturate addiction. Prior to the studies in this laboratory, the data were such as to suggest a definite no. The evidence for physical dependence was negligible. The findings presented in this paper are strong evidence for some definite physical dependence upon barbiturate in the rat despite the fact that the symptoms are not so dramatic as in higher mammals (Essig and Flanary, 1961 ; Seevers and Tatum, 1931 ; Fraser and Isbell, 1954). Nevertheless, the usual connotation of addiction is an appetite for the addictive agent. As can be seen above, the evidence on this point is at best equivocal with regard to the barbiturates in the rat. Addiction in the sense of craving for barbiturates remains to be demonstrated in the cat and dog as well. THEORETICAL SIGNIFICANCE

Two different notions of theory have been developed to attempt at least to account for the findings reported in this paper. These are what is essentially a statistical model of double action, on one hand, and an attempt to identify some sites of action where the postulated actions take place. However, these attempts at theory are quite limited as compared with the data now available. Not all that has been empirically determined has been given sufficient theoretical designation. Overall, the situation is one of more intuition than rigorous theory.

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A statistical concept of double action The curves obtained by varying dosages of hypnotic barbiturates when water ingestion is the measure are indicative of a double action. Two alternative concepts may be devised to explain this kind of finding: (1) two actions of the drug exist with regard to the same cell dependent upon dosage, and (2) the same action is manifested by a drug on all affected cells but a variation in dosage affects various cells. The latter a1ternative is assumed in the following discussion. Another notion to be assumed is that the outcome of the behavior of a group of cells is the net product of the number of cells firing and the frequency of their activity. Assume the simplest case of two pools of cells one of which activates a response, the other of which depresses the response. No presumption need be made at this point whether the sources of activation are one or many. To simplify the situation, assume that each pool is the same size and each element in each pool has an equal absolute effect whether plus or minus. A drug might act upon either pool or both by either inactivating or conversely activating elements in the pool. If a normal distribution of elements within a pool is assumed, the cumulative dosimetric function will be sigmoid ( S shaped) in shape. This may be identified as a sensitivity function. For the first case we can deal with a very limited likelihood of occurrence. Assume that the relative rates of inactivation of each pool are the same. Despite definite action at a cellular level, the response associated with the pool is unchanged. Another instance would be that of inactivation of the activating pool at a much greater rate than that of the inhibitory pool. Only depressive activity would be manifest in this situation since disinhibition without activation has no manifest effect. Finally, the case of inactivation of inhibitory elements may occur more rapidly than the inactivation of excitatory elements. This case gives rise to a double action in that there is progressively less and less resistance to residual activation to give a rise then excitation itself falls off to zero at which point the response disappears. The actual concept of the rate of excitation in this instance may be contrasted with that developed by Anand and Dua (1955) for eating. Those workers fee1 that the excitatory elements are a ways active and ready to initiate food ingestion but are restrained by inhibitory activities ostensibly arising in the ventromedial nucleus of the hypothalamus. The present conception is that of variations in excitatory level itself as well as restraint by inhibitory activity. Needless to say similar paradigms can be constructed for drugs having marked stimulating effects. Certainly, in the case of the barbiturates it is necessary to assume that the hypnotics depress the activity of cells, i.e. prevent them from firing. There is some evidence that this effect occurs more readily, i.e. at lower doses, upon inhibitory systems than upon excitatory systems (Machne et al., 1955). That the excitatory systems are progressively obtunded at high doses only requires the examination of an animal hypnotized by barbiturates. Various other things can be conceptualized within this scheme. For example, the differential facilitation of drinking by phenobarbital as compared with pentobarbital may be viewed as a change in slope of the rate of inactivation. Phenobarbital may relatively reduce inhibitory activity more rapidly than pentobarbital. Equally plausible RKferenrPS p . .783/284

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is the notion that phenobarbital has a slower effect, i.e. at relatively higher doses, upon the excitatory elements. Unfortunately, at present there is no way of distinguishing these alternatives. On the other hand, the antagonism of McN 481 (5-ethyl 5,1,3dimethyl-2-butenyl barbituric acid) and pentobarbital involves two drugs competing for sites of action. McN 481 may be presumed to initially activate the inhibitory sites antagonistically to the inactivation induced by pentobarbital. A concrete prediction might be made that McN 481 or other stimulant barbiturates have opposed effects to the hypnotics such that if convulsant actions are suppressed there will result first a falling, then rising drinking curves as a function of the dosage. The antisatiating and thirst threshold altering actions of barbiturates are not separately dealt with in this scheme. They can be considered only as separable actions which contribute to the reduction of inhibitory activity. The possibility that there is a positive correlation between reduction of the drinking threshold and magnitude of facilitation at doses producing maximum responding poses no contradiction but is not explicable within this conception. Sites and modes of action

As was pointed out above, the statistical concepts underlying the present theoretical thinking on this give rise to a very generalized scheme applicable to many drugs. There is, per se, no need to invoke the brain in the descriptions available to us at present as long as there is merely concern with measuring barbiturate activity. This can be done with great power and elegance using the water intake measures discussed in this paper. Yet, if there is concern with using drugs as a research tool, specification of sites and modes of action are needed. It is my opinion that our data, limited as they are and not specifically directed to the central nervous system and the brain as has been the case nevertheless implicate the brain, in general, and hypothalamus, in particular, as being involved in barbiturate actions upon drinking. It has been possible to conceptualize an antisatiating action of hypnotic barbiturates in the rat. This is probably a feature of all of the hypnotic barbiturates in that it seems identifiable in phenobarbital and is about the only possibility of action upon drinking with pentobarbital. Observation of anterior hypothalamic lesions indicates the development of diabetes insipidus in the rat when lesions are made in the vicinity of supraoptic nucleus. Whether this is a primary disturbance of water retention or ingestion is not altogether clear at this time but tends to suggest a potential failure of satiation. While reasoning from the damaged mechanism to the intact is hazardous it might be feasible to believe that the anterior hypothalamus has a role in identifying when satiation is reached. I have some evidence for this idea in that water deprived rats with a chronic intragastric cannula (Epstein preparation - Epstein, 1960) when given a large load of water demonstrate a marked bradycardia usually, though not invariably, quite transient. Phenobarbital will interfere with the development of this bradycardia. It is quite important to note that if water is placed very slowly into the rat’s stomach and time is allowed for absorption through the intestine this response does not develop. Thus, it is very likely to be a reflexive slowing of the heart mediated through the brain and in all likelihood the anterior hypothalamus which is quite

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significant in parasympathetic activity. Whether or not there is an actual functional significance to the slowing of heartbeat at this time is a moot point. The threshold altering action of phenobarbital must be considered. There is considerable disagreement as to the precise sites implicated in the initiation of drinking. Anderson and McCann (1955) find that medial lesions posterior to the paraventricular nucleus markedly reduce drinking in the dog. These are the same sites where Anderson (1 953) found placement of hypertonic saline to greatly increase drinking in the goat. Lesions in this region seem to have no great effect upon drinking in the rat. Nevertheless, Greer (1955) found electrical stimulation of this medial region of the rat’s hypothalamus gave rise to enhanced drinking in one rat. Montemurro and Stevenson (1 957) report that lateral hypothalamic lesions can result in aphagia and adipsia. Moreover, some animals die despite persistent intubation of both food and water. Perhaps most recently Morgane (1961) has reported similar findings to those of Montemurro and Stevenson (1957) as long as the lesions are fairly large and in the far lateral hypothalamus. More medial lesions though in the so-called lateral hypothalamic regions have less persistent effects (Morgane, 1961 ; Teitlebaum and Epstein 1962). Morgane (1961) distinguishes between a ‘metabolic’ significance of far lateral sites and a ‘motivational’ aspect of the medial lateral hypothalamus. The present report cites some data which bear upon Morgane’s distinction. Phenobarbital treated rats thrive during the course of medication gaining weight, eating well, and drinking copiously. All of these disappear transiently at the time of drug withdrawal. Most remarkable in this view is that the weight loss associated withphenobarbital withdrawal does not depend upon water loss or differential drinking alone. It is our present belief that this excess of weight loss is of the same kind as that associated with the cessation of ‘metabolic’ activity produced by far lateral hypothalamic lesions. I propose that this weight loss is a hypermetabolism resulting from ‘rebound hyperexcitability’ of anrinhibitor of this region. The supraoptic nucleus and lateral hypothalamus are the places where my coworkers and I are planning an electrographic inquiry into barbiturate action upon water ingestion. While it is true that the foregoing notions of sites of action are based upon educated guesses, they have some significance in terms of what is known both of drinking and barbiturates. Today, despite little that is known for sure of how the barbiturates enhance the drinking response, there are enough data to help us decide whether a given difference between a barbiturate treated rat and a control counterpart is related to alterations in drinking. SUMMARY

Various findings with respect to barbiturate actions upon drinking have been reviewed. The plan of the paper has been to discuss a fairly broad range of phenomena and to classify those phenomena in terms of variables and models which describe them. Since a substantially chronological plan was adopted, the models develop from relatively simple to relatively complex. The first model is based upon the notion that hypnotic dosage or some function References p. 2831284

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of it suffices to describe barbiturate facilitation of drinking. A hypnotic dimension of barbiturate action was described and some evidence obtained to support the existence of such a dimension. An implication of simple additivity of effects of various barbiturates follows from the postulated dimension of action which appears to obtain between barbiturate antagonists but not between barbiturate synergists. The finding that various hypnotic barbiturates facilitated drinking to differing degrees necessitated the postulation of at least one dimension of action other than the hypnotic dimension. Other phenomena were obtained which indicate at least one drug action other than the hypnotic. Among these were findings of drinking under a wide range of conditions while pentobarbital required more limited conditions to reveal facilitating activity upon drinking. Whether these various differential effects as a function of drug can be explained on the basis of only two dimensions of action is not clear at this time. A new much neglected area of psychopharmacological research was described, namely drug effects upon preference and acceptability. Phenobarbital generally tends to make a drinkable saline solution more acceptable without affecting the point of maximum acceptability. Chronic effects and withdrawal after a chronic barbiturate course were next considered. When phenobarbital is given with ad lib. food and water, water and food intake rise for a very short time and fall rapidly to control levels. Pentobarbital has no effect under these conditions. Withdrawal of phenobarbital results in a marked hypodipsia and hypophagia. A few days after withdrawal, the animals return to a state comparable to controls in terms of food and water intake. Comparable experiments done with water deprived animals show similar initial changes in water intake. However, there is no decrease in the amount of water ingested after phenobarbital during a 30-day course. Withdrawal results in a marked hypodipsia. The decrement in drinking is essentially constant in magnitude for all effective withdrawal conditions but varies in duration. Retreatment after a period of withdrawal and recovery gives evidence of sensitization to phenobarbital and occasional evidence of tolerance to the hypnotic activity of phenobarbital. Finally, two general theoretical schemes were advanced to partially account for the findings: one a statistical concept to describe the double action of barbiturates, the other an attempt to designate locus for future work. The statistical scheme posits one action at the cellular level advancing in a gradient which gives rise to response variations as a function of dosage. The attempts to identify loci of action point to the hypothalamus, probably both anterior and lateral. These sites seem to have properties associated with satiation and threshold regulation which appear to be the mechanisms upon which the barbiturates act to alter water intake.

ACKNOWLEDGEMENT

The present paper was written with the support of United States Public Health Service grant NH. 02755-05.

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SCHMIDT, H., JR., (1963b); Saline acceptance following phenobarbital in the rat. Amer. Psycho/., 18,454. SCHMIDT, H., JR., AND DRY,L., (1963a); Comparison of phenobarbital and pentobarbital actions upon water ingestion. J . comnp. phjlsiol. Psychol., 56, 179-1 82. SCHMIDT, H., JR., AND DRY,L., (1963b); Dose and time as variables in barbiturate action upon water ingestion. Amer. J . Physiol., 204, 817-820. SCHMIDT, H., JR., A N D MOAK,S. J . , (1957); Evidence that barbiturate facilitation of water ingestion is related to the hypnotic potency. Amer. Psychol., 12, 435. SCHMIDT, H., JR., A N D MOAK,S. J., (1959); Some drug effects influencing barbiturate facilitation of water ingestion. Amer. J . Physiol., 196, 307-310. SEEVERS, M. H., AND TATUM, A. L., (1931); Chronic experimental barbital poisoning. J . Pharmacol. exp. Ther.,42,ZI 7-23 1. STANTON, E. J., (1936a); Dihydromorphine hydrochloride (dilaudid): its tranquillizing potency, respiratory depressant effects, and addiction liability as tested on the rat. J. Pharmaco/. exp. Ther., 56,252-263. STANTON, E. J., (1936b); Addiction and tolerance to barbiturates? The effects of daily administration and abrupt withdrawal of phenobarbital sodium and pentobarbital sodium in the albino rat. J . Pharniacol. exp. Tlier., 57,245-252. TEITLEIIAUM, P., AND EPSTEIN,A. N., (1962); The lateral hypothalamic syndrome: Recovery of feeding and drinking after lateral hypothalamic lesions. Psycho/. Rev., 69, 74-90. WAYNER,M. J., JR., AND EMMERS, R., (1959); A test of the thirst deprivation trace hypothesis in thc hooded rat. J . comnp. physiol. Ps,vchol., 52, 112-115. WAYNER,M. J., JR., AND REIMANIS, G., (1958); Drinking in the rat induced by hypertonic saline J . comp.physio1. Psychol., 51,ll-15. WAYNER, M. J., JR., WETRUS,B., AND BLANK, D., (1962); Artificial thirst, serum Na, and behavioral implications in the hoodcd rat. Psycho/. Rep., 11, 667-674. WILLIAMS, D. R., AND TEITLEBAUM, P., (1956); Control of drinking by means of an operant conditioning technique. Science, 124, 1294-1296. WOLF,A. V., (1958); Thirst: Physiology of the Urge to Drink a/7dProb/ems of Water Lack. Springfield, Charles Thomas.

285

The Cerebral Circulation : Some Hemodynamic Aspects F. M. K N A P P Tliudichum Psychiatric Research Laboratory, Galesburg State Research Hospital, Galesbuug, Ill. { U.S. A . )

In the hierarchy of organs the position of the brain is a singular one. Interference with the blood supply to the cerebral tissue threatens the welfare of the animal, and may produce irreversible changes within minutes. For this reason a good deal of attention has been directed towards a better understanding of brain hemodynamics. In spite of extensive work in this area, obvious gaps still exist in our knowledge; the information pertaining to flow-pressure relationships in the cerebral structures being much less available than from other portions of the cardiovascular system. This deficiency is primarily due to the inherent difficulties of reaching the brain and to only a slightly lesser degree the heterogenous nature of the organ. Perhaps the ‘skull-brain barrier’, which denies rzady access to the intracranial structures with the tools necessary for precise measurements, is as significant in its own right as the much discussed blood-brain barrier. If one accepts the importance of maintaining an adequate cerebral blood flow (CBF), it then becomes difficult to believe that the blood supply to the brain, under most conditions, becomes a slave to the whims of the systemic blood pressure. Cerebral arteries are not passive tubes. They contain muscle fibers capable of performing work and responding to neural and/or hormonal influences. Alteration of the cerebral vascular resistance (CVR) to meet specific requirements is considerably more important in maintaining a sufficient blood flow. In the experience of the author, the CBF, except in exceptional cases, tends to follow the systemic pressure only so long as the integrity of the brain is not jeopardized. The primary controls imposed upon the CBF serve to maintain a blood flow which will be steadier than that to most other organs, to furnish optimal COZtension and hydrogen ion concentration in the local cellular environment, and to provide for local variations to meet the changes in regional requirements. A variety of methods for CBF determination have been utilized. To dicuss all of these would be beyond the scope of this paper, but some of the major approaches include the plastic calvarium which permits visual access to the pial vessels (Shelden et al., 1944; Bogumill and Settlage, 1955; Minard et al., 1954), thermostromuhrs (Gibbs, 1933; Ludwigs and Schneider, 1954; Sonnenschein et al., 1955), polarography (Meyer et al., 1954; Sonnenscheinet al., 1953; McLaurin et al., 1959), dyes (Gibbs References p. 2951294

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et al., 1947; McDonald and Potter, 1951 ; Shenkin et al., 1948), plethysmography (Feiris, 1941; Bridges et al., 1958), angiography (De la Torre et al., 1959; Kigstrom et al., 1958; Kuhn, 1962), isotope uptakes (Sapirstein and Hanusek, 1958; Oldendorf, 1961 ; Love et a/., 1961 ; Steiner et al., 1962), infusion methods (Geiger, 1958; Finesinger and Putnam, 1933; Sagawa and Guyton, 1961 ; Green and Denison, 1956), rheoencephalography (Shalit, 1963) and flowmeters (Symon et al., 1963). Perhaps the longest single stride towards obtaining quantitative values for the CBF occurred with the development of the nitrous oxide method of Kety and Schmidt (1948). This technic, in conjunction with certain variations (Munck and Lassen, 1957; Scheinberg and Stead, 1949; Nylin et a/., 1961 ; Lassen, 1959) has provided considerable insight, at least with respect to total flows. The procedure does not resolve the problem of securing quantitative flow data for the various anatomical and/or functional regions of the brain. The 1311-tagged trifluoroiodo methane experiments of Kety el al. (1953; 1955) have provided some valuable information along these lines. The technic, however, is severely limited because the animal must be sacrificed at some specific point and, therefore, continuous monitoring of the flow cannot be obtained. Attempts to localize the extent of carotid and vertebral blood supply to the brain have been made in this laboratory. Surgical preparation of exteriorized carotid and vertcbral loops (Fig. 1) permits injections of specific materials directly into these

i Fig. I . Diagram of the carotid-vertebral anastomosis in the dog (Himwich ef a/., 1960a). A Tnternal thoracic artery; B = subclavian artery; C = axillary artery: D = proximal vertebral artery, tied off; E carotid artery; F = vertebral loop; G = carotid-vertebral anastomosis; H - vertebral artery; I = vertebrae; J - distal common carotid artery, tied off.

principal supply vessels of the brain (Himwich et al., 1960a). Dye distribution studies in animals prepared in this fashion display distinct and definite patterns (Himwich and Inman, 1962). Tntracarotid injections remain ipsilateral, contrasting with vertebral loop injections in which the dye, while not proceeding to the anterior portions of the cerebral hemispheres, does appear on both sides of the brain (Fig. 2). Altera-

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287

Fig. 2. Dyeinjection into vertebral artery. Bilateral coloration is seen in all sections except the most anterior (Himwich and Inman, 1962).

tions in the normal flow patterns within the circle of Willis, induced by placing a clip on some part of the circle, significantly rearrange the dye distribution. Results of the dye studies have been correlated with behavioral responses to drugs injected in the same manner (Himwich et al., 1960b; Costa et al., 1959). Utilizing a method similar to that described by Rapela et al. (1961), we have also attempted to measure the CBF and oxygen utilization (CMR02) from more specific areas of dog brain by analyzing the venous flow from the sagittal and straight sinuses (unpublished data) (Fig. 3). This was done primarily on acute preparations, but in a few instances the dogs were allowed to recover after placement of the catheters in the respective sinuses. The latter has allowed daily sampling for a period of time before clotting occurred in the catheters or the animal pulled them out. Blood flow values and CMROz values were determined by the N20 method and blood sugar levels. Distinct differences in flow and oxygen utilization are seen, but their evaluation is somewhat difficult. The sagittal sinus drainage represents a virtually homogenous tissue. The straight sinus, however, drains several areas from the subcortical region, each with its own blood flow and metabolic requirements. Nevertheless, some indications may be derived regarding flow and metabolic activity in the two areas. Miniaturization of the electromagnetic flowmeter extends the possibility of approaching some of the desired values. A program has been initiated recently in our laboratory to attempt the measurement of blood flow with the electromagnetic flow meter in various major cerebral vessels, particularly those of the circle of Willis. References p . 2951296

288

F. M. K N A P P

Fig. 3. Acute preparation showing placement of cannulas into the sagittal and straight sinuses of the dog as described in the text.

At present, however, mechanical problems such as large and cumbersome transducers prohibit extensive use of this procedure. These transducers necessitate exposure of large areas of brain surface for implantation, a procedure which is surgically difficult and which therefore limits its usefulness. Modifications of the system are being instigated, however, to suit the flowmeter to this type of work. Data obtained at the level of the circle or just previous to it principally reflect changes in gray matter since this occupies the larger portion of the brain mass as well as being the most vascular. The real problem then, with respect to a better understanding of brain hemodynamics, involves obtaining information about flow, pressure and resistance beyond the circle of Willis. The acquisition of these data requiies that measurements be derived from discrete areas of brain without damage or distortion of the tissue involved. The tissue clearance technic utilized by Kety (1949) to evaluate skeletal muscle blood flow was applied to the study ofthe CBF by Boatman et al. (1950) and the author (Knapp et al., 1955). The method is based on the premise that the primary pathway for the removal of a properly selected radioactive material is via the local circulation.

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289

The small volume of necessary isotope permits it to be placed in most regions of the brain with a minimum of trauma. Certain requirements are mandatory in utilizing the clearance technic. The isotope cannot be bound by brain tissue but must move freely into the circulation. Secondly, the shape of the bleb must be symmetrical, thus permitting equal removal in all directions. If these and other conditions are met, the clearance of the isotope should reflect the blood flow in the specific region under consideration. The applicability of the method can be verified by at least two factors: (1) the clearance rate is readily altered, and ( 2 ) the alteration is reflected by simultaneous changes in carotid flow (Fig. 4). The clearance of I3lI from the caudate nucleus is not altered during the intra-

-

!AT. C * P I " L E

10 2 0 3 0

Time in min

Fig. 4. Effect of Hydergin upon clearance rate, mean arterial blood pressure and carotid blood flow in the rabbit.

arterial infusion of isotonic saline and the carotid blood flow remains constant. The clearance rate from the caudate nucleus in the rabbit increased 47% when nylidrin HCI* (1-5 pglkg) was infused in the same manner as the saline. Clearance from the internal capsule in a comparable experiment was 60 %. These values were accompanied by respective increases of 35 % and 63 % in carotid blood flow. Systemic pressure and apparent CVR declined. Corresponding values for COz, histamine and Hydergin" * are given in Table I. This method is fraught with serious unsolved problems, and, while the clearance technic is commonly utilized in other tissues, it has not been exploited to any degree in the brain. Finally, refinement of isotope uptake studies, which have left much to be desired in the past (Oldendorf, 1961 ; Lassen et al., 1963) portends a more active investigation

* Nylidrin HCI : 1-(-phydraxyphenyI)-2-( 1-methyl-3-phenylpropylamino)propanol hydrochloride supplied as Arlidin by U.S. Vitamin Corporation. * * Hydergin: dihydroergocornine methanesulfonate, dihydroergocristine methanesulfonate, and dihydroergocryptine methanesulfonate supplied by Sandoz Pharmaceuticals. References p . 2951.296

290

F. M. K N A P P

TABLE I C O M P A R A T I V E V A L U E S F O R C A T A N D R A B B I T I N R E S P O N S E TO V A R I O U S A G E N T S (SEE T E X T )

Per cent change in CVR relative except for C02 in the rabbit. Increase or decrease in values indicated by direction of arrows. Injection site

Agenr ___

~~

Animal

CHG. clearance rate

”/, CHG. % CHG. mean arterial carotid blood press bloodj7o w

CHG.

CVR

~

I- 16.9 1 50.0 i 60.1 i23.2 41.2 j ,31.9 41.5 4I 22.1

- 13.2 i 6.2 - 2.1 2.9 - 9.4 zt 4.5 - 4.1 1.3

Cat

t 59.6 i- 10.5

-

Rabbit

i 88.0

Cat Tnt. cap Rabbit Arlidin Cat

C. nuc. Rabbit

Int. cap. CO2

Cat C. nuc. Rabbit Cat Int. cap. Rabbit Histamine Cat C. nuc. Rabbit Cat Tnt. cap. Rabbit

+ +

& 45.3 1 61.5 + 36.8 I 72.2 4-18.3 -28. I f 7.6 -61.5 i 5.4 -41.5 1 14.2 - 55.8 i 53

30.6 *iI 49.6 9.3

5 28.8

Hydergin Cat C . nuc. Rabbit

+ 41.0

i 16.9 1 48.6 32.7

+

1.6 f 4.1 I 35.1 i 33.1 - 3.1 rt 1.3 -t 4.4 4z 17.1

+

4,

+ 35.6

.1

+ 63.4

- 13.1 i. 6.9

63.1 5 33.8

i 18.4

5 54.8

+ 51.6 zt 35.1

-31.4 18.5

- 92.0

t

*

- 10.4

i 1.6 - 12.1 L 1.2 - 16.6 13.1 - 1.9 1 9.6

4 3.6

-82.6 f 9.5

1

+ 59.8

.1

-1 73.4

4

-20.9

i 1.6 - 12.8 1. 10.2 - 4.2 & 10.3 - 8.1 i- 6.3

21.7

+ 32.9

of regional cerebral circulation by this method. The development of precise equipment such as the Gamma-camera will perhaps provide !more quantitative data from this technic. Thus stands the investigation of regional CBF at present. Present technics, perhaps in modified or combined form, may provide the necessary insight into the problem but the fact that so many methods and variations of methods have been tried is indicative that the acceptable method is yet to be developed. Although the CBF is altered independently of blood pressure (Fazekas et al., 1960)

THE CEREBRAL CIRCULATION

29 1

the latter cannot be relegated to a position of minor importance. It is still the arterial venous pressure differential that is responsible for the movement of blood through cerebral tissue. Closely associated yet distinct entities establish the base line for pressure dynamics within the brain. The first of these is the ‘flattening out’ of the systemic pressure due to the anatomical construction of the supply vessels and their torcular pathways (Cairney, 1924) prior to entering the circle of Willis. Second the circle itself acts as an equalizer, and finally there is the vasomotor activity of the cerebral vessels themselves. That the cerebral vessels do exhibit a reactivity to pressure has been demonstrated by Fog (1938), who observed that a rise in systemic pressure produced a constriction of the pial vessels whereas a decrease in pressure resulted in dilatation. It might be argued that pial vessels are not necessarily representative of vessels elsewhere in the brain, but the response loosely fits the results one might expect from the application of the Monro-Kellie Doctrine. The role of pressure in the brain is constantly being evaluated, probably because it is more accessible to investigation than other factors. Out of this have arisen several studies on the isolated cerebral circulation. An example is the work done by Sagawa and Guyton on the dog (1961), which disputes the hypothesis of an independent relationship between the CBF and the cerebral perfusion pressure (CPP). The fact that preparations of this nature can no longer be considered physiological casts some doubt upon the reliability of the results. The question of cerebral vascular tone is yet unanswered. Its presence is generally accepted, the dispute being over the matter of degree (Sokoloff, 1959; Potter, 1961; Fazekas and Alman, 1963). Arguments for a high level are substantiated by the relatively small reduction in size of these conduits in response to most types of stimulation (Rosenblum and Zweifach, 1963). In Sagawa and Guyton’s preparation and in similar experiments, the vessels may well be atonal with the result that the blood merely moves through unresponding tubes, producing the linear flow-pressure relationships reported. As previously stated, the blood pressure is the most readily determinable component of the cerebral hemodynamic factors. Values on either side of the circle of Willis may be readily secured as shown by Ayala and Himwich (1961). In addition we have been able to expose a sufficient length of the basilar artery in the dog to permit the recording of pressure in this vessel. Attempts will now be made to further refine the surgical procedures, allowing us to expose the circle of Willis and to obtain pressures from precise areas near to or within the circle. Continuing the work of Ayala, we have recorded pressures from lingual and middle cerebral arteries during occlusion of the supply vessels to the circle. This has consisted of tying, acutely and chronically, the common carotids, vertebrals, basilar and anterior spinal arteries in a variety of combinations. It is quite apparent that the collateral circulation in dog very quickly compensates for the normal blood supply to the brain. In the acute condition simultaneous occlusion of both common carotid and vertebral arteries rapidly jeopaidizes the integrity of the animal (Fig. 5 ) (unpublished data). Pressures in the middle cerebral arteries approach zero and death occurs in approximately 10% of the animals if the occlusions are not removed. If the animal survives References p . 2951296

292

F. M . K N A P P

M=

SYSTEMIC &---A = M I D . CEREBRAL W R. LINGUAL O---o L.LlNGUAL

-.

*

90-

CONTROL

I L.C.C.

I R.VERT.

I L.VERT.

I

R.C.C.

I BASILAR

VESSEL OCCLUDED

Fig. 5. Control animal No.425. Acute progressive occlusion of the primary supply vessels tothebrain. 45 sec intervals between successive clampings. Pressures recorded from femoral artery (systemic), right and left lingual arteries, and middle cerebral artery. The external carotid arteries are tied cephalad to the lingual arteries. The latter thereby record back pressure from the circle of Willis when the ipsilateral common carotid artery is closed. L.C.C. = left common carotid artery; R.C.C. = right common carotid artery; L. Vert. = left vertebral artery; R . Vert. = right vertebral artery.

I

+'Oi

CONTROL

L.C.C.

L.Exr.C.

R.C.C.

R.Exr.C.

VESSEL OCCLUDED

Fig. 6 . Control animal No. 338. Chronically occluded common carotid and vertebral arteries. Procedure as in Fig. 5. Drops in pressure upon clamping external carotid arteries are indicative of a retrograde collateral blood flow via these vessels. L. Ext. C. = left external carotid artery; R. Ext. C. = right external carotid artery.

20

/

-

SYSTEMIC

50.60. CONTROL

I

I

L.C.C.

L.VERT.

I

BASILAR

VESSEL OCCLUDED

Fig. 7. Control animal No. 398. Left common carotid and vertebral arteries chronically occluded. Right side normal. Procedure as in Fig. 5. Note significant difference in pressure values between the two sides. Evidence indicates that the collateral circulation is primarily established in relationship to the carotid artery and its branches.

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293

Fig. 8. Schematic drawing of the circle of Willis model showing diameter sizes and junction angles.

Fig. 9. Photograph of circle of Willis model. Not shown are the constant pressure reservoirs or the pumping system for filling the reservoirs (photo approx. 1/6 actual size). References p. 2951196

294

F. M. K N A P P

the first few minutes of occlusion, however, the recorded pressures start rising slowly and consistently. If the procedure is repeated a second time, the pressure drop gcnerally does not fall to the previous level. I n a dog with chronically cut common carotid and vertebral arteries, occlusion of the remaining supply vessels to the circle does not produce the pressure readings described for the acute preparation (Fig. 6). The animal rarely appears to be in serious difficulty. This relationship can be carried even further. If, following a chronic unilateral occlusion of the vertebral and carotid arteries, the corresponding vessels on the opposite side are clamped, a significantly higher pressure will be recorded on the chronically occluded side (Fig. 7). This response has two possible interpretations : ( I ) the build-up of collateral vessels can be unilateral, and (2) the circle of Willis may be more concerned with equalizing flow than pressurz. The interrelationships of the vessels in the neck and head region of the dog are, of course, amenable to collateral vessel development. Model: Confronted with the problems of flow-pressure relationships in the brain and thwarted because of the inaccessibility of the area, an attempt has been underway for the past 3 years to develop a workable model of the circle of Willis (Knapp et af., 1963) (Fig. 8). This project is being carried out conjointly with a group of fujd mechanicians who are investigating the dynamics of such a system. Observations have been made on a rigid, non-pulsatile model in which differential pressures, flow volumes, resistances and Reynold’s numbers can be predetermined and applied in a variety of combinations. Being a steady flow, rigid system limits its usefulness, but flow patterns and values are similar to those proposed by Jewel1 and Verney (1957) and others. Attempts are currently underway to convert the model into an elastic, pulsatile system that will more closely approximate conditions in vivo. The advantages of such a model are numerous. It permits the investigation of hemodynamic phenomena in the circle under carefully controlled conditions. It is not inferred that the results may be transferred directly to the animal in all instances, but general concepts can be derived to clarify the flow-pressure resistance patterns in the brain. SUMMARY

As previously suggested, investigations of the cerebral circulation leave much to be desired. The field still awaits a major technical break-through, particularly in the study of regional flows. Enough is known, however, to demonstrate that the brain, with great obstinacy, maintains its blood flow, and, to a lesser degree, the necessary blood pressure. It must be remembered, however, that these two are not synonymous. Certainly in the healthy animal a rapid realignment of factors occurs to re-establish the status quo when this is disturbed for either pressure or flow. The abnormal situation is not so clearly defined, but it appears that even in these instances cerebral hemodynamic patterns maintain their integrity in response to considerable stress before brcaking down. Investigations in brain hernodynamics in our laboratory are directed at this time

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295

toward a better understanding of pressure-flow problems, particularly in the region of the circle of Willis and a multiple-disciplined approach is being made in this direction. REFERENCES AYALA,G., AND HIMWICH,W. A., (1961); Hemodynamics of circle of Willis in the dog. Amer. J. Physiul., 201, 443-447. BOATMAN, J. B., KENDRICKS, T. R., FRANKE, F. R., AND MOSES,C., (1950); The use of radioactive iodine, radioactive phosphorus, and radioactive sodium in the determination of cerebral and muscle clearance. J . Lab. clin. Men., 36, 456459. BOGUMILL, G. P., AND SETTLAGE, P. H., (1955); A method of replacing portions of the calvarium with transparent plastic. J . Neuropathol., 14, 305-312. BRIDGES, T. J., CLARK,K., AND YAHR,M. D., (1958); Plethysmographic studies of the cerebral circulation : Evidence for cranial nerve vasomotor activity. J . clin. Invest., 37, 763-772. CAIRNEY, J., (1924); Tortuosity of the cervical segment of the internal carotid artery. J. Anat., 59, 87-96. COSTA,E., HIMWICH,W. A., AND HIMWICH,H. E., (1959); Effects of injections of bufotenin into various arterial sites. Neuro-Psychupharmaculugy.P. Bradley, P. Deniker and C. Radouco-Thomas, Editors. New York, Elsevier (pp. 299-303). DELA TORRE, E., NETSKY, M. G., AND MESCHAN, I., (1959); Intracranial and extracranial circulations in the dog: Anatomic and angiographic studies. Amer. J. Anal., 105, 343-382. FAZEKAS, J. F., AND ALMAN, R. W., (1963); Vasodilators in cerebral vascular insufficiency. Amer. J. med. Sci.,246, 410416. FAZEKAS, J. F., THOMAS, A., JOHNSON, J. V. V., AND YOUNG,W. K., (1960); Effect of Arterenol (norepinephrine) and epinephrine on cerebral hemodynamics and metabolism. A . M . A . Arch. NeuruL, 2,435438. FERRIS,E. B., JR., (1941); Objective measurement of relative intracranial blood flow in man. A.M.A. Arch. Neurol. Psychiat., 46, 377401. FINESINGER, J. E., AND PUTNAM, T. J . , (1933); Cerebral circulation. 22. Induced variations in volume flow through the brain perfused at constant pressure. A.M.A. Arch. Neurol. P.yychiat., 30,775-794. FOG,M., (1938); The relationship between the blood pressure and the tonic regulation of the pial arteries. A.M.A. J . Neurul. Psychiat., 1 , 187-197. GEIGER,A., (1958); Correlation of brain metabolism and function by the use of a brain perfusion method in situ. PhyJiol. Rev., 38, 1-20. GIBBS,F. A., (1933); A thermoelectric blood flow recorder in the form of a needle. Pruc. SUC.exp. Biol. ( N . Y.), 31, 141-146. GIBBS,F. A., MAXWELL, H., AND GIBBS,E. L., (1947); Volume flow of blood through the human brain. A.M.A. Arch. Neurol. Psychiat., 57, 137-144. GREEN,H. D., AND DENISON, A. B., JR., (1956); Absenceof vasomotor responses to epinephrine and arterenol in an isolated intracranial circulation. Circulat. Res., 4, 565-573. HIMWICH, W. A., COSTA,E., CANHAM, R. G., AND GOLDSTEIN, S. L., (1960a); Isolation and injection of selected arterial areas in the brain. J. appl. Physiul., 15, 303-306. HIMWICH, W. A., COSTA,E., AND HIMWICH,H. E., (1960b); Technics for the study of behavior induced by drugs using injections into selected arterial sites in the brain. Acta Znt. Meetings Techn. Study Psychotropic Drugs. Modena, Societa Tipografica Modenese (pp. 1-9). HIMWICH, W. A., AND INMAN, 0. R., (1962); Injection of dye into isolated arteries supplying the brain of the dog., In/. J. Neurupharmacol. 1,303-307. JEWELL, P. A,, AND VERNEY, E. B., (1957); An experimental attempt to determine site of the neurohypophysial osmoreceptors in the dog. Phil. Trans. toy. SOC.Lond. B, 240, 197-324. KAGSTROM, E., LINDGREN, P., AND TORNELL, G., (1958); Changes in cerebral circulation during carotid angiography with sodium acetrizoate (Triurol) and sodium diatrizonate (Hypaque). Acfa radiol.,50,151-159. KETY,S. S., (1949); Measurement of regional circulation by the local clearance of radioactive sodium. Amer. Heart J., 38, 321-328. KETY,S. S . , AND SCHMIDT, C. F., (1948); The nitrous oxide method for the quantitative determination of cerebral blood flow in man; theory, procedure, and normal values. J. din. Invest., 27,476483.

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KETY,S. S., LANDAU,W. M., AND FREYGANG, W. H., JR., (1953); Measurement of regional circulation in the#brainby the uptake of an inert gas. XIX Int. Congr. Physiol. Montreal. Abstracts, p.1511. KETY,S. S., LANDAU,W. M., FREYGANG, W. H., JR., ROWLAND.1.P., AND SOKOLOFF, L., (1955); Estimation of regional circulation in the brain by the uptake of an inert gas. Fed. Proc., 14, 85. KNAPP,F. M., CLARK,M. E., A N D WENGLAKZ, R., (1963); Dynamics of the circle of Willis as observed in a model. Fed. Proc., 22, 344. KNAPP,F. M., HYMAN,C., AND BERCEL,N. A., (1955); A method for the estimation of regional cerebral blood flow. Yale J . Biol. Med., 28, 363-371. KUHN,R. A,, (1962); The speed of cerebral circulation. New Engl. J . Med., 267, 689-695. LASSEN, N. A.,(1959); Cerebral blood flow and oxygen consumption in man.Physio1. Rev., 39,183-238. LASSEN,N. A., HOEDT-RASMUSSEN, K., SORENSEN, S. C., SKINHOJ, E., CRONQUIST, S., BODFORSS, B., AND INCVAR, D. H., (1963); Regional cerebral blood flow in man determined by R5Kr.Neurology, 13,719-727. Love, W. D., OMEALLIE,L. P., AND BURCH,G. E., (1961); Assessment of cerebral circulation by an external isotope technique. J . Lub. clin. Me& 58, 445454. LUDWIGS, N., A N D SCHNEIDER, M., (1954); Uber den Einfluss des Halssympathicus auf die Gehirndurchblutung. Pfliigers Arch, ges. Physiol., 259, 43-55. MCDONALD, D. A,, AND POTTER,J . M., (1951); The distribution of blood to the brain. J . Physiol., 114,356-371. MCLAURIN, R. L., NICHOLS, J . B., JR., AND NEWQUIST, R. E., (1959); Polarographic measurement of cerebral oxygenation using chronically implanted electrodes. J . appl. Physiol., 14,480-462. MEYER, J. S., FANG,H. C., AND DENNY-BROWN, D.,(1954); Polarographicstudy of cerebral collateral circulation. A.M. A.Arch. Neurol. Psychiat., 72, 296-312. MINARD, D., OSS~RMAN, E. F., A N D HowtLi., S. R., (1954); The lucite calvariumfor direct observdtion of the brain in monkey. Anat. Rec., 120,317-332. MUNCK,O., A N D LASSEN,N. A., (1957); Bilateral cerebral blood flow and oxygen consumption in man by the use of s5Kr. Circ. Res., 5, 163-168. NYLIN, G., HEDLUND, S., A N D REGNSTROM, O., (1961); Cerebral circulation studied with labeled red cells in healthy males. Acta radiol., 55,281-304. OLDENDORF, W. H., (1961); Measurement of cerebral blood flow by external collimation following intravenous injection of radioisotope. IRE Trans. Bio-Med. Electron., 8, 173-177. POTTER, J. M., (1961); Cerebral arterial spasm. World Neurology, 2, 576-588. RAI'ELA, C. E., MACHOWICZ, P., AND GREEN,H . D., (1961); Cerebral venous blood flow. Fea! Proc., 20, loo. ROStNRLUM, W. I., AND ZWEIFACH, B. w., (1963); Cerebral microcirculation in the mouse brain. A.M.A. Arch. Neurol., 9, 4 14-423. SAGAWA, K., AND GUYTON, A. C., (1961); Pressure-flow relationships in isolated canine cerebral circulation. Amer. J . Physiol., 200, 71 1-714. SAPIRSTEIN, L. A., AND HANUSEK, G. E., (1958); Cerebral blood flow in the rat. Anrer. J . Plzysio,.. 193,272-274. SCHEINBERC, P., AI\D STEAD,E. A., J R . , (1949); The cerebral blood flow in male subjects as measured by the nitrous oxide technique. Normal values for blood flow, oxygen utilization and peripheral restistancc, with observations on the effect of tilting and anxiety. J . clin. Invest., 28, 1163-1 171. SmLi-r, M. N., (1963); A method for the measurement of regional hemodynamics in the brain cortex. J . Neuropathol. exp. Neurol., 22,479487. SHELDEN, C . H., PUDENZ, R. H., RESTARSKI, J., AND CRAIG,W. M., (1944); The lucite calvarium: a method for direct observation of brain. J . Neirrosurg., 1, 67-75. SHLNKIN, H . A., HARMEL. M. H., AND KETY,S. S., (1948); Dynamic anatomy of the cerebral circulation. A . M.A. Arch. Neurol. Psychiat.. 60, 240-252. SOKOLOFF, L., (1959); The action of drugs on the cerebral circulation. Pharmacol. Rev., 11. 1-85. SONNENSCHEIN, R. R., S T t i N , S. N., AND PEROT,P. L., JR., (1953); Oxygen tension of the brain during hyperoxic convulsions. Amer. J . Pliysiol., 173, 161-163. SONNENSCHEIN, R. R., WALKER,R. W., PALMER, J. J . , AND KYLE,W., (1955); Continuous measurement of local cerebral blood flow: measurement and preliminary observations. Amer. 1.PliyJiol., 183,663. S T E I N ~S. R ,H., Hsu, K . , OLINER, L., AND BEIINKE, R. H., (1962); The measurement of cerebral blood flow by external isotope counting. J. clin. Invest., 41, 2221-2232. SYMON, L., ISHIKAWA, S . , LAVY,s., AND MEYER, J . S., (1963); Quantitative measurement of cephalic blood flow in the monkey. J. Neurosurg., 20, 199-218.

297

Multi-Channel Telemetry Systems F. T E R R Y H A M B R E C H T Biomedical Engineering, Johns Hopkins University, Baltimore, Md. (U.S.A.)

INTRODUCTION

Within the last few years the use of telemetry systems for the remote measurement of physiological parameters has been transformed from an experimental novelty to a reliable tool. The transistor with its reduced size and power consumption has permitted sophisticated electronic circuitry to be packaged in such a manner that it can be carried almost unnoticed by humans and animals. The greatest single advantage of telemetry is that it permits the subject to move about freely without restricting cables. In studies of physiological and pharmacological responses on the central nervous system this is quite important (Himwich and Hambrecht, 1963). Even if cables between the subject and the recording apparatus are permitted they can introduce artifacts by the change of the electrostatic charge of the insulating materials of the wire and induction of an emf in the wire loop as a result of its movement in the earth’s magnetic field. The use of multi-channel systems permits simultaneous recording of several different parameters. We have telemetered EEG, blood pressure and EMG, and with small modifications could include EKG, galvanic skin resistance, respiration and temperat ure.

DIFFERENTIAL AMPLIFIERS

MULTIPLEXER

PULSE SHAPER AND DISTRIBUTOR

r

DEMODULATORS

Fig. 1. Block diagram of basic telemetry system.

Basic system: The basic system used in our multi-channel systems is shown in Fig. 1. The low noise, broad band, high-gain differential amplifiers provide signals of the proper level for the multiplexer. The function of the multiplexer is to combine the Rcferpnrrs p.-300

298

F. T E R R Y H A M B R E C H T

different signals from the amplifiers into a single signal that then modulates an FM transmitter in the range 88-108 Mc.

I

N

P

TO AMPLIFIER DIFFERENTIAL

q BRIDGE

Fig. 2. Block diagram of carrier generator.

The received signal, after detection by an F M receiver, is divided into its original signals by the pulse shaper, the distributor, and the demodulators. The outputs can be connected directly to a pen recorder or an oscilloscope. In order to transmit parameters with an absolute level, for example blood pressure, a carrier system must also be added. The carrier generator is shown in block form in Fig. 2. If it is desired to telemeter temperature rather than blood pressure the strain gage is replaced by a thermistor bridge.

7:DIT

-

22

Fig. 3. Sine wave carrier oscillator.

Circuitry: The amplifiers, multiplexer, transmitter, distributor and demodulator circuitry have been discussed in previous publications (Hambrecht et al., 1963; Hambrecht, 1963). In two different systems constructed, one used pulse frequency, pulse duration multiplex for a two-channel system, and the other used pulse duration multiplex for a four-channel system. The latter can easily be extended to a maximum of 80 channels. Fig. 3 is the sine wave oscillator used in the carrier system. It has a frequency of 250 c/s and an output amplitude of approximately 1 V peak-to-peak. This particular

299

M U L T I - C H A N N E L T E L E M E T R Y SYSTEMS -4.5v

-4.5v

-4.5v

-4.5%-

-I.iV

-4.5V -4.5V

Fig. 4. Carrier demodulator.

circuit was used to excite a Statham P23Gb blood pressure transducer. Although a CK67B transistor is shown, almost any PNP transistor will suffice. Also needed if the carrier system is used is a carrier demodulator (Fig. 4). This follows the channel demodulator as shown in Fig. 1 and serves to separate the information-containing envelope from the carrier. DISCUSSION AND SUMMARY

A comparison of the direct measurement of blood pressure with telemetered blood pressure is shown in Fig. 5 (note that the calibration scale for the telemetered signal is logarithmic and the direct recording is linear). A similar comparison of EEG signals can be found in the article by Himwich et al. (this Volume). These records were obtained simultaneously from a n alert dog. An Offner Type R dynograph was used for write-out.

Fig. 5 . Blood pressure recordings. Rqferrnces p . 300

F. T E R R Y H A M B R E C H T

300

The specifications for the four-channel system are given in Table 1. TABLE I SPECIFICATIONS OF COMPLETE T E L E M E T R Y SYSTEM

Number of channels Type of multiplexing Frequency response Dynamic rangc of inputs Noise (referred to input) Cross talk between channels Transmitter radiofrequency Operating range Battery life Power rcquirernents

4 pulse duration 3 dB, 0.7-200 c/s (each channel) 5-250 pV (each channel) 5 ,uV pcak-to-pcak (0.7-2000 cis) less than 3 88-108 Mc (tunable) 200 feet 12 h between recharging -2.5 V a t 6 mA -l at mA telemetry package -12 V at 200 mA -6 V at 15 mA decoder t3Vat13mA

1

’

REFERENCES

F. T., (1963); A multichannel electroencephalographic telemetering system. Technical HAMBRECHT, Report 423. Massachusetts Institute of Technology, Research Laboratory of Electronics, Cambridge, Mass. HAMBRECHT,F.T., DONAHUE, P. D., AND MELZACK, R., (1963); A multiple channel EEG telemetering system. Electroenceph. elin. Neurophysiol., 15, 323-326. HIMWICH, W. A AND HAMBRECHT F. T., (1963); Telemetry systems in physiological and pharmacological research. 16th Annual Conference on Engineering in Medicine and Biology. Baltimore, Md., 19 November.

30 1

Electrical Activity of the Dog’s Brain: Telemetry and Direct Wire Recording W I L L I A M I N A A. H I M W I C H , F R A N C I S M. K N A P P * W I L L I A M G. STEINER**

AND

Thudichum Psychiatric Research Laboratory, Galesburg State Research Hospital, Galesburg, Ill. (U.S.A.)

INTRODUCTION

The literature on the electrical activity of the dog brain is not very extensive considering the suitability of this animal for laboiatory experimentation. The available data are difficult to correlate because of the differing conditions of the animals (anesthetized or conscious) and the various types of electrodes and recording conditions used. The early investigations of PrBwdicz-Neminsky (1925) and of Berger (1929) identified two basic rhythms for the dog of 10 to 16 c/s and of 20 to 32 c/s which Berger felt corresponded to the so-called a- (8-12 cis) and p- (18-30 c/s) rhythms in the human being. Somewhat later, Ito and Kitamura (1939) described a dominant rhythm for the dog of 7 to 9 c/s but this finding undoubtedly reflected their use of anesthesia during recording since this range is more in keeping with the general finding for the guinea-pig than for the conscious dog. The published figures of Dow et al. (1945) reveal no fast activity from the brains of dogs studied during nembutal anesthesia but a 20-32 cjs rhythm is apparent in the EEG tracings which they obtained with only local application of novocaine. Motokawa (1949) using scalp electrode in unanesthetized animals placed the dominant rhythm at between 10 and 16 c/s for the dog. N o fast activity is apparent in the EEG tracing which he presents for the dog but a small amount of fast activity is noted in his frequency histogram for the dog. The dominant rhythm as judged by the histogram, however, is unmistakably in the range reported. The differences in the results of Dow et al. (1945) and of Motokawa (1949) may stem from differences in recording technique. Dow et al. traced the activity from the dura using insulated phonograph needles while the recordings by Motokawa were made much in the manner of present day recordings from the human subject. Attenuation by the skull bone of the components of the brain rhythm might account for the absence of a second peak of activity at 20-32 CIS in the frequency histogram of Motokawa. The resistance capacity-coupled amplifier used by this investigator gave * Present address: Department of Biology, Duquesne University, Pittsburgh, Pa. (U.S.A.).

**

Present address: Department of Psychology, Yale University, 333 Cedar Street, New Haven, Conn. (U.S.A.).

References p . 316/317

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about a 50% reduction in recorded amplitudes at about 12 c/s which undoubtedly also restricted his evaluation of the faster components. This type of reduction does not normally occur in most amplifiers of current design until about 50-75 C/S. The publication of the frequency characteristics of his amplifier in this quantitative study of the brain activity of a variety of animals assists in the evaluation of Motokawa’s data. During this same period, Swank and Watson (1949) made an extensive study of the electrical activity of the dog brain. They distinguished between low and high levels of excitation. In the animal of low excitation, the dominant rhythm was found to be around 24 c/s with some activity in the 12 c/s range distributed posteriorly over the brain. These findings are in good agreement with the published results of previous workers. Of particular interest, however, is their identification of a fast component in the anterior region of the brain which greatly exceeds the 20-32 c/s activity generally considered to be the fast component for the dog brain. They found, particularly with anterior transhemispheric leads, activity ranging between 40 and 80 c/s with a peak around 50 c/s. Under enhanced levels of excitation such as pain stimulation, this very fast component dominated the electrocorticogram in the anterior region with lesser amounts being observed in the posterior region. Under lowered levels of excitation such as cessation of pain stimulation, the 50 c/s activity disappeared and was replaced with 25 and 12 cjs activity in the recording. It is of interest in this connection, that Jasper and Andrews (1938) in their early work with the human had noted a similar although slower component (35-45 c/s) in the precentral and frontal regions. They labelled this very fast activity as y-waves; a term not in common use today. The published reports appearing subsequent to the study by Swank and Watson (1949) are somewhat equivocal in regard to the presence of very fast activity. Goldensohn et al. (1950) considered the 10-16 c/s activity to be dominant in the posterior region with smaller amounts of 16 to 24 c/s, 8 to 10 c/s and a scattering of 6 to 8 c/s activity also distributed posteriorly. In the anterior region, low voltage 12 to 35 c/s activity was found to be dominant. Schallek and Walz (1953, 1954a, 1954~)in a series of pharmacological studies, consistently obtained activity for the dog in the 18 to 20 cjs range as judged from their published figures with an increase to 42 c/s with the administration of the stimulant d-amphetamine. Charles and Fuller ( I 956) undertook a developmental study of the electroencephalogram of the dog utilizing scalp recordings without anesthesia. Their recordings revealed 30 c/s activity by the 7th week of age which appeared to be dominant for the maturing pup. Other frequencies between 6 and 60 CISwere also noted by these workers. This report is the first clear-cut confirmation of the very fast activity reported by Swank and Watson (1949). A fast component is apparent in the left occipital but not in the right occipital or midfrontal region of their tracing from the pup (Fig. 5 , Charles and Fuller, 1956). This tracing seems to place the very fast component more posterior than the findings of Swank and Watson although this very fast rhythm may shift more anterior with increased maturity of the organism. It is difficult in this type of scalp recording, utilized to some extent also by Swank and Watson, to judge the extent of the electrical

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activity of muscle (action potentials, etc.) which might become superimposed on the activity recorded from the brain. Turning to the Russian literature, we find that Sakhiuline (1951, 1955) utilizing a novel procedure which involved the inqertion of steel sewing needles through the skin and head muscle directly into the bone, found a wide range of frequencies. The needles were removed at the end of each days recording. No anesthetic was administered at any time. In the early phase of study, 30-40 c/s activity appeared to be dominant. As the animal relaxed, 14 t o 18 cjs waves appeared in the cruciate region while more anterior areas produced tracings in the 20 to 25 c/s range. With drowsiness, the frequency range dropped to 3 to 5 cjs but upon stimulation, frequencies of from 30-50 c/s would appear. During the process of' conditioning, the electrical activity of the brain appears to accelerate and become stabilized at 30-35 cjs. Amplitudes increased three fold (40-60 pV to 140-160 p V ) during this period. Sakhiuline also described multiple rhythms appearing in the dog. In the relaxed animal, an 80 p V , 8 to 12 c/s rhythm appeared in the occipital region in conjunction with a 10-15 p V , 25 to 30 c/s or a 40 to 60 c/s rhythm. He also found alternating rhythms in the awake animal such as a 40 to 60 c/s activity rhythm succeeded by a 4 to 14 cis rhythm with a 40 to 60 c/s superimposed. As in the case of the reports of Swank and Watson (1949) and Charles and Fuller (1956), it is difficult to distinguish some of these faster components from the type of activity produced by muscle. Luria and Profimov (1956) also noted the variability of the rhythms recorded from the dog particularly during conditioning. The EEG concomitants of conditioning form too vast a subject to be dealt with here but we will mention these Russian findings in regard to the rhythms normally obtained from dog. In technical detail, their animal preparations were very similar to the type of chronic implant which is in current use in many laboratories of this country, i.e. lacquered silver or platinum wires inserted directly into tissue with recordings taken from a multiple pin housing which is permanently accessible. Luria and Profimov (1956) report that waves faster than 35 c/s were not very frequent in their recordings, being mainly superimposed over the slower components. Activity from the motor cortex (28 to 35 cis) was found to be the most stable. The auditory cortex was less rhythmic, of lower amplitude and of higher frequency (above 28 to 30 cis). The activity of the visual cortex was also found to be less regular with a low voltage 30-60 c/s rhythm superimposed on a 40-70 p V , 3 to 5 c/s rhythm. They also found that the nature of the EEG record not only changed during the duration of the experimental day but also throughout the period of investigation. Most of these changes, however, appear either to be a function of changes in sleep-wakefulness or to conditioning procedures. A series of studies dating back to the time of Adrian (1942) have been addressed to the analysis of electrical burst phenomena in various rhinencephalic structures. Rhythms having frequencies up to 50 c/s have been recorded in the amygdala, olfactory bulb, posterior hypothalamus, etc., which bear some relationship to respiration and also to EEG and behavioral arousal. Domino and Ueki (1960) published a fine paper in this regard dealing with the dog. They were able to record bursts of from 20 to 40 c/s from the olfactory stria, pyriform and prepyriform cortex Referenca p . 316131 7

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of the dog with amplitude and frequency being related to the degree of excitation or arousal of the animal ; the higher frequencies and higher amplitudes being associated with the higher levels of arouszl. It is not known whether this forebrain activity bears any relationship to the very fast component reported by Swank and Watson (1949) but the anterior distribution of both types of activity suggest a possible relationship. It is rather obvious from the foregoing review that the type of activity recorded in the EEG of the dog is very much a function of how the tracings were obtained. The presence of anesthesia, the habituation of the animal, the placement of the electrode as to scalp, skull bone, dura or tissue, the level of alertness, degree of experimental manipulation and even the level of amplification and Lharacteristics of the recording equipment have appeared as major influencing variables in the reports reviewed. It seems particularly futile, then, to speak of the electrical activity of the dog brain without at the same time specifying the behavioral state of the animal and the conditions which were operating at the time of the recording. These considerations have prompted us to make available some of the recordings we have obtained from dogs over the past few years in the hope that the published figures may serve as a useful reference for those persons interested in the dog as an experimental animal. Many of the recordings were obtained by means of a telemetry system which provides the animal with a greater degree of freedom than has hitherto been afforded by conventional direct cable methods. In addition, all of the animals are thoroughly habituated to our particular investigational procedures having been objects of study for upwards of two years. It is not our intent to offer a definitive study of the electrical activity of the dog brain but rather to present EEG tracings of the dog obtained by methods in current use. METHOD

Animals Adult mongrel dogs of both sexes were employed throughout all of the experiments to be described. The physical dimensions of the stereotaxic apparatus imposed a restriction on dog size and consequently, all of the animals weighed approximately 10 kg with very little variation in either direction. The dogs were free of obvious disease and each was handled for about 1 month prior to electrode implantation and an additional two weeks following the surgery before any EEG recordings were taken. Electroencephalography The multi-channel EEG telemetering system employed in these studies has been described elsewhere (Hambrecht et al., 1963; Hambrecht, 1963; Himwich and Hambrecht, 1963) and need not be dealt with in detail here. The system is capable of transmitting four channels of EEG with a bandwidth of 0.7 to 2000 cjs (& 3 db). The EEG potentials pass from transistorized differential amplifiers through a multiplexer into an FM transmitter. The potentials are then received by a Harman Kardon Citation IT1 FM receiver by means of a conventional dipole antenna. After demodulation, the potentials are led into the power amplifiers of an Offner dynograph recorder Type R. The frequency spectrum which can be recorded with this equipment

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Fig. 1. Dog No. 527 carrying telemetry package. The package is enclosed in foam rubber in a plastic box which fastens on the leather harness. The short cable plugs into the package and into the connector fixed in his head.

Fig. 2. Behavior room. Recording apparatus for telemetry is shown on the left.

is DC to 150 cjs ( & 10 %). The system can be used for 10 h between battery rechargings and it has an operating range of 200 feet. The total weight of the telemetering package with batteries is 212 g and is carried by the dog in a harness (Fig. 1). The telemetry studies are conducted in a room approximately 8 feet by 8 feet which is so References p . 3161317

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constructed as to allow the constant observation of the animal, the taking of motion pictures and the simultaneous recording of EEG and blood pressure (Fig. 2). Direct tissue recordings were routinely taken from anterior cortex, caudate nucleus, hippocampus and posterior hypothalamus. A dural electrode was located directly above each of these recording sites. All of the telemetered recordings were bipolar in nature with each of the four transmitting channels containing the potentials from a deep and a surface representation, e.g. posterior hypothalamus-dura, which was delivered to the Offner dynograph. The intra-electrode resistance for each pair of deep and surface recording electrodes ranged between 17,000 and 28,000 ohms as nicasured between the pins of the connector plug. The bipolar arrangement just described is a fixed property of our telemetry system in its present stage of design and consequently, conventional cable recordings of various types will be presented, when indicated, to clarify the individual contribution of each recording area. The recording electrodes were mide of 0.020 stainless steel wire covered except at the tip by PE polyethylene tubing which is bound to the wire by glue (Eastman 910 Monomer). The flexible portion of the electrode consisted of 24 to 26 gauge wire (Belden) soldered to the stainless steel shank. The solder joint is encased in No. 24 shrinkable tubing (Alphlex) (Fig. 3).

Fig. 3. Recording electrodes: unassembled parts center and assembled e!ectrocle at top and left side.

Electrode implantation Recording electrodes were chronically implanted under sterile surgical procedures. Each animal was pretreated with 1.0 mg of atropine sulfate prior to surgery to rcstrict the secretions of the mucosa. The animals were brought to an anesthetic level with intravenous pentobarbital sodium after which they were intubated to insure a n open air passage. They were maintained, thereafter, on a 5 % to loo/,

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dextrose-saline drip throughout the implantation. Pencillin was routinely given to each animal following the completion of all surgical procedures. Electrodes were implanted stereotaxically according to maps of Lim et al. (1960) for the dog brain. Coordinates were as follows: Anterior cortex, directly over the coronal suture; T 7-10, electrode inserted 1-2 mm below dura; caudate nucleus, R 25, T 7, V 11 ;hippocampus, R 13, T 10, V 12; and hypothalamus, R 20, T 2, V 30. The animal was positioned in the stereotaxic apparatus and the skull was laid bare from the sphenoid region to the occipital protuberance or in some cases a semicircular flap of scalp cut and laid back. The skull bone was then trephined for the insertion of the recording electrodes and hollow plastic screws, which had been fashioned from plexiglass tubing were anchored in each trephined hole in the bone. A dural and a subdural electrode were implanted individually within each plastic screw, both electrodes fastened into position and sealed with dental acrylic. The surface wires attached to each recording electrode were then brought forward to a plastic, multiple pin housing which was anchored with dental acrylic in a partially trephined indentation in the skull between the insertions of the masseter muscles and

Fig. 4. Sagittal section of electrode implantation: (a) ground electrode going to the neck, (b) plexiglass screw to hold electrodes, (c) plexiglass plug screwed into the bone, (d) layer of silastic (e) dental acrylic covering anchoring screws and lower threads of plug, (f) reference electrode to the nose, (g) dural electrode, and (h) electrode in brain tissue.

posterior to the sphenoid ridge (Fig. 4). Wires were also inserted subdermally in the direction of both the nose and the dorsal surface of the neck to serve in combinatiofi as a reference in the case of direct cable recordings and also as a ground. The entire area around the plastic housing was then covered with fluid silicone rubber (Silastic. 382, Dow Corning Corp.)" which served the double purpose of reducing irritation and also of filling the space between the skull bone and skin. The plastic pedestal housed a commercially available (Amphenol) 9 pin plug which served to connect the animal to the recording or telemetering instruments. After all plastic parts had been made

* The Silastic 382 and the Eastman adhesive 910 were kindly furnished by the Dow Corning Center for Aid to Medical Research, Midland Michigan, and Ethicon, Inc., Sommerville, N. J., respectively. ReferencPs p . 3161317

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secure and all openings sealed, the wound was closed and the animal was returned to his home cage. RESULTS

Simultaneous recording by telemetry and direct cable methods It is of immediate interest to compare the EEG rhythms obtained by the telemetry method of recording with those recorded by the conventional direct cable method. The two EEG tracings (Fig. 5) were recorded simultaneously by direct cable and by DOG NO. 522

9-4-63

DIRECT WIRE - EEG CORTEX

Fig. 5. Simultaneous recording by direct wire and by telemetry system. Electrode placement as described in the text.

the telemetry system employed in this study. In this particular recording, the signal was divided at the level of the multiple pin plug so that both recording systems could convey the same potentials simultaneously to the dynograph recorder. Both tracings appear to be quite similar on visual inspection although some differences may be noted in the finer detail of the recordings. The wave forms of low frequency reflect this lack of absolute correspondence particularly in the right hand portions of the tracings although, in general, the actual degree of correspondence is considered excellent for the purpose of this study. A complete frequency analysis would be required to determine if any substantial lack of correspondence exists at any point or points of the frequency spectra. Components of the bipolar recording. There is no wholly satisfactory electrode

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arrangement and particularly so in the freely moving animal. The intrasite (within structure) bipolar type of recording affords the greatest degree of specificity but the magnitude of the potentials is not always adequate in terms of the requirements of the recording system. The monopolar type of recording with an 'indifferent' electrode outside the brain affords the required degree of magnitude but the indifferent electrode is notoriously troublesome in the dog. Also, the indifference of the indifferent electrode is questionable, since an outside-the-head response can be recorded when the indifferent electrode is used as an active lead. The present bipolar arrangement which placed one electrode in the structure of interest and the other electrode on the dura directly above gave the required magnitude and stability of recording at the sacrifiLe of specificity. The three EEG tracings (Fig. 6) recorded by different combinations of recording electrodes demonstrate the tissue and dural components of the bipolar arrangement employed in this study. Since the bipolar and monopolar segments were

MONOPOLAR

- 1 DURA -SPHENOID

REF.

HIPPO. W i WJ

100 #V

1%

t

.IsEc.

40gWAH-56

Fig. 6. Components of bipolar recording. Recordings were taken in rapid sequence by direct cable. References p. 3161317

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W. A . H I M W I C H , F. M . K N A P P A N D W. G . S T E I N E R

not recorded simultaneously but in sequence any direct comparison of these tracings is tenuous. This illustration suggests, however, that sphenoid reference contributes a slow component which alters the appearance of the recorded rhythms (dura-tissue vs. tissue-sphenoid). For this reason, we consider the dura to be the better choice of reference. Length of time preparation remains usable. Dogs in whom electrode implantation was successful can be used for recordings for 6-9 months after the implantation. Control observations were made routinely each week and at the beginning of each experiment. The tracings are surprisingly uniform over the entire period as illustrated by recordings made under control conditions with the animal exhibiting the same behavior (Fig. 7). DOG NO. 503 CONTROL

N. CAUDATUS

-

1-24-63

w

-

1 SEC

CORTEX

100

,uv

I , 1 SEC

CONTROL N. CAUDATUS

- 2-21-63

T

,-I

100 p v

1 SEC

CORTEX -. 4OLWAH-6l

100

pv

1 SEC

Fig. 7. Telemetered rccord: control recordings taken approximately a month apart with the animal resting quietly in the behavior room. Electrodes were implanted on 9-20-62 (September 20, 1962).

EEG changes accompanying penfothal arid curare. We felt that it would be desirable in the interest of completeness, to present the recorded rhythms from a lightly anesthetized dog and from a curarized animal after recovery from anesthesia. These recordings were made in a quiet, dimly lighted room with the animal’s eyes lightly covered. The dog was anesthetized with the minimum amount of pentothal required for the insertion of an endotracheal tube and placed immediately on curare and artificial respiration. No more pentothal was given. Muscle relaxation was maintained with a slow infusion of curare. The EEG was telemetered throughout the procedure starting with actively moving animal before anesthesia was given. The characteristic slow activity of the cortical record with sleep spindles can be noted when the animal was under light pentothal anesthesia (Fig. 8). These wave forms gradually disappeared

IIf X31Y03

Fig. 8. Telenietered record. EEG changes accompanying light pentothal anesthesia and recovery from anesthesia with the animal maintained on curare.

to be replaced by the characteristic pattern of activity of the alert animal as the pentothal anesthesia wore off. Sleep-wakefulness shifts in recorded activity. The so-called ‘activation’ response is easily demonstrated in recordings from a sleeping dog subjected to peripheral stimulation (noise) (Fig. 9). The sleep portion of the EEG tracing contains low frequencies (1-3 cis) of high amplitude intermixed with faster components (6, 12, 22 c/s and upwards). The slower components are rarely seen in the recording of the dog in the waking state. At the point of peripheral stimulation indicated by the arrow, there is an immediate reduction in the recorded amplitudes with a pronounced shift towards the fast frequencies. The dark portions of the record contain 32 c/s activity which is the dominant component recorded from our dogs during the waking state. References p. 3161317

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W. A. H I M W I C H , F. M. K N A P P A N D W. ci. S T E I N E R

DOG NO. 522 SLEEP

9-12-63

AROUSAL

4OcWAH-SO

Fig. 9. Telernetered record showing sleep and arousal. Noise was due to opening bolt on behavior room door. Electrode placements as described in the text.

DIRECT CABLE RECORDINGS-DOG WALKING

8 SNIFFING

NO. 701

- 11-9-61 PANTING

Fig. 10. Direct cable monopolar record during sniffing and panting. RMC = right motor cortex, LMC-1 left motor cortex, RV- right visual cortex, LV= left visual cortex. Reference: nose electrode; ground-neck electrode (See Fig. 4).

Shifts in recorded activity during wakefulness. Marked spectral shifts are seldom encountered when recording from the dog in the waking state. There are two conditions, however, in which such shifts can be noted with ease; sniffing and drinking (Fig. lo). The slowing noted in the left portion of Fig. 10 is quite typical during sniffing, having been seen in the recordings from the rat and cat in addition to that of the dog (Personal experience, W.G.S.). The exact mechanism which produces this hypersynchronous type of EEG activity is unknown but this activity is certainly

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reminiscent of the type of EEG effect produced by hyperventilation in man. The right hand portion of this tracing taken when the dog was panting is a curiosity. The frequency of this rhythm is about 12 cjs which makes it a nice ‘a’ rhythm complete with some spindling. Usually, a simple rhythm such as this requires the attenuation of other frequency components by the intact skull. We have obtained this type of rhythm from skull bone recordings in the rat but normally not in direct tissue recordings from either the rat, cat, rabbit or dog. The EEG recording presented in Fig. 11 is of considerable interest because of its specificity. One of the major disappointments in recording from a variety of electrode sites in an animal subjected to widely changing conditions whether by stimulation, drugs or manipulation of behavioral conditions, is the lack of apparent specificity of the changes in activity at a particular recording site. In this figure, however, we have a clear instance of a marked change in the activity of the hypothalamus simultaneously with the drinking of water by the animal. We have not studied this phenomenon directly but the hypothalamus is known to be an important structure in the mediation of water regulation. Recorded activity associated with behavioral manipulation. Several tracings recorded from a dog under a variety of conditions are presented to indicate the apparent lack of shift in recorded activity consequent with a change in the conditions of the recording (Fig. 12). The top line presents the familiar ‘arousal’ response associated with a change from sleep to wakefulness. Line 2 was recorded shortly afterwards while the dog was chewing meat. In line 3, a fresh BenzedrexB inhaler (contains propylhexadrine, menthol and aromatics) was introduced directly into the nostril of the dog. This inhaler was judged to be potent by those working with this dog but without consequence in terms of the recorded rhythms of the dog brain even though the actions of the dog also seemed to suggest that he too found the inhaler to be potent. Line 4 was recorded while the dog was left alone in the observation room. A cat was intro-

Fig. 11. Telemetered record electrode placements as described in the text. Animal spontaneously drinking water. References p . 316/317

314

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STANDING

QUIETLY

duced into the room shortly thereafter and the recording of line 5 was taken. The dog reacted to this change in condition by making repeated jumps against the wall of the observation room but the EEG tracing remained singularly uninformative of this change in the situation even when the cat had been removed (line 6). Drug induced shifts in recorded activity. Changes are readily induced in the recorded rhythms of the dog brain with the administration of various drugs (Fig. 13). The tracing after tryptophan demonstrates a marked reduction in the incidence of fast DOG NO. 502 - ANTERIOR MOTOR CORTEX- 5 -7-63 CONTROL

IMMEDIATELY AFTER l . g / k g

I.V. ALCOHOL

Fig. 13. Telemetered record. The first two tracings were taken in sequence on 5-7-63 (May 7, 1963). The last recording was taken on another day immediately after the animal had received alcohol.

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activity with only a slight change in recorded amplitudes. In the tracing after alcohol the fast component is still visible in the recording but the amplitudes of all rhythms have increased. DISCUSSION

The electrical activity of the dog brain is complex and highly variable from one behavioral state to another as evidenced by the wide varieties of EEG activity described (Figs. 8-1 3). The experimental conditions also affect the patterns. This variety is nowhere in greater evidence than in recordings made during the resting state which normally shows patterns between relaxed inattentiveness and sleep in the dog. Close inspection of the recordings (Fig. 5) reveals activities at 5-6, at 10-12, at 18-22, and 28-32 c/s in the cortical tracings with some well defined 8 c/s activity in the hippocampal tracing and lesser amounts of the same frequency in the caudate and hypothalamic tracings. Faster and slower components than those mentioned above are also present in these subcortical areas. Slow frequencies of 1 to 2 c/s were recorded during sleep (Fig. 9) and 3 c/s immediately following the administration of alcohol (Fig. 13). Under light pentothal anesthesia (Fig. 8) frequencies of 3 to 6 c/s were traced and also during sniffing (Fig. lo). Five to 6 c/s activity occurs in the records 2$ h following tryptophan administration (Fig. 13) and some specific 6 c/s activity in the hypothalamus during drinking (Fig. 11). Fast frequencies approaching 32 c/s were recorded in a variety of naturally occurring (spontaneous) movements and also during deliberate attempts to excite the animal (Fig. 12). It is perhaps not surprising that a wide span of behavior as shown in Fig. 12 produced no differences in electrical activity under a variety of experimental circumstances. The animal was fully alert in the first line of the tracing and so could not become further aroused though recordings from subcortical structures might have yielded additional information. Our knowledge is insufficient at this stage to permit interpretation of the meaning of the cortical changes which are seen with drugs (Fig. 13). It will take more penetrating studies of the relations between behavior, drug administration and the electrical activity from many areas of the brain to answer this question. The present study merely demonstrates the feasibility of undertaking such investigations. In general, the rhythms from dog brain are mixed as to frequencies with faster and slower components forming patterns of alternation and superimposition. Fast and slow components may become more or less dominant under one condition or another so that the relative proportion of each has changed but it is particularly difficult to describe a specific pattern of tracings as being ‘slow’ or ‘fast’ when so many frequency components can be identified within a given tracing regardless of whether the recording was obtained under fear-inducing conditions or during a state of natural sleep. The series of tracings shown in Fig. 8 demonstrate to our satisfaction that the fast cortical activity recorded in the first line is not due to either muscle artifact or to visual stimulation. The characteristic activity reappeared in the animal after pentothal References p . 3161317

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had been dissipated and while the animal was maintained on curare and artificial respiration under conditions which reduced visual stimulation. This activity is similar to that described by Schallek and Walz (1954a, b and c) for the unanesthetized dog. The basic problems of the interpretation of the EEG still remain to be elucidated but the use of a telemetry system makes it feasible to attempt to correlate behavior and biochemistry with changes in the electrical activity of the brain. Such studies can be made satisfactorily only in the freely moving animal. The responsiveness of the animal to training, his rapport with his handlers and the great variety of behavioral studies which can be made render the dog ideal foi multidiscipline studies. SUMMARY

The electrical activity from the dog brain varies with recording conditions and the types of electrode used. Telemetry systems avoid many of the problems such as muscle artifacts involved in direct wire recording. The methods of electrode implantation for a telemetered system, comparison of direct cable and telemetered records and analysis of the contribution made by each component in the bipolar system are included in the material presented here. Analyses were made of tracings taken cortically and from the hippocampus, the posterior hypothalamus and the caudate nucleus under drugs, sleep and arousal, sniffing, panting and drinking. Recordings of this type are essential as a basis for the effect of drugs on the relation of behavior to the EEG under a variety of conditions. ACKNOWLEDGEMENT

The authors wish to thank Mr. E. C. Ginther for his generous and skilled assistance with electronic problems and the technicians and dog handlers for their able and interested help. REFERENCES ADRIAN, E. D., (1942); Olfactory reactions in the brain of the hedgehog. J . Physiol., 100, 4594’73. BERGER, H., (1929); Uber das Elektroencephalogramm des Menschen. 1. Arch. Psychiat. Nervenkr., 81, 527-510. CHARLES, M. S . , AND FULLER, J. L., (1956); Developmental study of the electroencephalogram of the dog. Electroenceph. elin. Neurophysiol., 8, 645-652. DOMINO, E. F., AND UEKI,S., ( I 960); An analysis of the electrical burst phenomenon in some rhinencephalic structures of the dog and monkey. Electroenceph. clin. Neurophysiol., 12, 635-648. Dow, R. S., ULETT,G., AND TUNTURI, A., (1945); Electroencephalographic changes following head injuries in dogs. J . Neurophysiol., 8, 161-172. GOLDENSOHN, E. S., BLJSSE, E. W., SPENCER, J. N., DRAPER, W. B., AND WHITEHEAD, R. W., (1950); Studies on diffusion respiration. VIT. The cortical electrical activity of dogs. Electroenceph. clin. Neurophysiol., 2, 3 3 4 0 . H A M B R ~ C F. H TT., , (1963); A multichannel electroencephalographic telemetering system. Technical report 41 3, November 6. Massachusetts Institute of Technology Research Laboratory of Electronics, Cambridge, Mass. HAMBRECHT, F. T., DONAHUE, P. D., AND MELZACK, R., (1963); A multiple channel EEG telemetering system. Electroencepli. clin. Neurophysiol., 15, 323-326. HIMWICH, W. A., AND HAMBRECHT, F. T., (1963); Telemetry systems in physiological and pharma-

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cological research. 16th Annual Conference on Engineering in Medicine and Biology, Baltimore, Md., November 19. ITO, G., A ND KITAMURA, K., (1939); Vergleichende Untersuchung iiber den Grosshirnaktionsstrom einiger Siiugetiere. Tohoku J . exp. Med., 37, 106-112. JASPER, H. H., AND ANDREW,H. L., (1 938): Electroencephalography. 111. Normal differentiation of occipita1 and precentral regions in man. Arch. Neurol. Psychiat., 39, 96-1 15. LIM,R.I<. S., Liu, C.,AND MOFFITT, R. L., (1960); A Stereoraxic Atlas ofihe Dog’s Brain. Springfield, Thomas. LURIA,R. N., A N D PROFIMOV, L. G., (1956); EEG recordings of various brain areas during prolonged experiments with dogs. Fiziol. Zh. SSSR, 17, 340-356. MOTOKAWA, K., (1949); Comparative studies on the brain waves of various animals in their natural resting states. Tohoku J . exp. Med., 50, 297-306. PRAWDICZ-NEMINSKI, W. W., (1925); Zur Kenntnis der elektrischen und der innervationsvorgange in den funktionellen Elementen und Geweben des tierischen Organismus. Pfliiger’s Arch. ges. Physiol., 209, 380-382. SAKHIULINE, G. T., (1951); A method of recording the electric brain potentials of dogs under conditions of prolonged (chronic) experiments. Zh. vyssh. nerv. Deyat. Pavlova, 1, 457461. SAKHIULINE, G. T., (1955); An analysis of the EEG pattern of dogs during the formation of a conditioned reflex. Dokl. Akad. Nauk SSSR, 104, 153-156. SCHALLEK, W., AND WALZ,D., (1953); Effects of d-amphetamine on the electroencephalogram of the dog. Proc. SOC.exp. Biol. Med. (N . Y . ), 82, 7 15-7 19. SCHALLEK, W., AND WALZ,D., (1954a); Effects of isoniazid and iproniazid on the central nervous system of the dog. Amer. Rev. Tuherc., 69, 261-266. SCHALLEK, W., AND WALZ, D., (1954b); Cardiovascular and central nervous system effects of morphinan series. Proc. SOC.exp. Biol. Med. ( N . Y . ) , 87, 233-236. SCHALLEK, W., AND WALZ,D., (1954~);Effects of drug-induced hypotension on the electroencephalogram of the dog. Anesthesiology, 15, 673-680. SWANK, R. L., AND WATSON, C. W., (1949); Effects of barbiturates and ether on spontaneous electrical activity of dog brain. J . Neurophysiol., 12, 137-160.

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1936 The effect of methylene blue, cystine and cysteine on the metabolism of the intact animal. Amer. J . Physiol., 117, 631 (with W. GOLDFARB AND J . F. FAZEKAS). 1936 The effect of zinc and aluminum on the hypoglycemic action of insulin. J . Pharmacol. exp. Ther., 58, 260 (with J. F. FAZEKAS). 1936 Effect of methylene blue, cystine and cysteine on the metabolism of isolated brain tissue. Amer. J . Physiol., 117, 631 (with W. GOLDFARB AND J. F. FAZEKAS). 1936 Blood sugar in experimental diabetes. N . Y. Acad. Med., 12,284. AND M. A. 1936 Studies on sodium loss. Proc. SOC.exp. Biol. ( N . Y.), 34,450 (with J. F. FAZEKAS SPIERS). 1936 The effects of inhalation of carbon dioxide on the carbon dioxide capacity of arterial blood. J. b i d . Chem., 113, 383 (with E. F. GILDEA, N. RAKIETEN AND D. DUBOIS). 1936 Studies on the physiology of lactation. V. The induction of lactation in depancreatized dogs. Anat. Rec., 66, 201 (with W. 0. NELSON AND J. F. FAZEKAS). 1937 The respiratory quotient of renal tissue of Houssay dogs. Amer. J . Physiol., 118, 297 (with J. F. FAZEKAS AND E. H. CAMPBELL). 1937 The formation of acetylcholine-like substances by excised tissues. Amer. J . Physiol., 119, 306 (with J. F. FAZEKAS). 1937 The effect of hypoglycemia on the metabolism of the brain. Endocrinology, 119,335 (with J. F. FAZEKAS). 1937 Effect of metrazol convulsions on brain metabolism. Proc. SOC.exp. Biol. ( N . Y . ), 37, 359 (with K. M. BOWMAN, J. F. FAZEKAS AND L. L. ORENSTEIN). 1937 Chronic adrenal insufficiency and pancreas diabetes. Proc. Soc. exp. Biol. ( N . Y.), 37, 361 (with J. F. FAZEKAS AND S. J. MARTIN). 1937 Brain metabolism during the hypoglycemic treatment of schizophrenia. Science, 86,271 (with K. M. BOWMAN, J. WORTIS A N D J. F. FAZEKAS). 1937 The effect of hypoglycemia on the metabolism of the brain. Amer. J . Physiol., 21, 800 (with J. F. FAZEKAS). 1937 Protamine-insulin and infection. Amer. J . med. Sci., 194, 345 (with J. F. FAZEKAS). 1938 Carbohydrate oxidation in normal and diabetic cerebral tissues. J . biol. Chem., 125, 545 (with Z . BAKER AND J. F. FAZEKAS). 1938 Respiratory quotient of diabetic liver. Proc. SOC.exp. Biol. ( N . Y . ) , 38,137 (with J. F. FAZEKAS). 1938 Carbohydrate metabolism. Ann. Rev. Biochem., 7, 143. 1938 Physiology and anatomy. The Popular Educator. 1938 Effect of adrenal vein ligation and pancreatectomy on metabolism of renal tissue. Proc. SOC. exp. Biol. ( N . Y.),38,499 (with Z . BAKER AND J. F. FAZEKAS). 1938 Syndromes secondary to prolonged hypoglycemia. Proc. Soc. exp. Biol. ( N . Y.), 39, 244 (with J. F. FAZEKAS, A. 0. BERNSTEIN, E. H. CAMPBELL AND S. J. MARTIN). 1938 Effect of acute anoxia produced by breathing nitrogen on the course of schizophrenia. Proc. SOC.exp. Biol. ( N . Y .), 39, 367 (with F. A. D. ALEXANDER AND B. LIPETZ). 1938 The effect of ligation of the lumboadrenal veins on the course of experimental diabetes in AND S. J. MARTIN). cats and dogs. Science, 87, 144 (with J. F. FAZEKAS 1938 Effect of nicotine on the oxidation of glucose. Amer. J . Physiol., 123, 6 (with Z . BAKER). 1938 The oxidation of various substrates by the diabetic kidney. Amer. J. Physiol., 123, 62 (with J. F. FAZEKAS AND Z. BAKER). 1938 Metabolism of depancreatized dogs and cats following bilateral ligation of the lumboadrenal AND S. J. MARTIN). veins. Amer. J . Physiol., 123, 100 (with J. F. FAZEKAS 1938 Syndromes produced in pancreatized cats and dogs as a result of bilateral ligation of the lumboadrenal veins. Amer. J . Physiol., 123, 142 (with S. J. MARTINAND J. F. FAZEKAS). 1938 The effect of bilateral ligation of the lumboadrenal veins on the course of pancreas diabetes. Amer. J. Physiol., 123,725 (with J. F. FAZEKAS AND S. J. MARTIN). 1939 The mechanism of the symptoms of insulin hypoglycemia. Amer. J . Psychiuf., 96,371 (with J. P. FROSTIG, J. F. FAZEKAS AND Z . HADIDIAN). 1939 Nitrogen inhalation therapy for schizophrenia. Preliminary report on technique. Amer. J . Psychiat., 96, 643 (with F. A. D. ALEXANDER). 1939 Clinical electroencephalographic and biochemical changes during insulin hypoglycemia. Proc. Soc. exp. Bio/. ( N . Y . ) , 40, 401 (with J. P. FROSTIG, J. F. FAZEKAS, H. HOAGLAND AND z. HADIDIAN). 1939 Effect of pyocyanin on cerebral metabolism. Proc. SOC. exp. Biol. ( N . Y . ) , 42, 446 (with J. F. FAZEKAS, H. COLYER AND S. NESIN).

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1939 Effect of cyanide on cerebral metabolism. Proc. Soc. exp. Biof. ( N . Y . ) , 42, 496 (with J. F. FAZEKAS AND H. COLYER). 1939 Effect of hypothermia on cerebral metabolism. Proc. SOC.exp. Biol. ( N . Y . ) , 42, 537 (with J. F. FAZEKAS). 1939, 1940, 1941 Brain metabolism. Transactions of the Kansas C i / y Academy of Medicine. Presented at the Kansas Citv Academv of Medicine. November 17. 1939. I939 Cerebral metabolism and electrical activity during insulin hypoglycemia in man. Anwr. J . Phy.siol., 125, 578 (with Z. HADIDIAN, J. F. FAZEKAS A N D H. HOAGLAND). 1939 The respiratory metabolism of infant brain. Amer. J . Physiof., 125, 601 (with Z. RAKERA N D J. F. FAZEKAS). 1939 Nitrogen inhalation therapy for schizophrenia. Amer. J . Physiol., 126, 535 (with F. A. D. ALEXANDER, B. LIPETA N D J. F. FAZFKAS). 1939 Effect of hypoglycemia and pentobarbital sodium on the electrical activity of the dog cerebral cortex and hypothalamus. Amer. J . Physiol., 126, 536 (with H. HOACLAND, E. H. CAMPBELL, I. F. FAZEKAS A N D Z. HADIDIAN). I939 The formation of acetylcholine by tissues of thc rat. Amer. J . Physiol., 127, 381 (with P. SYKOWSKI A N D J. F. FAZEKAS). 1939 The glucose and lactic acid exchanges during hypoglycemia. Amer. J. Physiol., 127, 685 (with J. F. FAZEKAS AND S. NESIN). 1939 Biochemical changes occurring in the cerebral blood during the insulin treatment of schizophrcnia. J . nerv. ment. Dis., 89, 273 (with K. M. BOWMAN, J. WORTISA N D J. F. FAZEKAS). 1939 Metabolism of thc brain during insulin and metrazol treatments of schizophrenia. J. Amer. J. WORTISA N D J. F. FAZEKAS). med. Ass., 112, 1572 (with K. M. BOWMAN, 1939 Electrocardiographic changes during hypoglycemia and anoxemia. Endocrinology, 24, 536 (with S. J. MARTIN,F. A. D. ALEXANDER AND J. F. FAZEKAS). 1939 Effects of hypoglycemia and pentobarbital sodium on electrical activity of cerebral cortex and hypothalamus (dogs). J . Neurophysiol., 2 , 276 (with H. HOAGLAND, E. H. CAMPBELL, J. F. FAZEKAS AND Z. HADIDIAN). 1939 The effect of cocarboxylase upon metabolism and neuropsychiatric phenomena in pellagrins with beribcri. Science, 90, 141 (with F. H. LEWY,J. P. FROSTIG A N D T. D. SPIES). W. GOLDFARB 1939 Cerebral metabolism during fever. Science, 90, 398 (with K . M. BOWMAN, A N D J. F. FAZEKAS). 1940 A study of the central action of metrazol. Amer. J . Psychiat., 97, 366 (with B. LIBETAND J. F. FAZEKAS). 1940 Oxygen consumption in the psychoses of the senium. Amer. J . Psychiat., 97, 566 (with D. E. CAMERON, S. R. ROSENAND J. F. FAZEKAS). 1940 Changes in cerebral blood flow and arterio-venous oxygen difference during insulin hypoglycemia. Proc. SOC.exp. Biol. ( N . Y.), 45,468 (with K . M. BOWMAN, C. DALY, J. F. FAZEKAS, J. WORTISAND W. GOLDFARB). 1940 Prolonged coma and cerebral metabolism. Arch. Neiirol. Psychiat. (Chic.),44, 1098 (with K. M. BOWMAN AND J. F. FAZEKAS). 1940 Cerebral carbohydrate metabolism during deficiency of various members of the vitamin B complex. Amer. J . med. Sci., 199, 849 (with T. D. SPIES,J. F. FAZEKAS AND S . NESIN). I940 Temperature and brain metabolism. Amer. J. med. Sci., 200, 347 (with K. M. BOWMAN, J. F. FAZEKAS A N D W. GOLDFARB). I940 Temperature and brain metabolism. J . Physiol. (U.S.S.R.), 29, 271 (with K. M. BOWMAN, AND J. F. FAZEKAS). W. GOLDFARB 1940 Cerebral metabolism in mongolian idiocy and phenylpyruvic oligophrenia. Arch. Neurol. Psychiar. (Chic.), 44, 121 3 (with J. F. FAZEKAS). 1940 Brain metabolism in mongolian idiocy and phenylpyruvic oligophrenia. Amer. J . ment. Defic., 45, 37 (with J. F. FAZEKAS AND s. NESIN). 1940 The effects of alcohol and pentobarbital on metabolism of excised cerebral tissues of adult and infant rats. Anier. J . Physiol., 129, 382 (with P. SYKOWSKI AND J. F. FAZEKAS). 1940 Control of electrical and oxidative activity of brain by temperature. Amer. J . Physiol., 129,404 (with B. LII~ET, J. F. FAZEKAS, A. M. MEIROWSKY AND E. H.CAMPBELL). 1941 The electrical response of the kitten and adult cat brain to cerebral anemia and analeptics. Amer. J. Physiol., 132, 232 (with B. LIBETAND J. F. FAZEKAS). 1941 A comparative study of excised cerebral tissues of adult and infant rats. Amer. J. Physiol., 132,293 (with P. SYKOWSKI AND J. F. FAZEKAS).

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1941 Comparative studies of the metabolism of the brain of infant and adult dogs. Amer. J . Physiol., 132, 454 (with J. F. FAZEKAS). 1941 Cerebral blood flow and brain metabolism during insulin hypoglycemia. Amer. J . Physiol., 132, 640 (with K. M. BOWMAN, C. DALY,J. F. FAZEKAS, J. WORTISAND W. GOLDFARB). 1941 Comparative effects of stimulants on infant and adult cerebral tissues. Amer. J . Physiol., 133, 325 (with H. C. HERRLICH AND J. F. FAZEKAS). 1941 Tolerance of the newborn to hypoxia and anoxia. Amer. J . Physiol., 133, 327 (with F. A. D. ALEXANDER AND J. F. FAZEKAS). A N D F. A. D. 1941 Hypoglycemia in the infant rat. Amer. J . Physiol., 133, 328 (with J . F. FAZEKAS ALEXANDER). AND 1941 Tolerance of the newborn to anoxia. Amer. J . Physiol., 134, 281 (with J. F. FAZEKAS F. A. D. ALEXANDER). 1941 The significance of a pathway of carbohydrate breakdown not involving glycolysis. J. biof. Chem., 139, 971 (with J. F. FAZEKAS). 1941 Effects of cyanide and iodoacetate on survival period of infant rats. Proc. SOC.exp. Biol. ( N . Y . ) , 46, 553 (with J. F. FAZEKAS AND F. A. D. ALEXANDER). 1941 Composition of the milk of the monkey ( M . mulatta). Proc. SOC.exp. Biol. ( N . Y . ) , 48, 133 (with G . VAN WAGENEN AND H. R. CATCHPOLE). 1941 Survival of infant and adult rats at high altitudes. Proc. SOC.exp. Biol. ( N . Y . ) , 48,446 (with H. C. HERRLICH AND J . F. FAZEKAS). 1941 Availability of lactic acid for brain oxidations. J. Neurophysiol., 4, 243 (with J. WORTJS, K. M. BOWMAN,W. GOLFDARB AND J. F. FAZEKAS). 1942 Action of insulin on pyruvate formation in depancreatized dogs. Fed. Proc., 1, 12 (with E. BUEDING AND J. F. FAZEKAS). I942 Effect of insulin on pyruvic acid formation in depancreatized dogs. Science, 95, 282 (with E. BUEDING, J. F. FAZEKAS AND H. C. HERRLICH). I942 A study of the comparative toxic effects of morphine on the fetal, newborn and adult rats. J . Pharmacol. exp. Ther., 75, 363 (with A. CHESLER AND G. C. LABELLE). 1942 The relative effects of toxic doses of alcohol on fetal, newborn and adult rats. Quart. J. Stud. Alcohol, 3, 1 (with A. CHESLER AND G. C. LABELLE). I942 Brain metabolism and the mental deficiencies. Amer. J . ment. Defic., 46, 302. 1942 Fundamental concepts in the treatment of diabetes niellitus and its complications. An interpretation of the symptoms of hypoglycemia. No. 21 of series published by The New York Diabetes Association, 2 East 103rd St., New York City, May, 1942. 1942 Hypoglycemic reactions. Proc. Arner. diab. Ass., 2, 161. 1942 Mechanisms for the maintenance of life in the newborn during anoxia. Amer. J. Physiol., 135,387 (with A. 0. BERNSTEIN, H. E. HERRLICH, A. CHESLER AND J. F. FAZEKAS). 1942 The metabolic effects of potassium, temperature, methylene blue and paraphenylenediamine J. F. FAZEKAS on infant and adult brain. Amer. J . Physiol., 137, 327 (with A. 0. BERNSTEIN, H. C. HERRLICH AND E. RICH). 1942 Effect of thyroid medication on brain metabolism of cretins. Amer. J. Psychiut., 98, 489 (with C. DALY,J. F. FAZEKAS AND H. C. HERRLICH). 1942 Factor of hypoxia in the shock therapies of schizophrenia. Arch. Neurol. Psychiat., 47, 800 (with J. F. FAZEKAS). 1942 Studies on the effects of adding carbon dioxide to oxygen-enriched atmospheres in low pressure chambers. 11. The oxygen and carbon dioxide tensions of cerebral blood. J . A v i d . Med., 13, 177 (with J. F. FAZEKAS, H. C. HERRLICH, A. E. JOHNSONAND A. L. BARACH). I942 Effect of iodoacetate on survival period during hypoxia. Fed. Proc., 1 , 4 0 (with H. C. HERRLICH, E. RICH,R. BARSTOW A N D J . F. FAZEKAS). I943 An evaluation of the factor of depression of brain metabolism in the treatment of schizophrenia. Psychiat. Quart., 17, 164 (with C. H. BELLINGER, C. F. TERRENCE AND B. LIPET). 1943 Effect of insulin on pyruvic acid formation on depancreatized dogs. J. biol. Chem., 148, 97 (with E. BUEDING, J. F. FAZEKAS AND H. C. HERRLICH). 1943 The glycogen content of various parts of the central nervous system of dogs and cats at different ages. Arch. Biochem., 2 , 175 (with A. CHESLER). 1943 Glycogen content of various parts of the central nervous system of dogs and cats. Fed. Proc., 2, 6 (with A. CHESLER). 1943 Effect of age on cerebral arterio-venous oxygen difference. Fed. Proc., 2, 21 (with J. F. FAZEKAS).

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I943 EiP:.st of anoxia and hypoglycemia on survival period of adult rats. Fed. Proc., 2, 23 (with E. HOMBURCER). 1943 Comparative toxicity of pentobarbital in the newborn and adult rat. J. Lab. clin. M d . , 28, 706 (with B. ETSTENAND F. A. D. ALEXANDER). 1943 Anaerobic survival of adult animals. Amer. J . Physiol., 139, 366 (with J. F. FAZEKAS). 1943 Electroshock. A round table discussion. Amer. J. Psychiuf., 100,361. 1943 The role of the vitamins in brain metabolism. Res. Publ. Ass. nerv. ment. Dis., 22, 33. 1943 Effect of neosynephrin on gaseous exchange of the brain. Proc. Soc. exp. Biol. ( N . Y . ) , 53, 78 (with C. DALYA N D J. F. FAZEKAS). I943 Brain metabolism and mental deficiency. Amer. J . menf. DeJic., 48, 137 (with J. F. FAZEKAS). 1943 Cerebral arterio-venous oxygen difference. I. Effect of age and mental deficiency. Arch. Neurol. Psychiat., 50, 546 (with J. F. FAZEKAS). 943 Effect of hypoglycemia and anoxia on the survival period of infant and adult rats and cats. Endocrinology, 33, 96 (with J. F. FAZEKAS AND E. HOMBURGER). 944 Carbohydrate metabolism in vitamin BI deficiency. J. biol. Chern., 153, 219 (with A. CHESLER AND E. HOMRURGER). 944 A comparison of the relationship of lactic acid and pyruvic acid in the normal and diabetic dog. J . biol. Chem., 155, 413 (with A. CHESLER). 944 Comparative studies of the rates of oxidation and glycolysis in the cerebral cortex and brain stem of the rat. Amer. J . Phyviol., 141, 513 (with A. CHESLER). 1944 Glycolysis in the parts of the central nervous system of cats and dogs during growth. Amer. J. Physiol., 142, 544 (with A. CHESLER). 1944 The cerebral arterio-venous oxygen difference. TI. Mental deficiency. Arch. Neurol. Psychiut., 51, 73 (with J. F. FAZEKAS). 1944 Effect of insulin hypoglycemia on glycogen content of parts of the central nervous system of the dog. Arch. Neurol. Psychiut., 52, I14 (with A. CHESLER). 1944 The effects of insulin hypoglycemia on the glycogen content of the various parts of the central nervous system of the dog. Fed. Proc. 3, 7 (with A. CHESLER). 1944 Carbohydrate metabolism in vitamin BI deficiency. Fed Proc., 3, 93 (with A. CHESLER AND E. HOMBURGER). 1944 The physiology of the 'shock' therapies. Psychiut. Quart., 18, 357. 1944 A review of hypoglycemia, its physiology and pathology, symptomatology and treatment. Amer. J. dig. Dis., 11, 1 . 1944 The oxygen content of cerebral blood in patients with acute symptomatic psychoses and acute destructive brain lesions. Amer. J. Psychinf., 100, 648 (with J. F. FAZEKAS). 1945 Energy metabolism. Ann. Rev. Physiol., 7 , 181. 1945 Cerebral metabolism in patients with depression. Amer. J . Psychiut., 101, 453 (with D. E. CAMERON, E. HOMBURGER AND F. FELDMAN). I945 Pyruvic acid cycle. Fed. Proc., 4,33 (with E. HOMBURGER AND W. A. HIMWICH). 1945 Chronic thiamin deficiency. Fed. Proc., 4, 155 (with W. A. HIMWICH). 1946 Stages and signs of pentothal anesthesia: physiologic basis. Anesthesiology, 7 , 536 (with B. ETSTEN). 1946 Similarity of cerebral arterio-venous oxygen differences in right and left sides of resting man. Arch. Neurol. Psychiut., 55, 578 (with G. YORKAND E. HOMBURGER). I946 Pattern of metabolic depression induced with pentothal sodium. Arch. Neurol. Psychiat., 56, 171 (with B. ETSTENAND G . YORK). 1946 The effect of hypoglycemia and age on the glycogen content of the various parts of the feline central nervous system. Amer. J . Physiol., 146, 389 (with S. FERRIS). I946 Effect of pentothal anesthesia on canine cerebral cortex. Amer. J. Physiol., 147, 343 (with E. HOMBURGER, W. A. HIMWICH,B. ETSTEN,G. YORKAND R. MARESCA). 1946 Criteria for the stages of pentothal anesthesia. J. nerv. ment. Dis., 104,407 (with B. ETSTEN). 1946 Pyruvic acid in exercising depancreatized dogs and diabetic patients. J. biol. Chem., 165, 513 (with W. A. HIMWICH). 1946 The effect of age on the hypoglycemic depletion of glycogen in the central nervous system. Fed. Proc., 5, 27 (with S. FERRIS). 1946 Effect of pentothal anesthesia on canine cerebral cortex. Fed. Proc., 5,47 (with E. HOMBURGER, B. ETSTEN,R. MARESCA, G. YORKAND W. A. HIMWICH). 1946 Organic phosphates and insulin. Fed. Proc., 5, 47 (with W. A. HIMWICH).

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1946 Brain metabolism in unanesthetized and anesthetized man. Fed. Proc., 5, 47 (with W. A. HIMWICH, E. HOMBURGER AND R. MARESCA). 1946 Pyruvic acid exchange of the brain. J. Neurophysiol., 9, I33 (with W. A. HIMWICH). 1947 The functional organization of the central nervous system as observed in pentothal anesthesia. Abstracts of Conitnunications of the Sevenleenth lnernational Physiological Congress, Oxford. 1947 The influence of some organs on the pyruvate level in the blood. Amer. J . Physiol., 148,323 (with W. A. HIMWICH). 1947 Brain metabolism in man: unanesthetized and in pentothal narcosis. Amer. J. Psychiat., 103, 689 (with W. A. HIMWICH, E. HOMBURGER A N D R. MARESCA). 1947 Factors influencing the susceptibility of rats to barbiturates. Fed. Proc., 6, 131 (with E. HOMBURGER A N D B. ETSTEN). 1947 Some factors affecting the susceptibility of rats to various barbiturates. J. Lab. clin. Med., 32, 540 (with E. HOMBURGER AND B. ETSTEN). 1947 The influence of vitamin B1 deficiency on the pyruvate exchange of the heart. Amer. Heart J., 33, 341 (with F. S. RANDLES, W. A. HIMWICH AND E. HOMRURGER). 1948 Management of anoxia during pentothal anesthesia. Amer. J . Surg., 76,268 (with B. ETSTEN). 1948 Anoxic survival and di-isopropyl fluorophosphate (DFP). Science, 108, 41 (with A. M. FREEDMAN). 1948 Correlation between signs of toxicity and cholinesterase level of brain and blood during recovery from di-isopropyl fluorophosphate (DFP) poisoning. Fed. Proc., 7 , 36 (with A. M. FREEDMAN). 1948 The effect of size, sex and pregnancy on the lethality of di-isopropyl fluorophosphate (DFP). Fed. Proc., 7, 36 (with A. M. FREEDMAN). I948 Di-isopropyl fluorophosphate (DFP) : site of injection and variation in response. Fed. Proc., 7 , 55 (with A. M. FREEDMAN). I948 Effect of age on lethality of di-isopropyl fluorophosphate. Amer. J. Physiol., 153, 121 (with A. M. FREEDMAN). I949 Experimental production of electrical major convulsive patterns. Amer. J. Physiol., 156, 1 17 (with A. M. FREEDMAN, P. D. BALES AND ALICE WILLIS). 1949 DFP: site of injection and variation in response. Amer. J. Physiol., 156, 125 (with A. M. FREEDMAN). 1949 Correlation between signs of toxicity and cholinesterase level of brain and blood during recovery from di-isopropyl fluorophosphate (DFP) poisoning. Amer. J. Physiol., 157, 80 (with A. M. FREEDMAN A N D ALICE WILLIS). 1949 Evidence for the evolution of the brain. Humanist, 8, 159. 1949 The brain and the symptomatology of the anoxias. Anesthesiology, 10, 663. I949 Effects of di-isopropyl fluorophosphate (DFP) on electroencephalogram and cholinesterase C . F. ESSIGAND ALICE WILLIS). activity. Fed. Proc., 8, 66 (with J. L. HAMPSON, 1949 Effect of tridione on convulsions caused by di-isopropyl fluorophosphate (DFP). Fed. Proc., 8, 75 (with C. F. ESSIGAND J. L. HAMPSON). 1949 Cholinergic nature of the vestibular receptor mechanism: forced circling movements. Fed. Proc., 8, 75 (with C. F. ESSIG,J. L. HAMPSON, P. D. BALES AND A. M. FREEDMAN). 1950 Effect of panparnit on brain wave changes induced by di-isopropyl fluorophosphate (DFP). Science, 111, 38 (with C. F. ESSIG,J. L. HAMPSON, P. D. BALES AND ALICEWILLIS). 1950 An experimental analysis of biochemically induced circling behavior. J . Neurophysiol., 13, 269 (with C. F. ESSIG,J. L. HAMPSON AND ALICEMCCAULEY). 1950 Effects of di-isopropyl fluorophosphate (DFP) on electroencephalogram and cholinesterase C. F. ESSIGA N D activity. Electroenceph. clin. Neurophysiol., 2 , 41 (with J . L. HAMPSON, ALICE MCCAULEY). 1950 Fat metabolism, medical aspects of. Cyclopedia of Medicine, Surgery, Specialties, 9, 85. 1950 Someevidenceon thefunctionalorganizationofthe brain. 3iochim.biophys. Acta (Amst.) ,4,118. 950 Effect of trimethadione (tridione) and other drugs on convulsions caused by di-isopropyl fluorophosphatc (DFP). Amer. J . Psychiut., 106, 816 (with C . F. ESSIG,J. L. HAMPSON, P. D. BALES AND A. M. FREEDMAN). 950 A central action of some antihistamines. Correction of forced circling movements and of seizure brain waves produced by the intracarotid injection of di-isopropyl fluorophosphate (DFP). Amer. J . Psychiuf., 107, 367 (with R. J. JOHNS). 950 Forced circling movements (adversive syndrome). Arch. Otolaryng., 51,672 (with M. SCHIFF A N D W. G. ESMOND).

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1951 The effec!s; of D F P on the convulsant dose of theophylline, theophylline-ethylenediamine and 8-chl~~rotheophylline. J . Pharmucul. exp. Ther., 101,237 (with R. J. JOHNSAND P. D. BALES). 1952 Mechanism of seizures induced by di-isopropyl fluorophosphate (DFP). Amer. J . Psychiut., 108, 847 (with W. F. BOUZARTH). 1952 Effect of shock therapies on the brain. Biology of Mental Ifealrh and Disease. Milbank Memorial Fund. Hoeber, New York (pp. 548-561). 1952 The functional organization of the central nervous system, an experimental analysis. Proc. Inst. Med. Chic., 19, 115. 1952 Report of committee on research. 111. Anticonvulsant and convulsant agents. Epilepsia Third Series, 1, 143. 1952 Comparative effects of antiepileptic and other drugs upon forced circling produced by the intracarotid injection of di-isopropyl fluorophosphate (DFP). Fed. Proc., 11, 70 (with I. H. WEINER,ALAYNE COOMBSAND EDITHCAMPBELL). 1953 Biochemically induced circling behavior. Confin. neurol. (Basel), 13,65 (with C. F. ESSIGA N D J. L. HAMPSON). 1953 Biological Foundations of Psychiatry (pp. 1-87). 1953 General neurophysiology (biochemical aspects). Progress in Neurology and Psychiatry. V111. E. A. Spiegel, Editor. New York, Grune and Stratton (pp. 14-39). 1953 Some effects of DFP (di-isopropyl fluorophosphate) and atropine on behavior. ArzneimittelForsch., 3 , 228. 1954 Brain acetylcholinesterase activities in rabbits exhibiting three behavioral patterns following the intracarotid injection of di-isopropyl fluorophosphate. Amer. J . Physiol., 177, 175 (with M . H. APR~SON A N D P. NATHAN). 1954 Relationship between ape and cholinesterase activity in several rabbit brain areas. Amer. J. Physiol., 179, 502 (with M. H. APKISON). 1954 A study of the relationship between asymmetric acetylcholinesterase activities in rabbit brain and three behavioral patterns. Science, 119, 158 (with M. H. APRISONAND P. NATHAN). 1955 Concentrations of a brain neurohornione, acetylcholine, associated with a syndrome resembling the motor aura of an epileptic episode. American League against EpilepAy, Abstract, Dec. 8 , (with M. H. APRISONAND P. NATHAN). 1955 Age and the water content of rabbit brain parts. Amer. J . Phjjsiol., 180, 205 (with JUANITA GRAVES). 1955 Basic research drugs used in psychiatry. Proc. Sci. Med. Con$, Marketing Con$, Amer. Pharmac. Manufact. Ass. (pp. 48-53). '. E. A. 1955 General neurophysiology (biochemistry). Progress in Neurology and Psychiutry, A Spiegel, Editor. New York, Grune and Stratton (pp. 16-56). 1955 The anatomy and physiology of the frontal lobes (metabolic considerations). Clinical Nearosurgery. Raymond K. Thompson, Editor. Baltimore, William and Wilkins (pp. 108-1 16). I955 The new psychiatric drugs. Sci. Amer., 193, 80. 1955 The use of fluids and electrolytes in the management of the neurosurgical patient (the bloodbrain barrier). Clinical Neurowrgery. Raymond K . Thompson, Editor. Baltimore, William and Wilkins (pp. 166-179). 1955 The permeability of the blood-brain barrier to glutamic acid in the developing rat. Biochemistry of the Developing Nervous System. H. Waelsch, Editor. New York, Academic Press (pp. 202-207) (with W. A. HIMWICH). 1955 The effect of age on cholinesterase activity of rabbit brain. Biochemistry of the Developing Nervous System. H. Waelsch, Editor. New York, Academic Press (pp. 301-307) (with M. H. APRISON). 1955, 1956 An analysis of the activating system including its use for screening antiparkinson drugs. Yale J . biol. Me[/.,28, 308 (with F. RINALDI). 1955 Some behavioral effects associated with feeding sodium glutamate to patients with psvchiatric AND W. A. HIMWICH). disorders. J . nerv. ment. Dis., 121,40 (with K. WOLFF,A. L. HUNSICKER 1955 Prospects in psychopharmacology. J . nerv. menf. Dis., 122, 413. 1955 The cerebral electropraphic changes induced by LSD and mescaline are corrected by Frenquel. J . nerv. menf.Dis., 122,424 (with F. RINALDI). 1955 A comparison of the effects of atropine with those of several central nervous system stimulants on rabbits exhibitinq forced circling following the intracarotid injection of diisopropyl fluorophosphate. Confin. neurol. (Basel), 15, 1 (with P. NATHAN AND M. H. APRISON).

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1957 The physiology of alcohol. J . Amer. med. Ass., 163, 545. I957 Tranquilizing Drugs. A.A.A.S., Washington, D.C. 1957 Analysis of thc action of benztropine rnethanesulfonate against parkinsonism. Tranquilizing Drugs. H. E. kiIMWICH, Editor. A.A.A.S., Washington, D.C. (pp. 47-57) (with F. RINALDI). I957 Therapeutic effects of azacyclonol in psychotic patients. Tranquilizing Drugs. H. E. HIMWICH, Editor. A.A.A.S., Washington, D. C.(pp. 1 15) (with F. RINALDI, E. E. HAYNES AND L. H. RUDY). 1957 Viewpoints obtained from basic and clinical symposia on tranquilizing drugs. Tranquilizing Drugs. H. E. HIMWICH, Editor. A.A.A.S., Washington, D.C., (pp. 183-192). 1957 General neurophysiology (biochemistry). Progress in Neurology and Psychiatry. XII. E. A. Spiegel, Editor. New York, Grune and Stratton (pp. 18-42) (with W. A. HIMWICH). 1957 The antiparkinson activity of benactyzine. Arch. int. Pharmacodyn. Ther., 110, 119 (with F. RINALDI). 1957 The effect of drugs on the reticular system. Brain Mechanisms and Drug Action. Wm. W. Fields, Editor. Springfield, Ill., Thomas (pp. 15-43) (with F. RINALDI). 1957 Brain composition during the whole life span. Geriatrics, 12, 19 (with W. A. HIMWICH). 1957 A comparative study of two central nervous system stimulants, MER-22 and SKF 5, on chronic, blocked and withdrawn psychotic patients. Amer. J . Psychiat., 113, 840 (with F. HAGENAUER AND L. H. RUDY). 1957 Clinical evaluation of two phenothiazine compounds promazine and mepazine. Amer. J. Psychiat., 113, 979 (with L. H. RUDYAND D. C. TASHER). I957 A clinical evaluation of L-glutavite in the treatment of elderly chronic deteriorated mental patients. Illinois med. J., 112, 121 (with T. T. TOURLENTES AND D. S. HUCKINS). 1957 Analysis of forced circling induced by DFP and ablation of cerebral structures. Amer. J . Physiol., 189, 513 (with R. P. WHITE). 1957 Circus movements and excitation of striatal and mesodiencephalic centers in rabbits. J. Neurophysiol., 20, 81 (with R. P. WHITE). 1958 Book review of: The Chemical Concepts of Psychosis. Herman C. B. Denber, Editor. New York, Mc-Dowell, Obolensky, (pp. 1-485). 1958 Designated discussion. Reticular Formation of the Brain. Herbert H. Jasper, Editor. Boston, Little,Brown, (pp. 169-176). I958 Introduction to the second round table discussion. Neurological and Psychological Dejicits of Asphyxia Neonatorutn. W. F. Windle, Editor. Springfield, Thomas (pp. 141-151). 1958 Prospects in psychopharmacology. The New Chemotherapy in Mental Illness. H. L. Gordon, Editor. New York, Philosophical Library (pp. 23-35). 1958 PsychopharmacologicaI drugs. Science, 127, 59. 1958 Reticular formation of the brain. International Symposiutn. Henry Ford Hospital. Boston, Little, Brown (pp. 169-173). 1958 Clinical evaluation of BAS (benzyl analog of serotonin), a tranquilizing drug. J. nerv. ment. Dis., 126, 284 (with L. H. RUDY,E. COSTAAND F. RINALDI). 1958 Brain disorders (trifluoperazine in mentally defective patients). TriPuoperazine Clinical and Pharmacological Aspects. Henry Brill, Editor. Philadelphia, Lea and Febiger (pp. 169-172) (with L. H. RUDY,E. COSTAAND F. RINALDI). 1958 A clinical evaluation of psychopharmacological agents in the management of disturbed mentally defective patients. Amer. J. ment. Defic., 62,855 (with L. H. RUDYAND F. RINALDI). 1958 Triflupromazine and trifluoperazine; two new tranquilizers. Amer. J . Psychiat., 114, 747 (with L. H. RUDY,F. RINALDI, E. COSTA,W. TUTEUR AND J. GLOTZER). 1958 Cortical and rhinencephalic electrical potentials during hypoglycemia. Arch. Neurol. Psycltiat., 80, 314 (with W. G . VANMETERAND HELENF. OWENS). 1959 Interactions of nionoamine oxidase inhibitors with physiological and biochemical mechanisms in brain. A discussion of the paper by H. E. Himwich, E. Costa, G. R. Pscheidt and W. G. Van Meter. Ann. N . Y .Acad. Sci., 80,614 (with B. B. BRODIE, S. SPECTOR AND P.A. SHORE). 1959 Effect of reserpine on urinary tryptamine excretion in man. Physiologist, 2, 19 (with G . W. BRUNE). 1959 Brain serotonin metabolism in insulin hypoglycemia. Biochemistry of the Central Nervous System. 111. 0 . Hoffman-Ostenhol, Editor. New York, Pergamon Press (pp. 283-290) (with E. COSTA). 1959 Behavioral changes following increases of neurohormonal content in selected brain areas. Fed. Proc., 8, 379 (with E. COSTA,W. A. HIMWICH, S. G. GOLDSTEIN AND R. G. CANHAM). 1959 Insulin hypoglycemia and rabbit brain norepinephrine. Fed. Proc., 18,123 (with G.R. PSCHEIDT).

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I959 An EEG analysis of psychotomimetic drugs. Neuro-Psychopharmacology. P. B. Bradley, Editor. Amsterdam, Elsevier (pp. 329-333) (with W. G. VANMETERAND HELEN F. OWENS). 1959 Biochemistry of the nervous system in relation to the process of aging. The Process of Aging in the Nervous System. J. E. Birren, H. A. Imus and W. F. Windle, Editors. Springfield, Ill., Thomas (pp. 101-112). 1959 Management of alcoholism. Mod. Med., 23. 1959 Some drugs used in the treatment of mental disorders. Amer. J . Psychiat., 115, 756. 1959 Book review of: Neuropharmacology : Transactions of the second conference. Amer. J. Psychiat., 116, 88. 1959 Book review of: The chemical concepts of psychosis. Amer. J. Psychiat., 116,286. 1959 Stimulants. Ass. Res. nerv. Dis. Proc., 37, 356. 1959 The functional organization of the brain, a reconsideration. Read at Regional Research Conference of A.P.A., Chicago, June. 1959 Correlationsbetweeneffectsof iproniazid on brain activating systemwith brain neurohormones. Biological Psychiatry. Jules H. Masserman, Editor. New York, Grune and Stratton (pp. 2-16) (with E. COSTA, G. R. PSCHEIDT AND W. G. VANMETER). I959 Drugs used in the treatment of the depressionc. Biological Psychiatry. Jules H. Masserman, Editor. New York, Grune and Stratton (pp. 27-52) (with W. G . VAN METERAND HELEN F. OUTENS). 1959 Triflupromazine (Vesprin) in the treatment of psychotic patients. Biological Psychiatry. J. Masserman, Editor. New York, Grune and Stratton (pp. 292-305) (with F. RINALDI, E. COSTA,L. H. RUDY,W. TUTEUR AND J. GLOWER). I959 Interaction of monoamine oxidase inhibitors with physiological and biochemical mechanisms in brains. Ann. N . Y . Acad. Sci., 80, 614 (with E. COSTA,G. R. PSCHEIDT AND W. G . VAN METER). I959 Triflupromazine and trifluoperazine in the treatment of disturbed mentally defective patients. Amer. J . ment. D e j c . , 64,711 (with E. COSTA,F. RINALDIAND L. H. RUDY). 1959 General neurophysiology (biochemistry). Progress in Neurology and Psychiatry. XIV. E. A. Spiegel, Editor. New York, Grune and Stratton (pp. 19-36) (with W. A. HIMWICH). 1959 Neurochemistry of aging. Handbook of Aging and the Individual. James E. Birren, Editor. Chicago, University of Chicago Press (pp. 187-216) (with W. A. HIMWICH). 1959 Insulin Treatment in Psychiatry. New York, Philosophical Library (pp. 1-380) (with M. RINKEL). 1959 Studies of the hypoglycemic brain. Amino acids, nucleic acids, total nitrogen, and side-group ionization of proteins in cat brain during insulin coma. Arch. Neurol. Psychiat., 81, 458 (with F. E. SAMSON, JR., D. R. DAHLAND NANCYDAHL). 1959 The effects of tofranil, an antidepressant drug, on electrical potentials of rabbit brain. Canad. psychiat. Ass. J., 4, Special Suppl. S113 (with W. G. VANMETERAND HELEN F. OWENS). 1960 Hemodynamic studies on the circle ofWillis in dogs. Tranmctionr of the American Neurological Association. Melvin D. Yahr, Editor. New York, Springer (pp. 187-188) (with G. F. AYALA AND W. A. HIMWICH). 1960 Effects of reserpine on urinary tryptamine and indole-3-acetic acid excretion in mental deficiency schizophrenia and phenylpyruvic oligophrenia. Reprinted from : Acta of the Znternational Meeting on the Techniques for the Study of Psychotropic Drugs, Bologna, June 26-27 (with G . BRUNE). 1960 Technics for the study of behavior induced by drugs using injections into selected arterial sites in the brain. Reprinted from: Acta of the International Meeting on the Techniques for the Study of Psychotropic Drugr, Bologna, June 26-27, (with W. A. HIMWICH AND E. COSTA). 1960 Brain concentrations of biogenic aniines and EEG patterns of rabbits. J. Pharmacol. exp. Ther., 130,81 (with E. COSTA,G. R. PSCHEIDT AND W. G. VANMETER). 1960 Biochemical and neurophysiological action of psychoactive drugs. Drugs and Behavior. L. Uhr and J. G. Miller, Editors. New York, John Wiley and Sons (pp. 41-85). 1960 Book review of: Differential treatment and prognosis in schizophrenia. Ment. Hyg. ( N . Y.), 44,593.

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I960 Functional organization of the brain, past and present. J . new. ment. Dis., 130, 505. 1960 Similarivies between tranquilizers and antidepressants. Memorial Research Monogruphs Naka. Committee on the celebration of the 60th birthday of Professor S. Naka, Osaka, Japan (pp. 125-142). 1960 Some drugs useful in the treatment of emotional disorders. Physiolog) , indications and contra-indications. Amer. Practit., 11, 687. 1960 Tranquilizers, barbiturates and the brain. (Brochure). (pp. 1-20). 1960 Neurochernistry of aginp. A Handbook of Aging and the Individual Psychological and3iological Aspects. J. E. Birren, Editor. Chicago, University of Chicago Press (pp. 187-215) (with W. A. HIMWICH). 1960 Effect of therapeutic doses of psychotropic drugs on clinical symptomatology and urinary amines. Physiologis/, 3, 126 (with G . R. PSCHEIDT AND G. G . BRUNE). 1961 Carbohydrate metabolism in mental disease. Chemical Pathology of the Nervous System. J. Folch-Pi, Editor. New York, Pergamon Press. 1961 Study of the EEG convulsant activity of pentylenetetrazol injected arterially into restricted areas of the rabbit brain. .T. Neuropsychiat., 2 , 138 (with G. F. AYALAAND W. G. VANMETER). 1961 Effects of methaqualone, an experimental CNS depressant, on electrical potentials of rabbit brain. Trans. Amer. neurol. Ass., 192. (with D. A. BAYLOR). 1961 Biphasic action of reserpine and isocarboxazid on behavior and serotonin metabolism. Science, 133, 190 (with G. G. BRUNE). 1961 Effccts of reserpine and isocarboxazid on behavior of mental patients and some urinary products. Symposium on /he Biology of Schizophrenia. V. A. Hospital, Battle Creek, Mich.: March 16-17 (pp. 22-34) (with G. G. BRUNEAND G. R. PSCHEIDT). 1961 Discussion to the first symposium: Thc problem of antagonists to psychotropic drugs. Neuropsychopharmacolo,oy. Vol. 2. E. Rothlin, Editor. Amsterdam, Elsevier (pp. 32-33). 1961 Correlations between behavior and urinary indole amines during treatment with reserpine and isocarboxazid, separately and together. Neuro-Psychopharmacology.Vol. 2, E. Rothlin, Editor. Amsterdam, Elsevier (pp. 465474) (with G. G. BRUNE). 961 5-HT content of brain structures of dogs given 5-HTP 6hydroxytryptophan) compared with the degree of behavioral and neurological changes. Neuro-Psychopharlacology. Vol. 2. E. Rothlin, Editor. Amsterdam, Elsevier (pp. 475-478) (with E. COSTAAND W. A. HIMWICH). 96 1 Brain serotonin in relation to imipramine interaction with a monoamine oxidase inhibitor. Neuro-Psychopharmacology. Vol. 2. E. Rothlin, Editor. Amsterdam, Elsevier (pp. 485-489) (with A. HIMWCH AND E. COSTA). 961 A laboratory manual for general hospital psychiatry. Frontiers in General Hospital Psychiatry. L. Linn, Editor. New York, International Universities Press (pp. 449-474) (with E. E. HAYNES). 1961 Carbohydrate metabolism in mental disease. Part 1: Studies on oral glucose tolerance tests and some associated phenomena. Chemical Pathology of the Nervous System. J. Folch-Pi, Editor. New York, Pergamon Press (pp. 47&496). 1961 Clinical and basic analyses of the similarities and differences in the actions of tranquilizing and antidepressant drugs. Symposium on the BioIogy of Schizophrenia. V. A. Hospital, Battle Creek, Mich., March 16-17 (pp. 11-12). 1961 Drugs and the Kefauver Bill. Science, 134, 1560. 1961 Uniform response of biogenic amines to psychotropic drugs in selected schizophrenic patients. Fed. Proc., 20, 305c (March) (with G . R. PSCHEIDT AND G. G. BRUNE). 1961 Effects of hallucinogenic drugs in man: Introductory remarks. Fed. Proc., 20, 874. 1961 Book review of: Handbook of toxicology. Vol. IV. Tranquilizers. Psychosom. Med., 23, 90. 1961 Pavlovian conference on higher nervous activity. Discussion. Part IT. Ann. N . Y . Acad. Sci., 92,978. 1961 Psychopharmacological aspects of the nervous system. Neurochemistry. 2nd Ed. K.A.C. Elliott, T.H. Page and J. H. Quastel, Editors. Springfield, Ill., Thomas (pp. 766-789). 1961 Research and its application to gerontology. Medicine. Proceedings of Joint Conference Cook County Community Aging (pp. 34-43), 1961 Similarities and dissimilarities between some tranquilizers and antidepressants. Recent Advances in Biolonical Psychiatry. Vol. 3. Joseph Wortis, Editor. New York, Grune and Stratton (p. 77). 1961 Correlations between the behaviour of patients with mental disturbances and effects of psychoactive drugs on some urinary products. The Third World Congress of Psychiatry. 1, 111. Presented at Montreal, Canada, June 4-10, (with G.G. BRUNEAND G. R.PSCHEIDT),

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1961 Correlations between amine metabolism and activity of psychosis in schizophrenic patients. The Third World Congress of Psychiatry. 1, 124 (Abstract). Presented at Montreal, Canada, June 4-10 (with G. G. BRUNE). 1961 Behavioral, EEG and biochemical variations after the administration of alpha-methyltryptamine (IT 403). Recent Advances in Biological Psychiatry. Vol. 3. Joseph Wortis, Editors. New York, Grune and Stratton (pp. 166-177) (with W. G. VAN METER,E. COSTA AND G. F.AYALA). 1961 Failure of ethanol to lower brain stem concentration of biogenic amines. Quart. J. Stud. AND B. ISSEKUTZ, JR.). Alcohol, 22, 550 (with G. R. PSCHEIDT 1961 A pilot study of the effects of pathcole, a serotonin antimetabolite, on schizophrenic patients. Amer. J . Psychiat.,117,1121(with G. VASSILIOU,E. COSTA, G. BRUNE, C. MORPURCO, G . AYALA AND V. VASSILIOU). 1961 Psychological effects of isocarboxazid and nialamide on a group of depressed patients. J. clin. Psychol., 17, 319 (with V. VASSILIOU). 1962 Effects of methionine loading on the behavior of schizophrenic patients. J. new. ment. Dis., 134,447 (with G. G. BRUNE). 1962 Indole nietabolites in schizophrenic patients. Arch. gen. Psychiat., 6, 324 (with G. G. BRUNE). 1962 Relevance of drug-induced extrapyramidal reactions to behavioral changes during neuroleptic treatment. I. Treatment with trifluoperazine singly and in combination with trihexyphenidyl. Comprehens. Psychiat., 3,227 (with G. G. BRUNE,C. MORPURGO, A. BIELKUS, T. KOBAYASHI AND T. T. TOURLENTES). 1962 Relevance of drug-induced extrapyramidal reactions to behavioral changes during neuroleptic treatment. 11. Combined treatment with trifluoperazineamobarbital. Comprehens. Psychiat., 3,292 (with G. G. BRUNE,T. KOBAYASHI, c. BULLAND T. T. TOURLENTES). 1962 Discussion. Research Approaches to Psychiatric Problems. T. T. Tourlentes, S. L. Pollack and H. E. Himwich : Editors. New York, Grune and Stratton (pp. 69-73). 1962 Emotional aspects of mind: clinical and neurophysiological analyses. Theories of the Mind. Jordan Scher, Editor. New York, MacMillan (pp. 145-180). Finds 2 processes lead to progress. Drug Trade News (pp. 4-5). (Jan. 22 and 1962 H. E. HIMWICH: Feb. 5). 1962 Questions and answers. J . Amer. med. Ass., 179, 476 (Feb. 10). 1962 Some specific effects of psychoactive drugs. Specific and Non-specific Factors in Psychopharmacology. Max Rinker, Editor. New York, Philosophical Library (pp. 3-82). 1962 Research in medical aspects of aging. Geriatrics, 17, 89. 1962 The reticular activating system-current concepts of function. Psychosoniatic Medicine. John H. Nodine and John H. Moyer, Editors. Philadelphia, Lea and Febiger (pp. 211-220). 1962 Tranquilizers, barbiturates, and the brain. Modern Med., 109 (Feb. 19). 1962 Tranquilizers, barbiturates and the brain. J . Neuropsychiat., 3, 279. I962 Drugs affecting rhinencephalic structures. J . Neuropsychiat., Suppl. 1, 3, S15 (with A. MoRILLO AND w. G. STEINER). AND F. M. KNAPP). 1962 Antagonists of serotonin action. Fed. Proc., 21,336f (with W. A. HIMWICH 1962 An electrocorticographic study of changes in mouse brain with age. Life Sci., 7, 343 (with T. KOBAYASHI). 1962 Central cholinolytic action of chlorpromazine. Science, 136,873 (with W. G. STEINER). 1963 Alcoholism. Encyclopaedia Britannica. William Benton, U . S. A. (pp. 547-551). 1963 Reserpine, monoamine oxidase inhibitors and distribution of biogenic amines in monkey brain. Biochem. Pharmacol., 12, 65 (with G. R. PSCHEIDT).. 1963 The psychoactive drugs. The Psychological Basis of MedicalPractice. H. I. Lief, V. F. Lief and N. R. Lief, Editors. Hoeber Medical Division, New York, Harper and Row (pp. 531-544). 1963 Biogenic amines and behavior in schizophrenic patients. Recent Advances in Biological Psychiatry. Vol. 5. J. Wortis, Editor. New York, Plenum Press (pp. 144-160) (with G. G. BRUNE. 1963 A multidisciplinary study of changes in mouse brain with age. Recent Advances in Biological Psychiatry. Vol. 5. J. Wortis, Editor. New York, Plenum Press (pp. 293-308) (with T. 0.INMAN AND w. BUNO). KOBAYASHI, 1963 An EEG and behavioral analysis of the anticonvulsant action of amphenidone in the rabbit. Arch. int. Pharmacodyn., 142, 1 (with W. G. SEINER). 1963 An electrographic study of psilocin and 4-methyl-alpha-methyl tryptamine (MP-809). J. Pharmacol. exp. Ther., 140, 8 (with J. F. BRODEY AND W. G. STEINER).

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1963 Different responses of urinary tryptamine and of total catecholamines during treatment with reserpine and isocarboxazid in schizophrenic patients. Znf. J . Neuropharmacol., 2 , 17 (with G. G . BRUNE AND G. R. PSCHEIDT). 1963 Influence of methodology on electroencephalographic sleep and arousal: studies with reserpine and etryptamine in rabbits. Science, 141, 53 (with W. G. STEINEK A N D G. R. PSCHEIDT}. 1963 Relevance of the N , N-dimethyl configuration to the pharmacological action of chlorpromazine. Biochem. Pharmacol., 12, 679 (with G. G. BRUNE,H. H. KOHLAND W. G . STEINER). 1963 Chicken brain amines, with special reference to cerebellar norepinephrine. Life Sci., 7, 524 (with G. R. PSCHEIDT). 1963 Alpha-ethyltryptamine (etryptamine) :An electroencephalographic, behavioral and neurochemical analysis. Psychopharmacologia, 4,354 (with W. G. STEINER, G. R. PSCHEIDT AND E. COSTA). 1963 An electroencephalographic study of the blocking action of selected tranquilizers as a function of terminal methyl amine group. Biochem. Pharmacol., 12,687 (with W. G. STEINER). 1963 Participants in psychiatric problems in non-psychiatric practice. Illinois med. J., 124, 217. 1963 Effects of antidepressant drugs on limbic structures of rabbit. J . nerv. ment. Dis., 137, 277 (with W. G. SEINER). 1963 Alcohol and evoked potentials in the cat. Nature, 200, 1328 (with A. R. DRAVID, R. DIPERRI A N D A. MORILLO). 1963 Dimethylamine configuration in chlorpromazine. Fed. Proc., 23, 2438 (with H. KOHL,G . BRUNE AND W. G. STEINER). 1963 Review of 'A decade of alcoholism research'; a review of the research activities of the Alcoholism and Drug Addiction Rescarch Foundation of Ontario, 1951-1961. R. E. Popham and W. Schmidt, Editors. Brookside Monograph, 3, 64. pp. Toronto, University of Toronto Press (Reviewed in J. Amer. nied Ass., 183, 980.) I964 Review of Oufline ofPsychiatry. L. Cammar, Editor. New York, McGraw-Hill (p. 338) in: Amer. J. Psychiat,, 120, 718-719. 1964 A comparative study of chlorpromazine and its demethylatcd derivatives: potency and tissue distribution. Biochem. Pharmacol., 13, 539-541 (with H. H. KOHLAND G . G. BRUNE). 964 Endogenous metabolic factor in schizophrenic behavior. Science, 144, 311-31 3 (with H. H. BERLET, C. BULL,H. KOHL,K. MATSUMOTO, G . R. PSCHBIDI', J. SPAIDE, T. T. TOURLENTES AND J. M. VALVERDE). 964 Non-uniform response of urinary indole compounds to variations of tryptophan intakc. Fed. Proc., 23,279 (with H. H. BERLEI, H. KOHLAND J. SPAIDE). 964 A pharmacological study of terminal methyl groups in animals : (1) Electrophysiological analysis of chlorpromazine, imipramine, and amitriptyline, and their demethylated congeners: (2) Behavioral evaluations of chlorpromazine and its demethylated congeners in relation to brain concentrations of phenothiazines. Recent Advances in BiologicaL Psychiatry, Vol. 6. J. Wortis, Editor. New York, Plenuin Press (Ch. 18) (with G . G. BRUNE, W. G. STEINER AND H. H. KOHL). 1964 An electroencephalographic and chemical re-evaluation of the central action of reserpine in the rabbit. J . Pharmacol. exp. Ther., 144, 37-44 (with G. R. PSCHEIDT A N D W. G . STEINER). 1964 An electrographic study of bufotenin and 5-hydroxytryptophan, J . Pharniacol. exp. Ther., 144,253-259 (with A. K. SCHWEIGERDT). 1964 Neurohistological studies of developing mouse brain. Progress in Brain Research, Vol. 8, Biogenic Amines. H. E. Himwich and W. A. Himwich, Editors. New York-Amsterdam, Elsevier (pp. 87-88) (with T. KOBAYASHI, 0. R. INMAN A N D W. BUNO). 1964 Summary. Progress in Brain Research, Vol. 8 , Biogenic Amines. H. E. Himwich and W. A. Himwich, Editors. New York-Amsterdam, Elsevier (pp. 227-240). 1964 An electroencephalographic study of some structural aspects of D-amphetamine antagonism in phenothiazine and related compounds. In/. J . Neuropharmacof., 2, 327-335 (with W. G. STEINER AND K. BOST). 1964 Electroencephalographic changes following administration of N-dimethylacetamide and other antitumor agents to rabbits. I n t . J . Neuropharmacol., 3, 327-332 (with W. G. STEINER). 1964 Variations of urinary creatinine and its correlation to tryptamine excretion in schizophrenic patients. Nature, 203, 1198-1199 (with H. H. BERLBT, G. R. PSCHEIDT AND J. SPAIDE). 1964 Excretion of catecholamines and exacerbation of symptoms in schizophrenic patients. J . Psychiat. Res., 2, 163-168 (with G. R. PSCHEIDT, H. H. BERLET, C. BULLAND J. SPAIDE).

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Author Index * Abood, L. G., 41 Abranis, A., 257 Adey, W. R., 250 Adkins, F. J., 115 Adrian, E. D., 303 Agar, W. T., 195 Aud, R. B., 97 Ajmone-Marsan, C., 101, 103 Akawie, R. I., 193, 199 Akert, K., 204 Albers, R. W., 142 Alexander, I. E., 264 Allan, J. D., 185 Allen, M. L., 198 Alman, R. W., 291 Ambache, N., 256 Amin, D. H., 185,245 Anand, B. K., 279 Anderson, B., 281 Andrews, H. L., 317 Aprison, M. H., 48430,245 Arakawa, T., 185, 186 Arduini, A., 169, 170,243, 253 Arduini, M. G., 169, 170, 253 Armstrong, M. D., 188, 190, 199, 200,202,203 Aronson, H., 90 Arrigoni-Martelli, E., 122 Artemev, V. V., 242 Asatoor, A. M., 196 Auerbach, V. H., 184, 186, 187, 189, 197, 203 Awapara, J., 153 Axelrod, J., 81, 82, 92, 200, 206, 207 Axiotis, A., 42 Ayala, G. F., 41,179,219 Babskii, E. B., 242 Bachtold, H. P., 122 Bach-y-Rita, P., 173, 180 Bagchi, B. K., 97 Bain, J. A., 143-145,158 Baird, H., 190 Bakay, L., 117 Baldridge, R. C., 190 Bales, P. D., 49, 57, 156 Balfour, W. M., 216, 217, 221-225 Balzer, H., 146, 158, 193 Bard, L., 185

Barkulis, S. S., 146 Barnard, G. L., 115 Barnes, C. D., 177 Baron, D. N., 186 Barron, D. H., 273 Barthel, C. A., 98 Baruk, H., 260 Bastian, J. W., 130 Batsel, H. L., 103 Baxter, C. F., 141, 143, 145, 221, 225 Baylor, D., 252-254 Bayne-Jones, S., 258 Beall, B. D., 98 Beaulnes, A., 240 Behnke, R. H., 286 Belavady, B., 196 Bell, F. R., 250,254 BeneSovB, O., 175 Benitez, D., 147, 150 Bennett, D. R., 149, 154 Benoit, O., 173, 179 Bentley, G. A., 58 Berard, E., 97 Bercel, N. A., 288 Berger, H., 301 Bergmann, E. D., 154 Berino, J., 90 Berkowitz, E. C., 157 k r l , S., 138, 139 Berlet, H. H., 42,44, 184-215 Bernheimer, H., 245 Bernsohn, J., 90 Bertino, J., 90 Bertler, A., 245 Besendorf, H., 122 Bhide, N. K., 268 Bickel, H., 188 Bielkus, A., 42, 122 Bircher, R. P., 118 Birkmayer, W., 245 Bixby, E. M., 185 Bjornson, J., 187, 188 Blank, D., 267 Block, R. J., 188 Blume, L., 190 Boatman, J. B., 288 Bodforss, B., 289

* Italics indicate the pages on which the paper of the author in these proceedings is printed.

334

AUTHOR INDEX

Bogdanski, 1).F., 63,74, 78,90, 185,202, 203, 245 Boggs, D. E., 190,195,196 Bogosch, S., 205 Boguniill, G. P., 285 Bohdanecki, Z., 175 Bolin, R. R., 122 Bolis, M. E., 197, 199 Bolling, D., 188 Bonamini, F., 116 Bonvallet, M., 179 Borden, M., 185 Borek, E., 188,194 Borkowski, W. J., 115 Boroff, D. A ., 256-262 Borofsky, L., 190 Borzelleca, J. F., 168 Boscott,R. J., 188,190 Bosquet, W. F., 268 BoszGrmCnyi, Z., 87 Bothe, A. E., 273 Bouisset, L., 257 Boulding, J. E., 205 Bouzarth, W. F., 49 Bovet, D., 156 Boyajy, L. D., 173,175 Boylen, J. B., 199 Bradley, P.B., 175, 177, 178, 241, 250 Brailowski, V. V., 242 Brecher, A., 188 Bremer, F., 173, 240 Brenchley, Y . , I96 Brenner, G., 115 Breuilland, J., 257 Bridges, T. J., 286 Brigham, M. P., 185, 189, 197 Brodie, B. B., 78,86,90, 164, 167, 185,245, 248 Brown, C. R., 29 Brown, D. D., 207 Brucke, F. Th., 177 Brune, G. G., 4, 40-43, 81--96, 122, 185, 187 Bryan, L., 122 Brygoo, E. R., 257 Buchel, L., 124 Buehler, H., 257 Buffa,D., 147 Bull, C., 39-47, 185 Bullock, D., 190 Bullock, T. H., 57 Bumpus, F. M., 88 Bunnell, S . , 5 Burch, G . E., 286 BureS, J., 113 Burknian, A. M., 128 Burn, J. H., 128 Busse, E. W., 302 Cadilhac, J., 242

Cairney, J., 219 Calhoun, H. D., 97 Calma, I., 112, 279 Canhani, R. G., 90,286 Cardon, W. P. V., 4,90,92, 185 Carlton, P. L., 128, 132 Carlton, R. A,, 173, 174 Carter, S. H., 136, 137, 146, 151, 153, 157, 1.58 Carver, M. J., 197 CdWte, J. E., 198 Cerletti, A,, 124 Chaillet, F., 179 Charles, M. S., 302, 303 Chatfield, P. O., 115 ChBvez-Ibarra, G., 240 Cheng, S. C., 225 Chenoweth, M. B., 149, 157 Childs, B., 185 Chirigos, M. A., 142, 193, 194 Christensen, H. N., 195 Claman, M. A., 185 Clark, K., 286 Clark, M. E., 294 Clark, W. G., 193, 199 Clarke, D. D., 138, 139 Closs, K., 188 Connamacher, R. H., 90 Coppock, H. W., 277 Conway, E. J., 219 Cook, L,127 Cooke, P. M,, 115 Cooke, R. E., 185 Cooper, J. R., 191 Cordeau, J. P., 240 Cortell. R., 57 Costa, E.,41,42,62,63,67,77,78,90,185,24.5, 286, 287 Courjon, J., 173, 179 Courvoisier, S., 123, 129, 130 Cragg, B. G., 37 Craig, W. M., 285 Craske, J., 196 Crawford, T. B. B., 185 Creveling, C. R., 202 Cronin, M. A,, 139, 143, 145 Cronquist, S., 289 Cusworth, D. C., 185 Dahl, D. R., 216,217, 221-226 Dahl, N. A., 216,221-224 Daigneault, E. A., 178 Dancis, J., 197, 199 Da Vanzo, J. P., 139, 141, 145 Davidson, E. A., 92 Davison, A. N., 193 Dawson, R. M. C., 147, 1.50, 155 De Balbian Verster, F., 57 De Duve, C., 223

AUTHOR INDEX

De Jonp, H., 260 DeLaTorre, E., 286 Dell, P., 179 De Maar, E. W. J., 169 Dempsey, E. W., 115 Denison, A. B., Jr., 286 Dennis, B. J., 250,254 Denniston, J. C., 198 Denny-Brown, D., 285 Dent, C. E., 185, 186, 198 De Ropp, R. S., 145, 155 Desmedt, J. E., 173, 178, 179 Deutsch, J. A., 37 Dews, P. R., 61,65, 125 Dickson, E. C., 256 DiCeorge, A. M., 185, 189, 197 Dikshit, B. B., 115 Di Perry, R., 250-255 Di Stefano, A. O., 44, 88 Dixon, M., 56 Dobbing, J., 117, 187, 220 Dobbs, J. M., 185, 189, 197 Dolce, G., 116 Doniae, A., 115-117 Domer, F. R., 131 Dominguez, A . M., 149 Domino, E. F., 303 Donahue, P. D., 304 Doshay, L. J., 122 Dow, R. S.: 301 Drabkin, D. L., 273 Drakontides, A. B., 248 Draper, W. B., 302 Dravid, A. R., 250--255 Drewes, P. A., 149 Drill, V. A., 116 Driscoll, K. W., 205 Dry, I.., 266, 269, 270 Dua, S., 279 Ducrot, R., 123, 130 Dusser de Barenne, J. G., 112, 114 Dutch, S . J., 197 Dutcher, T. F., 167 Eades, C . G., 240 Edelman, I. S . , 219 Efron, M. L., 185 Ehrlich, M., 195-197, 205 Eidelberg, E., 143, 145, 155 Eiduson, S., 193 Elkes, J., 175, 177, 178, 241, 250 Elliott, K. A. C . , 137,138,172 Ellison, Th., 202 Emmers, R., 266 Epstein, A. N., 280 Erickson, R. W., 57, 202 Erspamer, V., 82, 184 Essig, C . F., 49, 50, 57, 115, 156, 173, 177, 278

Euland, H. W., 257 Evarts, E. V., 250 Evered, D. F., 186 Everett, J. W., 230

Fabing, H. D., 87 Fabish, W., 98 Falk, J. L., 264 Fang, H. C . , 285 Faurbye, A., 121 Fazekas, J. F., 290, 291 FaLio, C., 113 Feldberg, W., 106, 112-117 Feldman, B. H., 195 Feldman, J., 57 Fellman, J. H., 193, 199 Fellows, E. J., 90, 124 Fenn, W. O., 112 Ferris, E. B., Jr., 286 Ferster, C . B., 61, 63, 66, 69-71, 73 Finesinger, J. E., 57, 286 Finger, K. F., 247,248 Fink, M., 98 Fischer, E., 44 Fischer, H. F., 151 Fischer-Williams, M., 97 Fitch, R. H., 273 Fitzpatrick, T. B., 186, 199 Flanary, H. G., 278 Fleischhauer, K., 112-114, 117 Flesner, A. M., 122 Floding, I.. 82 Fog, M., 291 Folkerth, T. L., 74 Folling, A., 188 Forman, D., 176 Forrer, G . R., 172, 178, 241 Forster, F. M., 115 Fournel, J., 130 Franke, F. R., 288 Franken, L., 179 Frankova, S., 6 Fraser, H. F., 278 Freedland, R. A., 192 Freedman, J. E., 49, 57 Frei, F., 101, 103 French, J. D., 171, 253 Freyganp, W. H., Jr., 286 Freyhan, F. A., 122 Friedhoff, A. J., 3, 206 Friedlander, W. G., 98 Friinpter, G. W., 185, 186 Frommer, G. P., 172 Fukuhara, T., 115-117 Fuller, J. L., 302, 303 Fulton, J. F., 176 Funderburk, W. H., 248

335

336

A U T H O R INDEX

Gaddum, J. H., 3,62,116, 185,245 Gal, E. M., 149, 191, 192,201 Gammes, T., 188 Gangloff, H., 179 Garello, L., 116 Gastaut, H., 103 Gebhard, 1. W., 29 Geiger, A., 146, 216,286 Geller, E., 193 Gellhorn, E., 57 Cendon, U. Z., 257 Georgii, A., 198 Gerrard, J., 202 Gerritsen, T., 185 Gershoff, S. N., 142 Gessa, G. L., 78 Ghadimi, H., 185 Giarman, N. J., 79, 172, 178, 192, 193, 195 Cibbs, E. L., 285 Gibbs, F. A., 285 Gibson, W. C., 5, 15, 187 Gillespie, L., Jr., 85 Gitter, S., 149 Glotzer, J., 41, 42 Goldensohn, E. S., 302 Goldman, D., 122 Goldktein, L., 175, 178, 179 Goldstein, S. G., 90 Goldstein, S. L., 286 Coodall, McC., 207 Goodman, A., 90 Gopalin, C., 196 Gosswald, R., 123 Govier, W. M., 128 Graff, R. A., 99 Graul, E. H., 195, 196 Green, A. A., 184 Green, H., 200,286,287 Green, J. D., 230 Green, R. H., 150 Greenberg, S. M., 202 Greengard, P., 142, 193, 194,219, 225 Greer, M. A., 281 Greig, M. E., 139, 143, 145 Grey-Walter, W., 29 Grizov, V., 257 Groff, S., 4 Gruber, L. M., 273 Grundfest, H., 79, 179 Gurdjian, E. S., 151 Curoff, G., 193, 194, 197, 198 Guyton, A . C., 256,286,291 Haase, H. J., 122 Haavaldsen, R., 142 Hable, K., 204 Haley, T. J., 116, 122 Hall, R. A., 121

Hambrecht, F. T., 297--?00, 304 Hampson, J. L., 49,50,57,156,173,177 Hance, A. J., 173, 178, 180 Hanrahan, G. E., 97 Hansen, S . , 191 Hanusek, G . E., 286 Hardy, E., 198 Harley-Mason, J., 2, 11 Harlow, H., 189, 204 Harmel, M. H., 286 Harrington, H., I93 Harris, H., 186 Hart, E. R., 78 Hart, E. W., 186 Hartman, W. J., 193, 199 Hasegawa, K., 97 Hassler, R., 176, 177 Hastings, A. B., 195 Haverback, B. J., 167 Hawkins, J. R., 87 Haymowitz, A., 185 Headlee, C. P., 277 Hearst, E., 132 Hedlund, S., 286 Henderson, W. R., 115 Hernindez-Peon, R., 240 Hersov, L. A., 186 Herz, A., 178 Herzet, J. P., 179 Hess, S . M., 85, 90 Hestrin, M., 191 Heyer, A. W., Jr., 266 Hickmans, E. M., 202 Hiebel, G., 178, 179 Hift, H., 149 Himwich, H. E., 4, 40-44, 49, 50-52, 57, 58, 82-91, 105, 122, 156, 157, 172-180, 185, 198, 216, 217, 221, 229, 238. 241, 245-249 Hirnwich, W. A., 78,90, 185, 198,286,287, 291, 297,301-317 Hird, F. J. R., 195 Hirsch, C., 78 Hobbs,R., 128 Hodgkin, A. L., 220 Hoedt-Rasmussen, K., 289 Hoffer, A., 204, 205 Hollister, L,. E., 98 Holmquist, B., 173 Holt, L. E., 199 Holtz, P., 146, 158 Hopper, S.,142 Harlein, U., 123 Hornlkiericz, O., 245 Horvath, N., 146 Horwith, M., 185, 186 Hottle, G. A., 257 Howell, S. R., 285 Hsia, D., 190, 193, 196, 198, 205

AUTHOR I N D E X

Hsu, K., 286 Huang, I., 193 Hube, P., 179 Hundeshagen, H., 195, 196 Hunsicker, A. L., 42 Hunter, A., 185 Hurd, D., 41 Hyman, C., 288 Ingvar, D. H., 173 Inman, 0. R., 15, 24, 30, 286 Inskip, W. M., 90 Isbell, H., 3, 278 Ishida, N., 206 Ishikawa, S., 286 [to, G., 301 Itoh, C., 206 Ttoh, H., 185, 186 Jackson, R. B., 121 Jacobs, R. J., 222 Jacobson, K. B., 155, 208 Jageneau, A. H. M., 128,129 Jakoby, W. B., 142 Janssen, P. A. J., 128, 129 Jarvik, M. E., 177 Jasper, H. H., 302 Jenkner, F. L., 124 Jennev, E. H., 122 Jepson, J. B., 186, 20-3 Jerris, G. A., 187 Jervis, G. A., 188 Jewel], P. A,, 294 Johannesson, Th., 175 John, E. R., 113 Johnson, J. V. V., 290 Johnson, L. B., 149, 154 Johnson, L. C., 103 Jones, M. R., 264,273 Jorgensen, R. S., 98 Jouvet, M., 173, 179 Julou, L., 123 Jung, R., 176, 177 KBgstrom, E., 286 Kaji, H. K., 145 Kakimoto, Y., 3,200, 206 Kanai, T., 118 Kandel, A., 149, 154, 157 Kaneko, Z., 3,200,206 Kanze, E., 188 Kappy, M., 190,195, 196 Kato, H., 115-117 Kaufman, S., 201,202 Kawakita, Y., 146 Kawashima, Y., 117 Kegeles, G., 257 Kellaway, P., 142

337

Kendricks, T. R., 288 Kennard, M. A., 265 Kety, S. S., 4, 90, 92, 185, 216, 280, 286, 288 Key, B. J., 254 Keynes, R. D., 217 Keyser, G. F., 273 Killam, E. K., 177, 179 Killam, K. F., 144, 145, 171, 179 Kind, W., 82-84 King, E. V., 169 King, J. B., 98 King, W., 193 Kirschner, N., 207 Kitamura, K., 301 Klee, G. D., 90 Klein, J. R., 151 Kline, N. S., 167 Kliiver, H., 37 Knapp, F. M., 185,285-296,301-317 Knauff, H. G., 194, 198 Knell, J., 193 Knox, W. E., 197,204, 205 Kobayashi, T., 42, 106-120, 115-1 17, 122 Koegler, S. J., 185, 197 Koella, W. P., 6 Koetschet, P., 130 Kohl, H., 44, 82, 86, 87, 91, 185 Kolsky, M., I30 Konigsmark, B., 179 Kooi, K. A., 97 Koster, R., 128 Koval, G. J., 142 Kramer, M., 122 Kreindler, A., 113 Kreiskott, H., 123 Kuhn, R. A., 286 Kumagai, H., 115-117 Kuntzman, R., 78, 167, 245 Kurbjuweit, H. G., 124 Kyle, W., 285 La Du, €3. N., 186 Lagravere, T. A. F., 44, 88 La Grutta, G., 173 Lahiri, S., 150 Laidlaw, A. E., 265 Laird, A., 11 Lairy-Bounes, G. C., 243 Lajtha, A,, 187, 194 Lamanna, C., 257 Landau, W. M., 286 Langford, H. G., 190 Lasagna, L., 128 Lassen, N. A., 286, 289 Last, S. L., 97 Lauer, J. W., 90 Lauppi, E., 122 Laurin, C., 240

338

AUTIIOR INDEX

Laursen, A. M., 176, 177 Lausen, H. H., 175 Lavy, S., 286 Lehman, H. E., 97 Lerner, A. B., 200,207 Lessin, A. W., 130 Lester, 1. H., 268 I.evin, B., 185 Levin, E. Y . , 201, 202 Levitt, M., 201 Levitz, M., 185, 197 Levy, C. K., 6 LCvy, J., 124 Lewandowsky, M., 114 Lifson, N., 151 Lim, R. K. S., 1 IS, 307 Lindgren, P., 286 Linnewch, F., 194-197, 205 Lishajko, F., 82 Litteral, E. B., 98 Liu, C . , 307 Livingston, R. R.. 172, 251 Loeb, C . , 169, 240, 253 Lolley, R. N., 218 London, D. R., 196 Longo, V. G., 156, 169-172, 177, 178, 240 Louttit, R. T., 185, 189, 203, 204 Lovc, W. D., 286 Lovell, R. A,, 137, 138, I55 Loventrerg, W., 202 Lowenthal, J. P., 257 Lucas, J., 90 Luria, R. N., 303 Lyberi, G., 97 Mabry, C. C., 198 Machnc, X., 172, 178, 279 Machowicz, P., 287 MacLean, P. D., 77, 78, 177 Macri, B. P., 259 Magni, F., 169, 253 Mapoun, H. W.. 121, 253, 279 Mahler, H. R., 149 Mahyuddin, F., 185, 197 Maling, H. M., 83 Manthei, It. W., 268 Marinesco, G . , 113 Marrazzi, A. S., 78, 180 Marsallon, A,, 173, 179 Marshall, F. D., 191, 192, 201 Marshall, V. F., I86 Martin, W. R , 169, 240 Massieu, G. H . , 145 Masuoka. D. T., 201 Matsumoto, K., 44, 185 Matveev, K . I., 256 Matzen, K., 198 Maxwell, H., 285

Maynert, E. W., 145 McCann, S. M., 281 McCann, W. P., 128 McCarter, R. H., 1 I 5 McCauley, A., 49, 173, 177 McDermott, E. E., 142 McDonald, D. A., 56 McDonald, M. A., 256,286 McElroy, 0. E., 257 McGeer, E. G., 187, 201, 205 McGeer, P. L., 187, 205, 206 McGregcr, L. L., 151 McIlwain, H., 219, 222, 225 Mclsadk, W. M., 4 McKean, C. M., 192-195 McKhann, G. M., 139, 150 McLaurin, R. L., 285 McLay, D., 190, 195, 196 McLennan, H., 172 McMillan, A., 200 McMillan, P. J . , 139 McMurray, W. C., 197 McNair, F. E., 187 Mead, J. A. R.,245 Medes, J., 186, 200 Melccr, J., 191 Melzack, R., 304 Merrilt, H. H., 115 Mrschan, I., 286 Messinper, E. C.; 205 Mettler, C. C . , 176 Mettler, F. A., 57, 176 Meycr, J. S., 285 Mejers, F. H., 177 Michaelis, M., 57 Milholland, R. J., 145 Miller, F. R., 242 Miller, N. E., 266 Miller, S., 185, 197 Milne, M. D., 196 Minard, D., 285 Miner, E. J., 3 Minvielle, J., 242 Minz, B., 179 Mitchell, E N., 147, 156 Mitoma, C., 81, 202, 203 Miura, Y.. 117 Mjya, T. S., 268 Miyamoto, M., 199 Moak, S. J., 265, 266 Moffitt, R. L., 307 Moir, W. M., 273 Montemurro, D. C., 281 Moore, S., 135, 141 Moreau, A,, 240 Morgane, P. J., 240, 281 Morillo, A ., 250-255 Morpurgo, C., 41, 121-134, 245, 247, 248

AUTHOR INDEX

Mortensen, R. A,, 139 Moses, C., 288 Motokawa, K., 301, 302 Munck, O., 286 Mufioz, C., 175, 178 Murakami, M., 117 Murphree, H. B., 122 Nachmansohn, D., 57 Nadler, H. L., 190, 198 Nagatsu, T., 201 Nash, C. B., 172, 176-178 Nathan, D. G., 155 Nathan, P., 51, 52, 57-59 Needhani, D. M., 56 Neill, K. C., 190 Nelson, T. L., 198 Netsky, M. G., 286 Newquist, R. E., 285 Nichol, C. A., 145 Nicholas, J. S., 273 Nichols, J. B.. Jr., 285 Nichols, I. R., 277 Niemegeer, C. J. C., 128, 129 Nirenberg, P. Z., 202 Nishimura, K., 196 Northfield, D. W. C., 97 Norton, P., 199 Nyhan, W. L., 185 Nylin, G., 286 Oates, J. A., 82, 202 Oberholzer, V. G., 185 Oghawara, S., 117 O’Kelly, L. I.. 264-267 Okumura, N., 155 Oldendorf, W. H., 286, 289 Oliner, L., 286 Olsen, N. S., 151 O’Meallie, L. P., 286 Ortega, B. G., 145 Osmond, H., 2 . 205 Osserman, E. F., 285 Ostfeld, A. M., 172, 178 Otsuka, Y., 115 Owen?, H. F., 156, 157 Paasonen, M. K., 78 Pace, J., 142 Page, I. H., 88, 184, 185 Palattao, L. G., 185 Palm, D., 146, 158 Palmer, G., 189, 203 Palmer, J. J., 285 Papez, J. W., 77 Pare, C. M. B., 190, 192, 204 Parkes, M. W., 130

339

Partington, M. W., 185 Pa?ka, R., 197 Pasner, H. S., 202, 203 Passouant, P., 242 Pattison, F. I.. M., 151 Pellmont, B., 122 Pepeu, G., 172, 178 Peretz, D., 5 Perot, P. L., Jr., 285 Perry, T. L., 191, 206 Peters, E. L., 142 Peters, R. A,, 147, 140, 151 Petersen, J. C., 197 Peterson, R. E., 82 Petriello, L., 201 Pfeiffer, C. C . , 122 Pletscher, A., 164, 167, 185 Poczik, M., 191, 192, 201 Pollack, S. L., 97-105 Pollin, W., 4, 90, 92, 185 Ponirovski, N. G., 242 Posner, H. S , 202, 203 Possanza, G. J., 172, 177, 178 Potter, J. M., 56, 291 Potter, V. R., 149, 286 Poulos, G. J., 71 Prawdicz-Neminsky, W. W., 301 Prevot, A. R.,257 Prockop, D., 86 Profimov, L. G., 303 Proler, M., 142 Pryles, C. V., 185 Pscheidt, G. R., 40, 44, 78, 82-84, 86, 90, 92, 147, 150, 185, 245-249 Pudenz, R. H., 285 Purkis, V. A , 15 Putnam, F. W., 257, 286 Quastel, J. H., 150, 199 Rabinovitch, R. D., 90 Ramold, M. C., 156 Ramwell, P. W., 268 Randall, A. H., 122 Rapela, C. E., 287 Raper, H. S., 200 Rapport, M. M., 184 Rathbeen, J. C., 197 Ravdin, I. S., 273 Raymond, K., 122 Raynaud, M., 257, 258 Redfield, B. G., 84 Regnstrom, O., 286 Reichle, F., 190 Reimanis, G., 267 Renson, J., 192 Restarski, J., 285 Richter, D., 155

340

AUTHOR INDEX

Riley, H., 130 Rinaldi, F., 41, 63, 78, 156, 172, 173, 175, 178180,229-244,245 Risse, KI. H., 123 Roa,P. D., 136, 138, 140, 146, 153 Roberts, E., 138, 140, 143, 145, 225 Robertson, M. G., 122 Robinson, K. S., 188, 190 Rodnight, R., 186 Rosen, F., 145 Rosenberg, R., 193, 195 Rosenblum, W. I., 291 Rosenkilde, H., 128 Rossi, G. F., 169, 253 Rothballer, A. B., 177-179 Rothenberg, M. A,, 57 Rowland, L. P., 286 Rudy, L. H., 41, 42 Rupe, B. P., 268 Russell, A., 185 Sacchi, U., 113, 116 Sagawa, K., 286 Sager, O., 113 Sakami, W., 193 Sakhiuline, G. T., 303 Sakuma, A., 115-117 Samiy, A. H., 195 Samson, F. E., Jr., 173,216-228 Sandler, M., 190, 192, 193, 204 Sano, I., 3, 200,206 Sapirstein, L. A,, 286 Sargent, T., 5 Sato, T., 206 Sawyer, C. H., 157,230 Sawyer, J. L., 202 Schabert, P., 194 Schadb, J. P.,22 1, 225 Schafer, I. A., 185 Schallek, W., 302, 316 Schanberg, S. M., 192-1 95 Schantz, E., 257 Schaumann, W., 124 Scheinberg, P., 286 Schlag, J., 178, 179 Schmidt, C. F., 216, 286 Schmidt, H., Jr., 263-284 Schneider, J. A., 248 Schneider, P., 285 Schott, H. E., 201 Schramm, H., 129 Schroeder, W. A., 206 Schubel, K., 256 Schueler, F. W., 132 Schumann, H. J., 82 Scriver, C.R., 185 Second, L., 251, 258 Seevers, M. H., 268

Segal, H. L., 142 Segundo, J. P., 254 Selving, B. T., 97 Sen, N. P., 206 Scttlage, P. H., 285 Shagass, C., 97 Shalit, M. N., 286 Sharp, D. G., 257 Shaw, C. R., 90, 185 Shaw, E., 3,62,78,204 Shaw, F. H., 58 Shaw, K. N. F., 188,200 Shelden. C. H., 285 Shenkin, 11. A,, 286 Sherlock, S., 92 Sherwood, S. L., 106, 113, 115, 116 Shevky, E., 256 Shideman, F. E., 149 Shnider, B. I., 101, 103 Shore, P. A., 78, 86, 164, 167, 245, 247 Shulgin, A. T., 5 Sidhu, G. S., 195 Sidman, M., 127 Siegel, P. S., 264 Silver, S. L., 164 Silverman, D., 99 Silvestrini, B., 169-172, 178 Sinclair, L., 185 Sines, .I. O., 103 Sjoerdsma, A., 82-85, 90. 202 Skinhoj, E., 289 Skinner, B. F., 61 Small, J. G., 97 Smythies, J. R., 1-38, 205 Snedeker, E. H., 145, 155 Snyderman, S. E., 199 Sokabe, H., 115-117 Sokoloff, L., 286, 291 Son, C. D., 198 Sonnenschein, R. P., 285 Sorensen, S. C., 289 Sourkes, T. L., I87 Spaide, J., 44, 185 Spencer, J. N., 302 Spencer, R. P., 195 Spector, S., 78, 85, 86,247 Spinks, A., 130 Spoerlein, M. T., 201 Sponholz, R. R., 189 Sprince, H., 187 Srikantia, S. G., 196 Stacey, R. S., 190, 192,204 Stanbury, J. B., 186 Stanton, E. J., 273 Stawraki, G. W., 242 Stead, E. A . , Jr., 286 Stein, S. N., 285 Stein, W. H., 135, 141

AUTHOR INDEX

Steiner, F. A., 122 Steiner. R. E., 92 Steiner, S. H., 286 Steiner, W. G., 82,86,91, Y7-105, 174, 175, 179, 301-317 Stern, J. A., 103 Stern,L.,113 Sterne, M., 257 Stevens, J. R., 177 Stevenson, J. A. F., 281 Steward, L. F., 98 Stewart, W. C., 57 Stone, W. E., 135-163 Stoupel, N., 240 Straub. R. W., 225 Stumpf, Ch., 177 Sturtevant,F. M., 116 Suh, T. H., 115 Sulser, F., 90 Summerskill, W. H. J., 96 Sundstein, J. W., 157 Sutton, H. E., 90 Swain, J. M., 98,121 Swank, R. L., 302-304 Sykes, E., 11 Symon, L.,286 Szara, St., 87 Szatmari, A., 97 Tada,K., 186 Taeschler, M., 124 Takagaki, G., 138,139 Takesada, M., 3,200,206 Tallan, H. H., 154 Tamura, H., 115-117 Taniguchi, K., 203 Tannenbaum, S.,190 Tapia, R., 145 Tatum, A. L., 268 Taylor, N. F., 149 Tedeschi, D. H., 127 Tedeschi, R. E., 90,127 Teitlebaum, P., 263 Temperley, H. N. V., 37 Terry, L. L., 167 Tews, J. K., 135-163 Theobald, W., 123, 124, 131 Ther, L., 129 Thomaq, A., 290 Thompson, J. H., 205 Timo-Iaria, C., 240 Tischler, B., 191 Tissier, M., 124, 130 Titus, E., 82, 164 Toman, J. E. P., 121 Tomchick, R., 200,207 Tomich, E. G., 167 Tornell, G., 286

341

Toth, J., 194 Tourfentes, T. T., 42,44, 122, 185 Tower, D. B., 135, 139 Towler, M. L., 98 Troll, W., 205 TschLgi, R. D., 113 Tunturi, A., 301 Tuohy, J. H., 101, 103 Turner, M., 97 Tuteur, H. E., 41 Twarog, B. M., 185 Tyler, F. H., 199, 202 Udenfriend, S., 63, 67, 78, 81-85, 90, 142, 164, 175, 192-194, 196, 198, 201-203, 245 Ueki, S., 303 Uhrbrand, L., 121 Ulett, G. A., 103, 301 Unna, K. R., 124, 169, 172, 178 Utley, J. D., 145 Valcourt, A. J., 40, 164-168 Valverde, J. M., 44, 185 Van der Hoeven, T., 204 Van der Schoot, J. R., 202 Van der Wende, C., 201 Van Gelder, N. M., 137, 138 Van Meter, W. G., 78, 90, 157, 179 Van Winkle, E., 3, 206 Vasquez, A. J., 44,88 Vassiliou, G., 41 Vassiliou, V., 41,42 Vaughn, J. G., 185 Velluti, R., 240 Verhave, T., 61 Verney, E. R., 294 Vernier, V. G., 124, 172, 178 Verzeano, M., 253 Vincent, H., 260 Vitek, V., 6 Vogt, M., 116,245 Vojtechovsky, M., 6 Von Berger, G. P., 156 Von Euler, U. S., 82, 115,245, 247 Votava, Z . , 175 Wada, Y.,185, 186, 206 Wadzinski, I. M., 192 Waelsch, H., 138, 139, 154, 188,225 Wahbe, V. G., 223 Waisman, H. A., 185, 189, 190, 192, 195, 196, 203,204 Walaszek, E. J., 112, 113, 115 Walker, R. W., 285 Wallach, D. P., 140, 146, 158 Walz, D., 302, 316 Wang, C. H., 115 Wang, H. L., 189,204

342

A U T H O R INDEX

Wang, S. C., I18 Ward, A., 124 Ward, J. W., 176 Warren, K. S . , 155 Watson, C. W., 302-304 Wayner, M. I., Jr., 266,267 Webster, J. E., 151 Weidley, E., 127 Wcidman, H., 124 Weil-Malherbe, H., 150, 190, 198 Weinberg, S. J., 116 Weiss, H. H., 264-267 Weissbach, H., 78, 81, 82, 90, 164, 192 Wells, c. E., 101, 103 Wenglarz, R., 294 Wentzel, I,. M., 257 Wenzel, B. M., 113 Westall, R. G., 185, 197 Westerbeke, E. J., 172, 173, 175, 177, 178 Westermann, E., 193 Wetrus, B., 267 Wherrett, 3. R., 139, 150 White, R. P., 169-183 Whitehead, R. W., 302 Whittaker, V. P., 223 Wiechert, P., 194 Wikler, A., 103, 171, 172, 241 Williams, D. R., 263 Williams, H. L., 143, 158, 263 Willis, A., 49, 156 Wilson, T. H., 187

Wilson, V. K . , 185 Wilson, W. C., 115 Windsheinier, F., 198 Winnick, R. E., 154 Winnick, T., 154 Winters, W. D., 173, 180 Winterstein, H., I12 Wirth, W., 123 Wittson, C. L., 197 Wolbach, A. R., Jr., 3 Wolf, A. V., 263 Wolf, M. A., 74 Woolf, L. I., 188 Woolley, D. W., 3, 62, 78. 164, 185, 204 Woonton, G . A., 242 Wright, S., 112 Wulff, M. H., 98 Wqckoff, L. B., 203 Yahr, M. D., 286 Yamamoto, S., 117 Yoshinaga, K., 206 Young, W. K., 290 Yuwiler, A., 185, 189, 193, 203, 204 Zaltzman, P., 82, 202 Zeller, E. A., 90, 188 Zickgraf, H., 194 Zier, A., 122 Zinsser, H., 258 Zweifach, B. W., 291

343

Subject Index Acetylcholine, brain level, and physostigniine, 172 Acetylchohe chloride, behavior changes, 110, 115, 116 Acetylcholinesterase activity, functional levels, 56-61 inhibition by. DFP, 49-61 Adrenergic agents, EEG activation, 175, 179 reticular formation, 177 Adversive syndrome, studies, 49-61 Amino acid, blood-brain barrier, function, 198 blood-brain exchange, competitive inhibition, 193-195 brain uptake, interference, 193 metabolism, and cerebral dysfunction, 184-209 in schizophrenia, 205-208 Amino acidopathy, amino acid nitrogen, homeostatic control, 198 classification, 186 and mental retardation, 184-187 Amino-oxyacetic acid, effect on cerebral constituents, 139-142 Ammonium chloride, infusion, effect on cerebral constituents, 136, 138, 139 Amygdala, pattern of bouton distribution, 18, 20, 21, 23 Anoxia, effect on cerebral constituents, 137 Anticholinergic drugs, and behavior, 178 Anxiety, degree, and excretion of catecholamines, 86 severity, degress>82 ATP, cerebial concentration, and anoxia, 218 and brain energy flow, 21 7-226 and hypothermia, 224 and sodium iodoacetate, 217-225 ATP, nerve fibers activity, and energy cost, 225 Atropine, convulsive discharges, and ieticular stimulation, 242, 243 and EEG rhythm, 170, 171, 175, 176, 179 effect on cerebral cortex, and contralateral desynchronization, 241, 242

and homeostatir regulation, 242 spontaneous electrical activity, 229-243 and reticular formation, EEG activation, 173 synchronized activity, and retici!lar stimulation, 210,241 topical application, convulsant effect, 242,243 Barbiturates, dose-drinking response curves, 265-269, 275277 doublc, action, theoretical scheme, 278-280 drinking threshold, and response latency, 266-269, 275 long term effects, and dosage, 272 and preference tests, 270-272,278 repeated treatment, and sensitization, 274, 277 reticular formation, evoked potentials 252-254 saline acceptability, effect on, 270-272, 278 tolerance, studies, 272-274,277 water ingestion, sites of action, 280, 281 variations, 263-282 withdrawal symptoms, 272,273,276 Behavior, anticholinergic agents, effects, 178 changes , and EEG rhbthm, telemetry, 313, 314 and experimental phen) Iketonuria, 203,204 after IAT of acetylcholine chloride, 110, 115, 116 aftei TA1 of chlorpromazine hydrochloride, 111, 116, 117 after IAI of L-epinephrine bitartrate. 110, 114, 115 after IAI of potassium chloride, 108, 112, 113 after IAT of serotonin creatinine sulfate, 110, 111, 116 after I A I of strychnine sulfate, 109, 114 after IAI of D-tubocurarine chloride, 109, 110, 113, 114 cholinergic agents, effects, 178 criteria, 49 and chemical techniques, 49 disturbances, and indole metabolites, 40-45 drug-induced changes,

344

SUBJECT INDEX

biochemical interpretation, 89-92 and EEG pattern, effect of drugs, 177 and isocarboxazid and methyl donors, effects of combined administration, 92-94 and MAOI, effect of treatment, 92-94 and niethionine, effect of treatment, 92-94 studies, fixed-interval component (FI), 64-67 fixed-ratio per fcrmance (FR), 6 4 6 7 and tryptamiiie excretion, 40 Biogenic amines, and degree of psychotic activity, 82.83 excretion patterns, and psychotic activity, 83-85 metabolism, and brain function, 90 and psychotic agents, 81 paper chromatography, 87-89 Boutons, distribution, 16-20 niorphology, 23-27 qualification, 15, 16 quantification, 16 types in human brain, 15 Brain, acetylcholine level, and physostigniine, 172 biogenic aniines, and tryptophan, 43 chicken, hiogenic arnines, content, 245-248 norepinephrine level, 245-248 serotonin level, 245-248 circuiation, heniodynamic aspects, 285-294 electrical activity, excitation level, 302 and telemetry, 301-316 energy, and ATP concentration, 217-226 metabolism, 21 6-226 and power consumption, 217 and active surfaces, 222 energy flow, and hypothermia, 224 and ratio of mitochondria, 222, 223 transport mechanisms of electrons, 21 6-21 9, 222 homeostatic regulation, and atropine, effect on cortex, 242 metabolism, and glutamic acid, 226 regions, hiochcmical specification, 15,16 research, and neurochemical-behavioral studic:, 49 Bright phase patterns,

hy pothsses, 34-37 types, 31, 34 CAR (conditioned avoidance responsc), and mescaline, 11-14 and physostigniine, 177 Catecholamines, and brain function, 39, 81, 86 excretion, and psychotic activity, 86, 87 metabolic changes, 82 and phenylalanine, competitive inhibition, 198-201 CBF, bee cerebral blood flow Cerebral blood flow, and blood pressure, 285,291-294 and CVR, 285,291 determination, 285-287 and oxygen utilization, 287 and tissue clearance technic, 288, 289 Cerebral vascular resistance, and CBF, 285,291 Chlorproma7ine hydrochloride, and behavior changes, 111, 116, 117 Cholinergic drugs, and behavior, 178 and EEG activation, 175-178 Cholinergic system, and behavioral changes, 50 brain, and adversive syndrome, 49-61 brain, and behavior, 49-61 Clostridium botulinrinr, bacterial neurotoxins, 256-261 toxin, composition, 257 toxin, immunological studies, 258-260 Convulsant drugs, effects on brain tissue metabolism, 1.75-158 effects on free amino acids metabolism, 135158 Convulsion, and atropine, topical application, 242, 241 CVR. see cerebral vascular resistance Dark phase patterns, types, 29, 31 Depression, induced by drugs, 4 DFP (di-isopropylfluoro-phosphate), effect on acetylcholinesterase, 49-61, 177 Dimethoxyphen ylethylamine, and mescaline, 3, 5

EEG activation, and adrenereic agents, 175, I79 cholinergic drugs, 175, 176, 178 neuropharmacology, 179 and physostigmine, 172-174, 176-179 reticular formation, and atropine, 173

SUBJECT I N D E X

EEG pattern, and behavior, effect of drugs, 177 EEG record, evaluation, in medicated patients, 101 EEG rhythm, abnormally slow -, drug-induced, 102, I03 atropine, effect of, 170, 171, 175-177, 179 physostigmine, 172, 177 and concepts of brain function, 103, 104 diagnostic use, 103, 104 and drug action, 169-1 80 and drug-free recording, 100 and imipramine, 175 telemetry, activation response, 311,312 behavior changes, 313, 134 comparison with direct cable method, 308310 effect of curare, 310, 311 effect of pentothal, 310, 311 'tranquilized' and brain patbology, 103, 104 and tranquilizing medication, 97-104 Emotion, anatomical substrate, and indole metabolism, 93 L-Epinephrine bitartrate, and behavior changes, 110, 114, 115 Escherichia coli, toxins, and mental disorders, 260 Extrapyramidal system, effects of neuroleptic drugs, 121-132

Fluoro compounds, effect on cerebral constituents, 146-154 Globus pallidus, pattern of bouton distribution, 22, 23 Hallucinogenic drugs, chemical formula, 4 Hippocampus, pattern of bouton distribution, 18, 19,22 5-Hydroxyindole acetic acid, excretion, effect of reserpine, 146-1 67 5-Hydroxytryptaniine, see Serotonin Hypothalamus, pattern of bouton distribution, 18 IAI, see intra-arachnoid injection Imipramine, and EEG rhythm, 175 Indole, derivatives, role in brain function, 40-45 metabolism, and psychotic activity, 41, 4 3 4 5 Interaction, of drugs, and mescaline, 9

345

Intra-arachnoid injection, distribution of drugs, 117, 118 of neurotropic drugs, 106-119 Iproniazid, and brain 5-HT levels, 69-73 and brain M A 0 activity, 69-73 LSD-25, reticular formation, evoked potentials, 252254 spinal cord potentials, 252-254 Melanin, formation, and tyrosine system, 200 Mescaline, ana10gues, activity, 14 chemical formula, 2, 5 effects, comparison with other drugs, 6 effects, on CAR, 11-14 on electrical activity of the brain, 6-9 and mescaline-saline (M-S) score, 11-14 interaction of drugs, 9 metabolism, 10, 11 neuropharniacological tests, 6 psychological effects, 2, 3 tolerance effect, 14 Methionine, clinical effects, 91-94 Methionine sulfoximine, effect on cerebral constituents, 142, 143 Monoamine oxidase inhibitors, role in brain function, 39-45 and serotonin brain level, 89, 90 and urinary tryptamine, 85 Neurohumoral agents, and behavior studies, 48-78 Neuroleptic drugs, antagonism with antiparkinson drugs, 121-1 32 effects on extrapyraniidal system, 121-1 32 Neurotoxins, bacterial -, Closfrirliuinboldinurn, 256-261 Escheyichia coli, and mental disorders, 260 Norepinephrine level, brain regiors, 245-248 and MAOT, 247,248 and reserpine, 247, 248 Nucleus caudatus, effect of unilateral (electrostimulation), 176, 177 Parkinsonism, depletion of brain amines, 43 Pattern, analysis, 3 1 Pentobarbital, effect on EEG rhythm, 170-172 Pentylene tetrazol,

346

SUBJECT INDEX

effect 011cerebral constituents, 146 Phenylalanine, brain level, neurotoxic effects, 202,203 and intectinal absorptlon, 195 metabolism, and nil elination, 204 metabolism, in phenylketonuria, 188-198 plasma level, and CNS secondary defects, 201-203 and serotonin, hrain level, 191, 192 tryptophan metabolism, interaction, 188-1 98 tyrosine metabolism, interaction, 198-201 Phenylethylamine, urinary excretion in phenylkctonuria, 202 Phenylketonuria experimental -, and behavior chanqcs, 203, 204 mctabolism, effect of MAOT, 202 and phenylalanine nletabolism, 188-198 Physostigmine, and acetylcholine, brain Icvcls, 172 and CAR, 177 and cortical activity, 179 and EEG activation, 172, 174, 176-179 effect on EEG rhythm, 170, 171 Picrotoxin, effect in cerebral constituents, 146 Potassium chloride, and behavior chanEes, 108, 112, 113 Psychiatry, biclogical -, advances, 1 research. 1 , 2 Psychopathology, level of urinary amines, 82 Psychosis, symptoms, degrees of severity, 82 Psychotic activity, and excretion of catecholamines, 86 urinary excretion of indole derivatives, 83 Reserpine, and serotonin brain Icvel, 89 effect on serotonin metabolism, 167 effect on urinary excretion o f 5-HIAA, 164167 Reticular formation, and adrenergic agents, 177 EEG activation, and atropine, 173 evoked responses, anesthctics, classification, 172 and anticholinesterases, 173 and barbiturates, 252-254 and drug effects, 170-174 hypnotics, classification, I72 and LSD-25, 252-254 sleep regulation and atropine, 240,241 and cholinergic agents, 240,241 and spinal input,

pharmacological investigation, 250-254 Schizophrenia, aetiology, hypotheses, 4 and amino acids, metabolism, 205-208 biochemical explanation, 2 biochemical patterns, 81 metabolism, abnormal, 3 chemical mechanism, 5 studies, 40-45 psychotic behavior, and biogenic amincs, 81-95 and serotonin metabolism, 4 Seizures, neurocheniical claasification, 155-1 58 Serotonin, assay, behavioral effects, 67, 68 and iproniazid, 69,70 and limbic system, 77, 78 and M A 0 activity, 69 brain -, and associated enzyme sqstenis, 48 effect of phenylalanine, 191, 192, 200, 201 level, brain regions, 245-248 and MAOT, 85, 89, 90 and reserpine, 89, 247,248 <ex difference,, chicken, 247,248 chemical formula, 4 LSD-25, antagonistic action, 62 metabolic steps, 62, 63 metabolism, 4 and LSD-25, 3 effect of ieserpine, 167 relationship with schizophrenia, 4 role in brain function, 39-45 tissue preparations, 73-76 Serotonin creatinine sulfate, and behavior changes, 110, 1 I I , I 16 Serotonin-monoamine oxidase system, and behavior, 62-78 Stroboscopic patterns, 28-37 Strychnine sulfate, and behavior changes, 109,114 Synapses, human brain, various tSpes, 15 Telemetry, basic system, 297, 298 circuitry, 298, 299 and direct wire recording, 301-316 EEG rhythm, activation response, 31 1, 3 12 bchavior changes, 313, 314 comparison with direct cable method, 308310 effect of curare, 3 10,311 effect of pentothal, 310, 311

SUBJECT INDEX

systems, multi-channel, 297-300 Thalambs, pattern of bouton distribution, 21 Thiosemicarbazide, effert on cerebral constituents, 143-146 Tranquilizers, medicaticn. and EEG slowing, 9 7-104 Tryptamine, excretion, and behavior studiec, 40 and MAOI, 85 and L-tryptophan, 85

psychogenic derivatives, effects, 92-94 Tryptophan, metabolism, and phenylalanine interaction, 188-198 D-Tubocurarine chloride, and behavioral changes, 109,I10,113,114 Tyrosinase system, and melanin formation, 200 Tyrosine, metabolism, and phenylalanine interaction, 198-201 Water ingestion, variations, and barbiturates, 263-282

341

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