Progress in Brain Research Volume 09 The Developing Brain

PROGRESS I N BRAIN RESEARCH VOLUME 9 THE DEVELOPING BRAIN

PROGRESS IN BRAIN RESEARCH

ADVISORY BOARD W. Bargmann

E. De Robertis

J. C . Eccles J. D. French

H. Hydtn J. Ariens Kappers

S. A. Sarkisov

Kiel Buenos Aires Canberra Los Angeles

Goteborg Amsterdam Moscow

J. P. Schadt

Amsterdam

T. Tokizane

Tokyo

H. Waelsch

New York

J. 2. Young

London

PROGRESS I N B R A I N R E S E A R C H VOLUME 9

THE DEVELOPTNG BRAIN EDITED BY

W I L L I A M I N A A. H I M W I C H AND

H A R O L D E. H I M W I C H Galeshuvg State Research Hospital, Galeshuvg, Ill. ( U .S.A .)

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1964

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TABLES

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List of Contributors

K. AKERT,University of Wisconsin Medical School, Madison, Wisc. (U.S.A.). P. K . ANOKHIN, Academy of Medical Sciences, Moscow. M. H. APRISON, The Institute of Psychiatric Research and Departments of Psychiatry and Biochemistry, Indiana University Medical Center, Indianapolis, Ind. (U.S.A.). K. D. BARRON,Neuropsychiatric Research Laboratory, Veterans Administration Hospital, Hines, Ill. (U.S.A.). S. BERL, Department of Neurological Surgery, College of Physicians and Surgeons, Columbia University, New York, N.Y. J. BERNSOHN, Neuropsychiatric Research Laboratory, Veterans Administration Hospital, Hines, 111. (U.S.A.).

D. F. BOGDANSKI, Laboratory of Chemical Pharmacology, National Heart Institute, Bethesda, Md. (U.S.A.).

B. B. BRODIE,Laboratory of Chemical Pharmacology, National Heart Institute, Bethesda, Md. (U.S.A.). W. BUNO,Thudichum Psychiatric Research Laboratory, Galesburg State Research Hospital, Galesburg, Ill. (U.S.A.).

M. M. COHEN,Division of Neurology, University of Minnesota Medical School, Minneapolis, Minn. (U.S.A.).

R. DIPERRI,Thudichum Psychiatric Research Laboratory, Galesburg State Research Hospital, Galesburg, Tll. (U.S.A.).

A. R. DRAVID,Thudichum Psychiatric Research Laboratory, Galesburg State Research Hospital, Galesburg, Ill. (U.S.A.).

R. J. ELLINGSON, Nebraska Psychiatric Institute, University of Nebraska College of Medicine, Omaha, Nebr. (U.S.A.). J. FISCHER, Physiological and Pathological Institute, Faculty of General Medicine, Charles University, Prague.

E. V. FLOCK, Mayo Clinic and Mayo Foundation, Rochester, Minn. (U.S.A.). A. FOURMENT, Centre de Recherches Neurophysiologiques, HBpital de la Salpktrikre, Paris.

G. GUROFF,Laboratory of Clinical Biochemistry, National Heart Institute, Bethesda, Md. (U.S.A.). K . HABLE,University of Wisconsin Medical School, Madison, Wisc. (U.S.A.).

V1

LIST O F CONTRIBUTORS

A. R. HESS,The Department of Neurology and Psychiatry, Northwestern University School of Medicine, Chicago, Ill. (U.S.A.).

H. E. HIMWICH,Thudichum Psychiatric Research Laboratory, Galesburg State Research Hospital, Galesburg, Ill. (U.S.A.). W. A. HIMWICH,Thudichum Psychiatric Research Laboratory, Galesburg State Research Hospital, Galesburg, Ill. (U.S.A.).

M. K. HORWITT,L. B. Mendel Research Laboratory, Elgin State Hospital, Elgin, Ill. (U.S.A.). 0. R. INMAN, Thudichum Psychiatric Research Laboratory, Galesburg State Research Hospital, Galesburg, Ill. (U.S.A.).

L. J ~ L E KPhysiological , and Pathological Institute, Faculty of General Medicine, Charles University, Prague.

J. J. KABARA,Division of Biochemistry, Department of Chemistry, University of Detroit, Detroit, Mich. (U.S.A.). T. KOBAYASHI, Thudichum Psychiatric Research Laboratory, Galesburg State Research Hospital, Galesburg, Ill. (U.S.A.).

L. KRULICH, Physiological and Pathological Institute, Faculty of General Medicine, Charles University, Prague. A. LAVELLE,University of Illinois College of Medicine, Chicago, Ill. (U.S.A.). D. B. LINDSLEY, University of California, Departments of Psychology and Physiology, Los Angeles, Calif. (U.S.A.).

R. E. MCCAMAN, The Institute of Psychiatric Research and Departments of Psychiatry and Biochemistry, Indiana University Medical Center, Indianapolis, Ind. (U.S.A.). J. M. MILSTEIN,Division of Neurology, University of Minnesota Medical School, Minneapolis, Minn. (U.S.A.). C. A. OWEN,Jr., Mayo Clinic and Mayo Foundation, Rochester, Minn. (U.S.A.).

E. G. PASCOE, Central Institute for Brain Research, Amsterdam. J. PETERSEN, Thudichum Psychiatric Research Laboratory, Galesburg State Research Hospital, Galesburg, Ill. (U.S.A.).

G. R. PSCHEIDT,Thudichum Psychiatric Research Laboratory, Galesburg State Research Hospital, Galesburg, Ill. (U.S.A.). J. P. SCHAD~, Central Institute for Brain Research, Amsterdam. A. SCHEIBEL, Departments of Anatomy and Psychiatry, University of California Medical Center, Los Angeles, Calif. (U.S.A.). M. SCHEIBEL, Departments of Anatomy and Psychiatry, University of California Medical Center, Los Angeles, Calif. (U.S.A.).

LIST OF CONTRIBUTORS

VI1

J. SCHERRER, Centre de Recherches Neurophysiologiqucs, HBpital de la Salpttrikre, Paris. K. F. SWAIMAN, Division of Neurology, University of Minnesota Medical School, Minneapolis, Minn. (U.S.A.). S. TROJAN, Physiological and Pathological Institute, Faculty of General Medicine, Charles University, Prague. G . M. TYCE,Mayo Clinic and Mayo Foundation, Rochester, Minn. (U.S.A.).

S. UDENFRIEND, Laboratory of Clinical Biochemistry, National Heart Institute, Bethesda, Md. (U.S.A.). A. VERNADAKIS, Department of Pharmacology, University of Utah College of Medicine, Salt Lake City, Utah (U.S.A.). University of Wisconsin Medical School, Madison, Wisc. (U.S.A.). H. A. WAISMAN, H. L. WANG,University of Wisconsin Medical School, Madison, Wisc. (U.S.A.).

C. D. WITHROW,Department of Pharmacology, University of Utah College of Medicine, Salt Lake City, Utah (U.S.A.).

D. M. WOODBURY, Department of Pharmacology, University of Utah College of Medicine, Salt Lake City, Utah (U.S.A.).

Other volurnes in ihis series:

Volume 1 : Brain Mechanisms Specific and Unspecific Mechanisms of Sensory Motor Integration Edited by G. Moruzzi, A. Fessard and H. H. Jasper

Volume 2 : Nerve, Brain and Memory Models Edited by Norbert Wiener and J. P. Schade

Volume 3 : The Rhinencephalon and Related Structures Edited b y W. Bargmann and J. P. Schade

Volume 4 : Growth and Maiuration of the Brain Edited by D. P. Purpura and J. P. Schade Volume 5 : Lectures on the Diencephalori Edited by W. Bargmann and J. P. Schade Volume 6 : Topics in Basic Neurology Edited by W. Bargmann and J. P. Schade

Volume 7 : Slow Electrical Processes in the Brain b y N. A. Aladjalova

Volume 8 : Biogenic Aniines Edited by Harold E. Himwich and Williamina A. Himwich

Volume 10: Structure and Function of the Epiphysis Cerebri Edited by J . Ariens Kappers and J. P. Schade

Volume 1 I : Organization of the spinal Cord Edited by J . C . Eccles and J. P. Schade

Volume 12: Physiology of Spinal Neurons Edited by J. C . Eccles and J. P. SchadC

Volume 13 : Mechanisrns 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. SchadC

Preface

Approximately 10 years ago the first Neurochcmical Congress (Oxford, 1954) had as its theme the developing brain. The volume of papers from that meeting which contained the latest work and ideas on the subject had mostly biochemical or anatomical studies of the brain with very few references to the development of electrical activity. Three years later, the International Congress of Neurological Sciences devoted one session to the ontogenesis of the EEG. In the last few years, as is obvious from the papers contained in this volume, the interest in developing brain has grown enormously, has extended not only into the anatomical but also into the physiological, the electrical and the biochemical aspects of maturation. This field of investigation is truly an interdisciplinary one with all facets of the problem being actively followed. For these meetings leaders from all over the world have been invited to present their papers. The program was arranged so that the chairman of the opening session was a leader in the field with a broad background and capable of presenting an overall synthesis of the subject. This role was ably filled by Dr. Donald Lindsley, whose talk set the proper tenor for the entire conference. Chairmen for the other two sessions, Dr. Alfred Pope and Jordi Folch-Pi, were also chosen from men known for their breadth of knowledge in this area. Their contributions were unique and unfortunately they are only indicated in the unstructured discussion which closed each session. It is hoped that this volume will find a wide use not only among persons working actively in research in maturing animals, but also among neurologists, pediatricians and others interested in the function of the brain. Two monumental pieces of work, that of Dr. Ellingson and of Professor Anokhin, complete with many illustrations have been included. In the case of professor Anokhin, this volume probably represents the most complete publication in English of his theory of systemogenesis. A conference such as this one requires enormous attention to detailed planning in advance. The meeting would not have been possible without the aid, encouragement and active participation of the Superintendent of the hospital, Dr. Thomas T. Tourlentes, during the entire period of preparation. The overall planning of the meetings, the social occasions, the many opportunities for interpersonal discussions were largely due to his interest and cooperation. Although it is impossible to thank here all of the other personnel of the Galesburg State Research Hospital who assisted us so ably at the time of the meeting, a few must be singled out because of their unique contributions: these include Miss Florence 0. Johnson, Assistant Superintendent in charge of Nonmedical Affairs, Mr. Lloyd Tenneson and his staff, Mr. Gilbert Salter and his staff and Mrs. Ned Wilmont and her secretarial staff. The entire conference could not have taken place if the foundations and drug companies had not been sufficiently altruistic to furnish the necessary financial assistance : National Science Foundation; Manfred Sake1 Foundation ; Abbott Laboratories ; Burroughs Wellcome and Company; Ciba Pharmaceutical Company; Eli Lilly and Company; Geigy

X

PREFACE

Chemical Corporation ; Hoffmann-LaRoche, Inc. ; Merck, Sharp and Dohme Postgraduate Program; Pitman-Moore Company ; Sandoz, Inc. ; Schering Corporation ; Searle and Company; Smith, Kline and French Laboratories, Inc.; Squibb and Sons; Sterling-Winthrop Research Institute; Strasenburg Laboratories; Wallace Laboratories, and Wyeth Laboratories. To all of these persons and organizations as well as to all of our colleagues in the laboratory and the Galesburg State Research Hospital we wish to extend our appreciation. If the many compliments we have received on the Symposium are deserved, it is only because of their untiring efforts.

W. A. HIMWICH

Con tents

List of Contributors

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

V

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1X Bram development and behavior: Historical introduction D. B. Lindsley (Los Angeles, Calif.) . . . . . . . . . . . . . . . . . . . . . . . . . 1 Some structural and functional substrates of development in young cats M. Scheibel and A. Scheibel (Los Angeles, Calif.) . . . . . . . . . . . . . . . . . . . 6 Studies of the electrical activity of the developing human brain R. J. Ellingson (Omaha, Nebr.) . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 Systemogenesis as a general regulator of brain development 54 P. K. Anokhin (Moscow) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Neurohistological studies of developing mouse brain T. Kobayashi, 0. R. Inman, W. Buno and H. E. Himwich (Galesburg, Ill.) . . . . . . . 87 The evolution of the developing brain of the dog R. DiPerri, W. A. Himwich and J. Petersen (Galesburg, Ill.) . . . . . . . . . . . . . . 89 Critical periods of neuronal maturation A. LaVeIle (Chicago, Ill.) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93 General discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 Electrocortical effects of sensory deprivation during development J. Scherrer and A. Fourment (Paris) . . . . . . . . . . . . . . . . . . . . . . . . . 103 The reaction of the brain to stagnant hypoxia and anoxia during ontogeny L. Jilek, J. Fischer, L. Krulich and S. Trojan (Prague) . . . . . . . . . . . . . . . . . 113 Maturational changes in cerebral cortex 111. Effects of methionine sulfoximine on some electrical parameters and dendritic organisation of cortical neurons 132 J. P. Schade and E. G. Pascoe (Amsterdam) . . . . . . . . . . . . . . . . . . . . . Brain cholesterol. The effect of its development on incorporation of acetate-2-3H and gl~cose-U-~~C 155 Jon J. Kdbara (Detroit, Mich.) . . . . . . . . . . . . . . . . . . . . . . . . . . . Esterase activity and zymogram patterns in developing rat brain J. Bernsohn, K. D. Barron and A. R. Hess (Chicago, Ill.) . . . . . . . . . . . . . . . 161 Interrelationships of glucose, glutamate and aspartate metabolism in developing rabbit brain K. F. Swaiman, J. M. Milstein and M. M. Cohen (Minneapolis, Minn.) . . . . . . . . . 165 Biochemical studies of the central nervous system of the dog during maturation A. R. Dravid and W. A. Himwich (Galesburg, Ill.) . . . . . . . . . . . . . . . . . . 170 Effect of acute and subacute administration of diphenylhydantoin on electroshock seizure threshold in developing rats A. Vernadakis and D. M. Woodbury (Salt Lake City, Utah) . . . . . . . . . . . . . . 174 Postnatal changes in amino acid metabolism of kitten brain S. Berl (New York, N.Y.) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178 General discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183 The uptake of aromatic amino acids by the brain of mature and newborn rats 187 G . Guroff and S. Udenfriend (Bethesda, Md.) . . . . . . . . . . . . . . . . . . . . Tryptophan metabolism in the brain of the developing rat G. M. Tyce, E. V. Flock and C. A. Owen, Jr. (Rochester, Minn.) . . . . . . . . . . . . 198 Tissue acid-base changes during maturation C. D. Withrow and D. M. Woodbury (Salt Lake City, Utah). . . . . . . . . . . . . . 204 Some ultrastructural changes in thebrain of phenylketonuric rats and monkeys H. A. Waisman, K. Hable, H. L. Wang and K. Akert (Madison, Wisc.) . . . . . . . . . 207

XI1

CONTENTS

Effects of reserpine and isocarboxazid in the frog G. R. Pscheidt (Galesburg, Ill.) . . . . . . . . . . . . . . Effect of diet on lipid composition of brain M. K. Horwitt (Elgin, Ill.) . . . . . . . . . . . . . . . . The synthetic and catabolic enzyme system for acetylcholine and areas of the developing rabbit brain R. E. McCaman and M. H. Aprison (Indianapolis, Ind.) . . . Biogenic amines and drug action in the nervous system of various B. B. Brodie and D. F. Bogdanski (Bethesda, Md.) . . . . . General discussion . . . . . . . . . . . . . . . . . . . . Summary W. A. Himwich (Galesburg, Ill.) . . . . . . . . . . . . . Author index. . . . . . . . . . . . . . . . . . . . . . Subject index . . . . . . . . . . . . . . . . . . . . . . .

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serotonin in several discrete

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Brain Development and Behavior: Historical Introduction D. B. LINDSLEY University of California, Departments of P.gvcliology and Physiology, Los Angeles, CaliJ (V.S.A.)

Man's curiosity about his brain has a very long history. The early Greeks, as we all know, variously located the seat of the soul in the gut, heart and finally in the brain. During the course of the next several centuries the concept of the ventricles constituting a source of vital spirits was one of the dominating features. As a matter of fact, it was not until the 17th century that Descartes began to conceive of the nervous system as an instrument for the production and the propagation of animal spirits. Descartes had the concept of the seat of the soul in the pineal body, the container of animal spirits or of the vital fluid so to speak of both sensory and motor nature. It was not really until 150 years later that Galvani and Volta developed their interest in animal electricity, a concept which Galvani and his wife seemed to have discovered by hanging frog legs on their iron balustrade and the contact between two dissimilar metals in an electrolyte actually serving as a good electrical stimulus. The ideas which were engendered by Galvani and Volta during this time, later led DuBois-Reymond to develop the concept of the nerve impulse. But it was not until the early part of the 19th century when Marshall Hall, Bell, DuBois-Reymond and others seriously got to work i n the direction of demonstrating and measuring the nerve impulse. As we move along to the next period, we find anatomists beginning the study of the structure of the nervous system, physiologists beginning to understand something of the initial reflex nature of the sensory and motor roots and the spinal cord and Flourens developing ablation and operating technics to disprove the phrenological concepts of Gall and Spurzheim. During that early period there was a great deal of effort spent in attempting to envisage the nervous system as a system that is irritable, that is capable of conduction and that is indeed the recipient of the input information. The period 1850-1900 was one of very great activity with respect to research on the brain. We see the culmination, or rather the beginning, of a serious effort to improve the technology which brought to us electrical measuring instruments and microscopes of greater refinement, staining technics, chemical application to the nervous system, electrical stimulation, electrical recording and all the rest. One can only marvel at the amount of progress made during that 50-year period from 1850-1900. To mention only a few of the outstanding names one must, of course, from the structural point of view remark about Ram6n y Cajal and Golgi, who were awarded the Nobel Prize in 1906 for their efforts during this period and whose work today, as

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we will see from this conference, is still very much with us in a form which has been very stimulating to those who have been trying to understand the details of histological structures of the brain. In addition, the improvements of operative technics and, as I have said, the development of electrical stimulating devices started a whole series of investigations of the brain in the 1870’s and these were, of course, the basis for determining some of the specific landmarks upon brain. The work of Fritsch and Hitzig followed subsequently by Ferrier, Sherrington and others certainly demarcated the site of the motor cortex. The work of Caton in 1875 was the first report of the electrical potentials of the brain, a forerunner of things to come, but an advantage that was not really profited from very greatly until nearly 55 years later. During this period also we find the intensive study of the structure of the brain going on and many of the German and Swiss investigators who were concerned with the architectonics of the brain trying to differentiate the structures of different parts of the brain in terms of architectonic maps. Some of these maps we have with us today in very much the same form in which they were then developed. One can only remark upon the fact that the technical developments, which are so important to us today in the electron microscope or in the applications of the computer were very important indeed then. Each new development seems to have stimulated some new aspects of the investigative procedure, such as the concern with the study of behavior and the relation of behavior to the structural changes which had been observed during the past 40 to 50 years and to the functional changes that were being demonstrated by electrical stimulation of the brain. Here we have to seek some forerunners of these developments. One hundred years ago Sechenov, in his book: Reflexes 05the Brain, described central inhibition and certainly stimulated Pavlov and others to begin the behavioral studies which they eventually undertook. Pavlov, as we know, was awarded the Nobel Prize in 1904, but not for his work on conditioned reflexes. At the age of 55 he began a whole new career in this field of endeavor and his work, although not so greatly concerned with the brain, developed the interest about the function of the brain from the study of behavior by conditioned reflex methods. Also at this time Thorndike introduced new methods of testing and training animals in discrimination procedures. Lashley and Franz, studying both animals and human subjects in a behavioral setting and relating these studies to lesions of the brain constituted a great inspiration to people who are carrying out such studies today. But it was not until 1929 that the electrical recording from the brain, which Caton had first initiated in 1875, was again brought to our attention by Hans Berger. I think we must attribute to him the resurgence of interest and the upsurgence of a new interest in the brain. The neurophysiologist of the day had conceived of very little other than a classical spike or action potential. However, Berger’s work, more than anyone else’s perhaps, convinced the world that there was another type of electrical activity; namely, a wave like phenomenon which could be recorded from the brain. We are speaking about the development of the brain and must go back just a moment as an antecedent of this to mention the work of Flechsig begun in the 1870’s and culminating in his monumental publication in 1896. Flechsig, influenced by Meynert, had developed an

HISTORICAL INTRODUCTION

3

interest in myelogenesis. He studied the myelin formation in the projections to the cortex and in the cortex called attention to the differential rate of development based on myelogenesis of the particular regions of the brain. I think this was the first instance in which this type of attack was made. Flechsig quite correctly, noted that the motor area is myelinized earlier than the sensory area, a result which was later substantiated and extended by Conel in his monumental series of books extending up to the present day. Another person who was concerned with the developmental approach to the study of the brain was Von Gudden, who applied this technic to animals. We see, therefore, in the late 19th century a concern with the developmental structure of the brain which is also receiving considerable interest at the present time as we will hear subsequently from Dr. Scheibel's paper and others in this program. To come back then to the use of the electrical recording technic as a means of getting further information about the brain, we are reminded that Berger did many of these studies during the early 30's. He investigated the electroencephalogram in children and found indeed that the characteristics of the a-waves or Berger waves as they were called were of lower frequency than they were in adults. He did not pursue this problem at any great length and it was subsequently taken up by myself in 1935, and by J. R. Smith, working at the Baby and Childrens Hospital in New York with M. B. McGraw who was studying, as was A. Gesell, the behavioral development of the human infant. This work gives us some clues, 1 think, to certain functional and behavioral characteristics as well as to the structural ones which Flechsig, Conel and others have brought to us. In trying to envisage how this historical past has contributed to our interest in applying our present day technics, of which you will hear a good deal at this meeting and in particular the development in the field of neurochemistry, I can't help but feel that if one remarks that neurophysiology has had a heyday in the past 25 years, that the next 25 years are going to be ones of peak activity by the biochemists and the neurochemists. This will not mean that the other technics which I have mentioned will not be utilized during the period but it does mean, I think, that we will have a much greater emphasis upon the chemical changes going on concurrently with the behavioral changes or eIectrical changes than has been shown in the past. So, I think, it is incumbent upon us to try to envisage how it is that chemistry and the findings which are certain to emerge from the activities of the neurochemist can be related to some of these other technics and the changes which will be revealed there. For example, is it possible to designate by a careful study of behavior, certain stages at which critical changes occur? Could one identify then certain alterations in the chemical pattern which could be correlated with these behavioral changes? Are there electrical changes that come in at certain stages or disappear at certain stages that could be correlated with the chemical phenomena as well as behavior? These, I think, are some of the points that we must look toward. We must take note of the work of Sherrington, Adrian, Fobbes and subsequent classical neurophysiologists; rather rapidly coming up to the period of the 20's and 30s I would like to speak of forerunners in the development of new brain concepts. The work of Hess, Ranson, Bremer, Morison and Dempsey led up to the

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D. B. L I N D S L E Y

discovery by Moruzzi and Magoun in 1949 that the reticular formation of the lower brain stem had a significant role in modifying the electrical activity of the cerebral mantle and in addition to that had a good deal to do with behavior in the form of arousal, alerting, attention etc. I would like only to comment now on the distinction that was made between the classical or primary sensory pathways and the so-called nonspecific sensory fields because this will come up time and time again, I think, as we begin to see the unfolding of the electrical activities as a function of the developing brain. If one records from the lemniscus and from the reticular formation during a stimulation given peripherally we see a response in both of these structures. Under ether the response disappears in the reticular formation but is still maintained i n the sensory pathways leading to the cortex. But under this condition an animal trained to discriminate something behaviorally or an individual who is undergoing anesthesia will not be able to discriminate or report upon the environment which he faces. Therefore, it seems inevitable in attention and perception that one consider the specific sensory system and the so-called nonspecific sensory system of the lower brain stem and its connections with the nonspecific nuclei of the thalamus as closely linked together in their functions and, I think, we will see subsequently from the electrical recordings that this is true. Some years ago I had the opportunity when my own family was developing to record fetal EEG’s during the 7th fetal month. I discovered to my surprise that there was some electrical activity which could be recorded over the abdominal wall. Also on birth of the infant I was able to demonstrate, as Smith had already done, that over the motor region of the brain, during a relaxed almost drowsy state one could find the same kinds of rhythmical activity, somewhere between 5-7/sec exhibited. As Flechsig pointed out, and as Conel demonstrated later, the motor area of the brain leads in structural development and Conel names some 8 or 10 of these characteristics which were more advanced in the motor area than in the sensory and other association areas of the brain. We can also see this in behavior of an infant; certainly the arms or the extremities begin to move in a somewhat uncoordinated fashion before the other parts of the body are active. Our studies on children constituted an investigation of the development of the a-rhythm and we have shown a change of frequency with age. We see then that the a-rhythm is a function of activity in the dendritic system and it may be activated and modified by axodendritic connections; that is, by the nonspecific connections from the reticular formation which I mentioned earlier. At 3 or at 4 months in human infants the a-rhythm appears as a persistent phenomenon. What the nature of the chemical changes is we have not yet been made aware, but frequency of the rhythm then is only 3 or 4 waves/sec, by 12 months it is 5-6/sec7 by 10 years it is lO/sec. There is definitely a growth function in the change i n frequency with age. There is no growth change in frequency from 10 years to 21 years. Structurally also in the visual area, considerable changes take place in the first 3 months of life. This is indeed a gross correlation between the structural change and the onset of the a-rhythm7 but it also corresponds to certain behavioral accomplishments of the 3-month-old infant.

HISTORICAL INTRODUCTION

5

In the behavioral repertoire at 3 months of age a child will begin to use the visual apparatus, in which he has a-rhythms and has had these structural changes in the brain, by focussing upon a ring and following it with his eyes. Not only does the human have a developing a-rhythm but in some work with Caveness, who has done most of his work on monkeys, we were able to show that a very similar thing takes place in this species. By the age of about 15-20 days we see the u-waves present in the electroencephalogram of the monkey. The onset and frequency growth of the a-rhythm appears to be very similar to that we have seen in the human, considering overall differences in life span and rate of maturation. Now, let’s briefly comment on the work that C. Rose and I have been doing on the developing response of the kitten cortex which confirms the work of Dr. Scherrer and his group in Paris, Drs. Marty, Contamin and others, and that we think we can extend in certain ways. We have done this on a longitudinal basis in kittens, studying evoked responses to a flash of light from 4 to 42 days of age and recording from both contralateral and ipsilateral cortex. We see that the first component that comes in at 4 days is a long-latency response of negative character with peak latency of 170 msec. By 10 days of age the latency is reduced to 140 msec and an earlier negative resporise of short-latency has appeared. Frequently a positive wave comes in between 10 and 15 days. As we follow this pattern along, a first positive-negative complex and a second negative response, we see that there is a coalescence between this early longlatency negative response, and the later appearing positive-negative complex of short-latency. In other words, we see a positive wave and a coalescence of the two negative waves to form the overall pattern which we see in the more mature and even in the adult cat, Now we are going to propose, as perhaps others have done, thinking about the concepts of the nervous system in terms of the nonspecific systems, that the first developing long-latency negative wave is associated with the nonspecific systems, and that the later appearing positive and negative waves of short-latency are associated with the specific sensory system. The coalescence of these into one pattern may be the very thing, which, as I said earlier, makes possible the perceptual discriminations which are not possible when one removes the influence of the reticular formation by anesthesia. It appears that the development and maturation of the cortex and its integration with subcortical centers is in some measure reflected by the developing potentials as a function of age, with specific and nonspecific systems maturating at different times. Hopefully, further correlations of chemical nature will appear. This conference will bring out some of the latest results of structural, functional, behavioral and chemical correlations i n the brain and nervous system.

6

Some Structural and Functional Substrates of Development in Young Cats M A D G E SCHEIBEL

AND

A R N O L D SCHEIBEL

Departments of Anatotny and Psychiatry, University of California Medical Center, Los Angeles, Calif. ( U . S . A . )

The material that we are presenting in this communication developed within the framework of a long-term study of neuropil patterns in brain stem reticular core and related structures, and was geared toward elucidating certain aspects of the problems of inhibition and facilitation. We found that structural data derived from Golgi impregnations of neonatal or young material were being used to explain adult neural phenomena. We initially asked ourselves the question as to when reticular activation of the cortex could first be demonstrated. It turned out that a ‘simple’ question of this sort led to a multifaceted solution and a number of other problems also lent themselves to studies of the chronic mobile neonatal preparation. A discussion of several of these problems forms the basis of this paper. Three general topics will be considered : (1) some structuro-functional correlates of brain wave maturative patterns with particular reference to cerebral cortex, cerebellum and reticular formation; (2) the maturation of cortical activation mechanisms including cortical ‘following’ reactions ; and (3) the development of certain discriminative conditional responses including habituation-like phenomena. The data to be discussed in this communication are drawn from a large body of material gathered over several years from a group of approximately 80 kittens chronically implanted within a few hours to a few days of birth. Although the neurophysiological material and related structural correlative data are derived from this source, a larger body of structural information rests on some 12 years of experience with several thousand brains of laboratory animals examined by Golgi and related techniques. Wherever possible, kittens were operated within 3 to 12 h of birth. Minute amounts of intraperitoneal Nembutal (30 mg/kg) in saline were used as basal anaesthesia and supplemented by open drop ether as needed. Under sterile precautions, animals were placed in a specially constructed head-holder which was suspended within the frame of a standard Johnson stereotaxic apparatus. Three adjustable arms shaped to the bridge of the nose and the mastoid prominences held the head motionless without injury to sensitive tissue of mouth, eyes or ears. On the basis of previously prepared and averaged control measurements on a group of kitten heads, fine tripolar electrodes were introduced through burr holes, using the

SOME SUBSTRATES O F DEVELOPMENT I N CATS

7

electrode carriers of the Johnson instrument. All electrode wire tips were sharpened to points of 60 to 100 p and distance between the tips averaged 0.75 mm. The central shaft of each depth electrode reaching to a point within 5 to 8 mm of the electrode wire tips served as a local ground to minimize artifactual current spread on stimulation. Recording-stimulating sites sought with these electrodes included mesencephalic reticular formation, non-specific thalamic (reticular) nuclei, specific thalamic relay centers, hippocampus, entorrhinal cortex and caudate nucleus. Location of these placements was histologically verified upon sacrifice of the animals. Electrodes were held in position with quick-drying dental cement in the usual manner. Cortical sites, usually comprising posterior, ‘middle’, and anterior locations were almost always situated over posterior supra-sylvian, mid ectosylvian, and sigmoidal stations, and were recorded through stainless steel watchmakers’ screws. All lead-off wires were soldered to microminiature Winchester 7 and 14 pin female plugs also fixed to the skull with dental cement. Kittens were returned to their litters when all ether was blowii off and in most of these cases, the mother continued to care for them. When this was not possible, they were kept alive in electrically warmed boxes and fed a formula through doll baby bottles. Because of the nature of some of the auditory conditioning methods that we have used, we are presently trying to compare the groups raised artificially with those raised until natural weaning by their own mothers. Histological and neurochemical data were obtained from litter mates sacrificed at appropriate intervals and impregnated by modified rapid Golgi methods, controlled by Nissl or Kluver stained sections. Several technical problems of especial relevance to this study deserve mention. In some animals, electrodes remained in place for 5 months or more and we were concerned with the expected shift of electrode tips as the skull and brain grew in size. Histological studies of Nissl and Kluver stained tissue failed to reveal evidence of large-scale shift. Glial reactions were minimal and usually difficult to recognize, and even the most obvious growth-withdrawal tracks seldom exceeded 2 to 4 mm. Furthermore, unless grossly malpositioned in the initial procedure, electrode tip positions were usually close to the predicted stimulating-recording stations suggesting, once more, minimal displacement with head growth. Another point deserving emphasis in interpretation of results is the very appreciable variation, structurally and functionally, in animals of presumed similar age. Many kittens a t term showed well-developed gyrencephalic cortices which were in every respect miniature replicas of the adult hemisphere, and the general level of neonatal behavior reflected this level of relative maturity. Other kittens at term showed essentially smooth lissencephalic cortices of jelly-like consistency and the behavioral and electrophysiological performance again showed similar levels of attainment. Even among litter mates, the range of variation was impressive, ruling out the possibility of prematurity, errors of calculation i n conception time, etc. There was insufficient evidence to indicate that the surprising extent of intra-litter variation might depend solely on preferential in utero positions, localized relative hypoxic states, etc. Although we cannot yet marshal1 convincing evidence for the position, we are strongly persuaded that such variations in maturation are at least in part, the result of individual idioReferences p. 24/25

8

M. S C H E I B E L A N D A. S C H E I B E L

syncratic developmental patterns - possibly genetic - and as such, represent an entire area of their own for elucidation. Many investigators have made similar observations on this kind of variation (Ellingson and Wilcott, 1960; Dreyfus-Brisac, 1959; etc.) From the point of view of t h s report it underlines the need for caution in matching the functional and electrophysiological capabilities of one kitten against the degree of structural development in litter mates. The maturation of the cortical EEG has been traced in various anin-.als and in the human infant by a number of investigators starting with Lindsley (1936) and the reader is referred to the initial review article in this volume by Dr. Lindsley and to a review of neural substrates of development (Scheibel and Scheibel, 1963b) for a list of appropriate references. We will allude to certain components in the maturation of the cortical EEG which appear to bear reference to identifiable structural correlates. In general, records of the first 2 or 3 days consist of irregular 4 to 6/sec rhythms alternating with slower patterns, probably a function of varying sleep levels although clear-cut differentiations between sleeping and waking states are difficult to make before the 2nd to 5th days of life. Amplitudes vary but seldom exceed 50 pV. The actual process of maturation appears to include a number of phenomena, only a few of which have thus far been recognized. By the end of the 1st week, there is a gradual increase in the frequency spectrum though specific amplitude and frequency patterns remain haphazard. A second change which also begins to be seen between the 5th and 10th days of age is the appearance of isolated spindle bursts, and by the end of the 2nd week, appreciable lengths of cortical trace are occupied by intermittent and sometimes continuous spindling (Fig. 1). We have come to interpret this change as a significant index of cortical maturation, noting that in some kittens born with apparently precociously developed cortices, spindling may become evident as early as the 2nd to 4th day. Petersen and Himwich (1959) have reported finding sleep spindles as early as the 1st or 2nd day in well-cared-for pups under curare while SchadC (1959) correlated the appearance of spindle bursts with a virtually mature cortex. It seems likely that the difference in timing and interpretation of these spindling phenomena is due to species differences and to variations in recording technique (i.e. Schade’s rabbits were recorded under light urethane anaesthesia, etc.). Initial appearance of alpha activity usually follows spindling by 2 to 3 days, making itself initially evident over posterior (occipital) cortical stations at about the time that the eyes begin to open. Frequency patterns approximate those of the adult by the end of the first month although complete organization of waking and sleeping patterns may not be achieved until the middle or end of the 2nd month. Structural attributes of neonatal cortex have been described by Cajal (1955) and by Conel (1 939-1 959) and more recently, physiologically oriented investigators have added information (Purpura et a]., 1960; the Scheibels, 1959, 1961; etc.). Here we wish to call attention only to a few facets of the vastly complex sequence of structural maturation, realizing the risks attendant upon singling out any items from so difficult, and poorly-understood a process. Exclusive of the precociously developed 1st layer, the large pyramids of the 5th layer show a relatively high order of maturity with recognizable basilar dendrites, apical dendrites reaching toward, and in some cases

9

SOME SUBSTRATES O F DEVELOPMENT I N CATS

cx 2-
24 weeks old) were injected simultaneously with 14C- and 3H-labeled precursors and killed at various time intervals later by decapitation and exsanguination. Food was removed from the cages 24 h prior to isotopic injection. The organs of the dead animals were removed, washed twice in isotonic saline solution and quick frozen by a mixture of dry ice and acetone. Samples were kept in the frozen state until required for analysis. The procedure for the isolation and assay of 14C-labeledcholesterol was a modification of earlier methods (Kabara, 1957). Tissue cholesterol was extracted with acetone alcohol ether (4 : 4 : 1); and the free sterol isolated as the tomantinide. The tomantine-cholesterol complex was then dissolved in acetic acid. Colorimetric determination of cholesterol content and the radioassay of amount of isotope corporated were made on the same sample (Kabara et al., 1961). Resulting data are reported in terms of disintegration/min/g (wet) tissue. The radiochemical purity of the isolated tomantinide has been previously established (Kabara and Okita, 1961; Kabara et al., 1961). RESULTS

A~etate-2-~H incorporation: Animals representing the three groups previously mentioned, were starved for 24 h before intraperitoneal injection with 20.0 ,uC of a~etate-2-~H and 1.O ,uC of uniformly labeled glucose. Mice were killed at 15, 30, 45, 60, and 90 min intervals after isotope injection. Five animals were killed at each time interval. Liver, spleen, and brain were excised and prepared for analysis as previously

4o ol 3000

Fig. 1. Simultaneous incorporation of (a) g l u ~ o s e - U - ~(1.0 ~ C pC) and (b) a~etate-2-~H (20.0 pC) into liver free cholesterol.

157

I N C O R P O R A T I O N O F ISOTOPES I N T O C H O L E S T E R O L

mentioned. Animals in the youngest group exhibited a bi-phasic incorporation of acetate into liver cholesterol (Fig. 1). An incorporation peak was measured at a 15 min interval with lower values recorded for samples taken at the 30 and 45 min intervals. A second peak was measured after 60 min, while a significantly lower value was again registered after 90 min. Curves obtained for the second age group (8-10 weeks old) did not exhibit this bi-phasic phenomenon. In these animals, the amount of acetate incorporated at 15, 30, and 45 min, was essentially the same. In animals killed 60 and 90 rnin after isotope injection, less activity was measured. Mice representing the oldest group (more than 24 weeks old), seemed to exhibit a lag in regard to their incorporation of acetate into liver sterol. Maximum labeling in these animals took place somewhere between 30 and 60 inin with a definite lowering of radioactivity measured at the end of 90 min. The decrease at 90 min seemed to signify that rapid turnover was occurring in the liver of animals older than 24 weeks. The spleen, like the liver, showed the familiar bi-phasic curve in the youngest group with a similar pattern of labeling i n the other two age groups (Fig. 2). Brain cholesterol labeled with w Iiiil ..:. ~... 3 - 4 wk9.01d

5540

a

(I1 g )

8 -10 uk8.old

(27 g)

24 wks.old

(31 p )

Fig. 2. The simultaneous incorporation of (a) gluco~e-U-'~C (1 .O pC) and (b) a~etate-2-~H (20.0 pC) into spleen free cholesterol.

tritiated acetate in the three age groups confirmed previous reports using this precursor, i.e. increasing lower specific activity with age (Fig. 3). When the same data were recalculated on disintegrations per mg per g tissue basis, one could make some rather interesting observations. Now, the difference in incorporation between liver and brain is not quite as exaggerated as when compared on a specific activity basis. This is due to the high dilution factor (sterol concentration) within the brain for newly labeled cholesterol. Comparing the radioactivity at 45 min for the three age groups, we find that the amount of precursor incorporated is 2210 disintegrations/ References p . 160

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J O N J. K A B A R A

min/g tissue (3-4 weeks old); 2276 disintegrations/niin/g tissue (8-10 weeks old); and 1273 disintegrations/min/g tissue (more than 24 weeks old) respectively. If the amount of isotope incorporated was again corrected for a ,uC per g dose basis, the coiiipsrisoii would then be 2210; 3132; and 31 32 disintegrations/min/g tissue, respectively for eech of the age groups. As can be seen, the measure of radioactivity incorporated

4000

3000

20001

2000

I

lool~

~

0

15 W i n

30 Mln

4 5 PI"

60 Mi"

90

IUl"

Fig. 3. The simultaneous incorporation of (a) glucose-U-lC (1.0 pC) and (b) a~etate-2-~H (20.0 pC) into brain free cholesterol.

into brain cholesterol of animals, is partially a function of the basis used for calculating the expzrimental results. When the weight of the animal and the various corrections for pool size are considered, there seems to be little or no lowering of the synthetic capacity in older mice as compared to young animals even when acetate was used as a precursor. Glucose-U-14C incorporatior?: When uniformly labeled glucose was injected into animals of the three age groups, a completely different pattern of labeling was measured froin that obtained with a ~e ta te -2-~H. With glucose, there is a uniform delay in the incorporation of the carbohydrate into animals' tissue sterol. The peak of incorporation being reached at 45 min, regardless of age. As the animals become older, there seems to be a decrease in incorporation of glucose into liver cholesterol (Fig. 1). Qualitatively, incorporation of uniformly labeled glucose into spleen cholesterol was also low, whether the comparison was made on the specific activity or activity per g of tissue basis (Fig. 2). When the incorporation of unifornily labeled glucose into brain cholesterol of the three groups was compared on a tissue weight basis, it was seen that the exaggerated differences noticed between the age groups were not as apparent as with acetate incorporation (Fig. 3). If the isotope dose is considered on a

I N C O K P O K A T I O N OF ISO’IOPhS INTO CHOLESTEROL

159

&/kg basis, theii the comparison of glucose incorporated into sterol of the three groups, 45 niin after injection, would bs I121 ; 3428; and 3472 disintegrations/min/g tissue respectively. Again, as with the acetatc incorporation data, the older animsls incorporate more, rather than less radioactivity into brain sterol, under the described experimental conditions. DISCUSSION

It is generally accepted that the brain represents an unusual organ in so far as the study of its inetabolism is concerned. This uniqueness is due in part to the protection afforded to the brain by a mechanism which restricts the entry and exit of substances. Because of this ‘barrier’ phenomenon, biochemical studies concerned with investigating metabolic patterns in this organ have bzen difficult to interpret. The earliest experiments in this field had shown that while the brain possessed synthesizing ability during early development, once myelinization had occurred, this capacity in the young or adult brain either ceased or was insignificant (Waelsch et al., 1940; Srere et nl., 1950). In the past few years, however, using special injection techniques (intracerebral and intracisternal) it has been demonstrated that the adult mouse or rat is capable of synthesizing brain sterol (McMillan et al., 1957; Nicholas and Thomas, 1959). In our own laboratories we have shown that the brain of young adult mice is capable of incorporating a variety of cholesterol precursors into cholesterol (Kabara et al., 1957, 1958; Kabara and Okita, 1959, 1961). Evidence for synthesis in the adult animal was made more obvious by restricting food intake and calculating the data on an activity per g tissue basis rather than specific activity (Kabara and Okita, 1961). Our present findings have shown that the synthetic capacity of young and old animals is partially a function of calculating the data to different denominators. The calculation of glucose and acetate incorporation data on a specific activity basis shows that there was a decrease rate with age. However, where differences in sterol concentration were taken into account as a diluent of newly synthesized cholesterol, a closer comparison between liver and brain synthesizing ability was noted. Further, by taking the differences in total body weight of the animals in the various age groups as another factor, there s e e m to be little if any difference in neural metabolism between younger and older animals. Any difference which may exist, suggests a greater rather than a lower capacity for adult animals. A fourth correction factor could also be introduced. If the cell density of the tissues was examined, the total number of cells, (neurons, glia, and vascular cells) i n the brain is calculated to be ten times less per unit wet weight than in liver (Nurnberger and Gordon, 1956). Using such a correction factor, the brain compares even more favorably with liver as an active site for cholesterol synthesis. Experiments reported here indicates that the brain of adult mice seems to have the same or greater synthesizing ability as younger animals only when proper correction factors are applied to the data. It is emphasized therefore that data from tracer experiments must be considered on several bases before comparison between groups can be made. Failure to appreciate the changing base-lines in the Rrfrrencrs p

160

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JON J . K A B A R A

developing animal in the past, has led to different interpretation of the same experimental data. SUMMARY

Animals representing three age groups were studied by simultaneous injection of acetate-2-3H and glucose-U-14C. Free cholesterol was extracted from the liver, spleen, and brain at various times after precursor injection. These studies indicate that there is a general decrease in incorporation of isotopes into cholesterol of the various tissues. when calculated on a specific activity basis. When the pool size of the animal is considered, the older animals incorporate as much or more radioactivity. Because of the pattern and degree of labeling exhibited by these animals, it has been made obvious that age, sex, time after injection, isotope, and pool size must be considered in the interpretation of experiments concerned with cholesterol metabolism. ACKNOWLEDGEMENT

The work presented was supported in part by grants from the National Institute of Health. Division of Neurological Diseases and Blindness (B-2286) ; Muscular Dystrophy Association of America and the Multiple Sclerosis Society (Grant Nr. 226). REFERENCES AZARNOFF, D. L.. CURRAN, G. L., AND WILLIAMSON, W. P.. (1958); Incorporation of acetate 1J4C into cholesterol by human intracranial tumors in vitro. J . nut. Cancer Inst., 21, 1109-1 114. KABARA, J. J., (1957); A quantitative micro method for the isolation and liquid scintillation assay of cholesterol. J. Lab. din. Med.,50, 146-151. KABARA, J. J., (1961): Brain cholesterol 111. Effect of starvation on the incorporation of acetate2-3H and G l ~ c o s e - U - ~ ~VII C . Transactions International Congress of Neurology, Rome. KABARA, J. J., MCLAUGHLIN, J. T., AND RIEGEL, C. A., (1961); Quantitative microdetermination of cholesterol using tomatine as a precipitating agent. Analyt. Chern., 33, 305-307. KABARA, J. J., AND OKITA,G. T., (1959); Incorporation of select precursors into brain cholesterol. Fed. Proc., 18, 1610. KABARA, J. J., AND OKITA,G. T., (1961); Brain cholesterol: biosynthesis with selected precursors in vivo. J . Neurochem., 7 , 298-306. KABARA, J. J., OKITA,G. T., AND LEROY,G. V., (1957); Cholesterol metabolism in normal and tumor mice. Proc. Am. Assoc. Canc. Res., 2, 219. KABARA, J. J., OKITA,G. T., AND LEROY,G. V., (1958); Simultaneous use of 3H and 14Ccompound to study cholesterol. Liquid Scinti//ation Counfing. C. Bell. Editor. London, Pergamon Press fpp. 19 1-197). MCMILLAN, P. J., DOUGLAS, G. W., AND MORTENSEN, R. A., (1957); Incorporation of 14C- of acetatelJ4C and pyruvate-2-14C into brain cholesterol in the intact rat. Proc. SOC.e x p . Biol. (N.Y.), 96, 738-741. MOSER,H. W., AND KARNOVSKY. M. L., (1959); Studies on the biosynthesis of glycolipides and other lipides of the brain. J . 6ioZ. Chem., 234, 1990-1997. NICHOLAS, H. J., AND THOMAS, B. E., (1959); The metabolism of cholesterol and fatty acids in the central nervous system. J . Neurochem., 4, 4249. NURNBERGER, J. I., AND GORDON,M. W., (1956); Effects of brief stress on the ribonucleic acids and the labile nitrogen pool of brain and liver in the rat. Book of Neurochemistry. S. R. Korey, and J. I. Nurnberger, Editors. London, Cassell (p. 124). PAOLETTI, R., PAOLETTI, P., AND VERTUA, R., (1960); Aspects of the biosynthesis of cholesterol and fatty acids. Panminerva med., 2, 306-309. SRERE, P. A., CHAIKOFF, I. L., TREITMAN, S. S., AND BURSTEIN, L. S., (1 950); The extrahepaticsynthesis of cholesterol. J . biol. Chem., 182, 629-636. SPERRY, W. M., (1963); Quantitative gravimetric analysis of brain lipids. Clinical Chem., 9, 241-249. WAELSCH, H., SPERRY, W.M., ANDTSTOYANOFF, V. A., (1940); Lipid metabolism in brain during myelination. J . biol. Chem.. 135, 297-308.

161

Esterase Activity and Zymogram Patterns in Developing Rat Brain J. B E R N S O H N , K. D. B A R R O N

AND

A. R. HESS

Neuropsychiatric Research Laboratory, Veterans Administration Hospital, Hmes, Ill. ( US.A .) and The Departtnent of Neurology and Psychiatry, Northwestern University School of Medicine, Chicago, 111. (U.S.A.)

It has been reported that as many as 20-25 different esterase bands can be demonstrated in human brain by electrophoretic separation of the enzymes on starch-gel (Barron, Bernsohn and Hess, 1963). Despite the lack of knowledge of the properties of the esterase isozymes which produce such a complex array of enzymes, it is of interest to ascertain whether this variety of molecular species of esterase is present in the developing brain. To relate brain maturation and morphological changes to alterations in enzymatic patterns, might provide correlations as to the uniqueness of the isozymes and provide information on the isozyme types associated with brain maturation. METHODS

White albino rats (Sprague-Dawley) were obtained commercially and the litters produced were used. The animals were sacrificed by vascular perfusion with chilled saline. Both 1 and 5-day old animal brains were pooled, but assays of individual brains were performed on the older animals. A 1:3 homogenate with distilled water was prepared from these brains. The homogenate was frozen and thawed 6 times, a n aliquot removed for quantitative assay and the preparation was centrifuged at 20,000 x g for 1 h at 2". The supernatant was decanted and this was used for quantitative assay as well as for starch-gel electrophoresis. Starch-gel electrophoresis was carried out by the vertical technique as described by Smithies (1959) except that a 0.04 A4 borate buffer was employed. The pH of the gel and buffer vessels was 8.35. The bands of esteratic activity were visualized with a-naphthyl esters as substrates as described by Hunter and Markert (1957). The method of Nachlas and Seligman (1948) with some slight modification was used to quantitate the esterase activity of total homogenate and supernatant. RESULTS

The zymogram patterns of esterase isozymes from whole brain obtained from 1-, 5-, References p. 164

162

J. B E R N S O H N . K . D. B A R R O N A N D A. R. H E S S

10- and 90-day old rats are shown in Fig. 1.Onlyanodal-migrating enzymes are present, but a number of differences between young and older brain appear. In the 1-day old animal, the area (designated by A) demonstrates very little activity but becomes denser as the animal maturates and in the 90-day brain resolves into 4 distinct bands. The

Fig. I . Esterase isozymes in developing rat brain. The numbers refer to the age of the animal in days for each pair of zymograms. The letters are explained in the text.

band (B) in the 1-day-old animal tends to diminish and in the older animal at least 2 bands appear in this area with the lighter one in the same position as in the younger animal, but in addition a dense band of enzymatic activity distal but contiguous with this band develops. Area C consists of a light band in the young animal which disappears on ageing, and area D has a dense zone in the 1-day-old brain which progressively decreases in activity with age. In the 90-day-old animal a dense band appears distal to the origin which is not present in the young brain (area D). To ascertain the degree to which the supernatant may reflect the total activity, quantitativeassays on homogenateand supernatant were done and are shown inTable1. It can be seen that the total esterase activity of brain is relatively constant from 1-10 days of age but increases markedly to 20 days of age and increases more slowly to maturity. However, in the supernatant the amount of extractable enzyme is constant at all age levels. Thus, the increase i n esterase activity occurs in the ‘bound’ state, but

163

ESTERASE ACTIVITY A N D ZYMOGRAM PATTERNS

the ‘free’ enzymatic activity does not change over this period of time. This applies particularly to a-naphthyl acetate activity. The amount of soluble enzyme activity TABLE 1 ESTERASE A C T I V I T Y I N D E V E L O P I N G R A T B R A I N

Age (days)

NA*

1 5 10 20 30 Mature

257.5 295.2 284.2 547.5 494.4 590.8

Hoiriogenate NP* * NB*** ( p M / g wet ii.ww/ii)

106.4 155.1 184.4 361.6 31 1.4 390.8

30.4 34.0 36.4 59.8 81.8

70.6

NA

SNpernatant NP (EcMlg wet tissuelh)

NB

171.3 186.9 130.0 122.4 113.7 157.8

48.6 64.8 44.1 57.2 43.4 76.3

20.4 24.0 13.6 23.1 23.6 21.8

* NA = a-naphthyl acetate * * NP = a-naphthyl proprionate * * * NB = a-naphthyl butyrate responsible for a-napthhyl acetate hydrolysis ranges from about 66% in the 1-day brain to 26% in the mature brain.

DISCUSSION

It is apparent that the supernatant preparation which is subjected to starclvgel electrophoresis represents only a fraction of the total esterase activity, particularly in the adult animal. It is not possible to ascertain how accurately the zymogram pattern represents the total activity. Attempts to extract a larger part of the ‘bound’ enzyme by sonic disruption produced zymograms in which most of the esterase activity remained a t the origin, and otherwise yielded no qualitative differences from that of the nonsonicated preparation. That the ‘free’ esterase activity represents enzyme which may be released from the ‘bound’ state during homogenization seems unlikely, since the ‘free’ esterase is constant throughout brain development though the total esterase activity is more than doubled during the same period. The variations in isozyme pattern as well as the changes in esterase activity occurring in the ‘bound’ state show the greatest transition between 10-20 days of age. This period of brain development in the rat is associated with active myelin deposition (McIlwain, 1959). The heterogeneity of esterase isozymes may be related either to the diversity of cell types found in the brain, or may be associated with the variety of structures within the cell. That the latter is the more reasonable explanation stems from the large number of esterase isozymes found in liver (unpublished observation) where the number of cell types is not as large. Refrrences p . I 6 4

164

J. B E R N S O H N , K. D. B A R R O N A N D A. R. HESS

SUMMARY

In the developing rat brain, the esterase isozyme pattern becomes more complex with time and at maturity at least 14 separate isozyme bands can be identified under the conditions described. The total esterase activity of the brain doubles in value during this period of time, but the increase occurs in the ‘bound’ enzyme only, since the ‘free’ esterase activity is relatively constant during development. ACKNOWLEDGMENT

This work was aided in part by a grant (No. 277) from the National Multiple Sclerosis Society. REFERENCES BARRON, K. D., BERNSOHN, J., AND HESS,A. R., (1963); Separation and properties of human brain esterases. J. Histochenz. Cyrochem., 11, 139-155. C. L., (1957); Histochemical demonstration of enzymes separated by HUNTER, R. L., AND MARKERT, zone electrophoresis in starch-gels. Science, 125, 1294-1295. MCILWAIN,H., (1959); Biochemistry and rhe Central Nervous System. Boston, Little Brown Co., p. 187. NACHLAS, N. M., AND SELIGMAN, A. W., (1948); Evidence for specificity of esterase and lipase by use of three chromogenic substrates, J. biol. Chem., 181, 343-355. O., (1959); Advances in Protein Chemistry. New York. Academic Press XIV. p. 65. SMITHIES,

165

Interrelationships of Glucose, Glutamate and Aspartate Metabolism in Developing Rabbit Brain * K E N N E T H F. S W A I M A N . J E R R O L D M. MTLSTEIN

AND

M A Y N A R D M. C O H E N

Division of Neurolop. University of Minnesota Medical Scliool, Minneapolis. Minn. ( U . S . A . )

Studies in this laboratory have demonstrated that gluta!iiate and aspartate have different metabolic effects than glucose when utilized as substrate for the in vitro metabolism of cerebral cortex slices prepared from mature animals (Chain et al., 1962; Cohen et al., 1962). Clinical observations of newborn infants have revealed apparent normal central nervous system activity despite exceedingly low blood glucose concentration (Rapoport, 1959). The distinct possibility arises that glucose plays a different and less pivotal role in immature brain metabolism. This study was undertaken to assess the relative contribution of glucose, glutamate and aspartate to oxidative decarboxylation in i mmature rabbit brain. MATERIALS A N D M E T H O D S

2-4 litters composed of 8 rabbits were employed i n each series. Two littermates of each age group were sacrificed at a time. Two slices 0.3 m m in thickness were prepared from each cerebral hemisphere of each rabbit employing a glass guide and an elongated razor blade after the technique of McIlwain (1951). The slices were kept cold during preparation and subsequent handling. They were weighed after removal of excess moisture and incubated for 2 h in Warburg flasks within 30 min of sacrifice. Saline solutions The following concentrations of constituents were present in the oxygenated media: 0.98 M NaCI, 0.027 A4 KCI, 0.0012 M MgS04, 0.004 M KHzP04, and 0.0175 M NaZHPOI. Glucose, glutamate and aspartate when present were 0.005 M , 0.0067 M and 0.0067 iM respectively. pH was carefully adjusted to 7.4. The flasks were prepared and incubated in the manner previously described (Swaiman et al., 1963).

* This investigation was supported by grant number B-3364 (Neurological Research Center in Cerebrovascular Disease) from NINDB. References p . 1681169

I66

SWAlMANetd.

K. F.

Radioactive materials [U-14C]glucose, ~-[U-'~C]glutamate"and L-[ U-'4C]aspartate* were employed. To 29.8 nil of the saline solution 0.2 ml of a stock solution of the intended substrate solution containing a total of 5.0 ,uC was added. In each flask 0.5 ,uC of radioactivity was present in 3.0 ml of media. Incubation of the slices and subsequent counting of the center well NaOH was carried out as previously reported (Swaiman et al., 1963). Titration Titratioii of the center well NaOH under nitrogen was carried out with an automatic titrator** utilizing 0.030 N HCl. The center well NaOH was prepared for titratioii by quantitative removal with Pasteur pipettes, including 3 rinsings with a total of 0.9 ml of COs-free water. The solution was then brought to a 5.0-ml volume. A 1.0-ml aliquot was removed for counting and a 3.0-ml aliquot was removed for titrating. RESULTS

Oxygen uptake Studies of oxygen uptake (Fig. 1) reveal an increase as the animal ages when glucose 100

-

90 -

SUBSTRATE(S1

.

GLUCOSE L- GLUTAMATE L- GLUTAMATE t GLUCOSE 0 L- ASPARTATE L-ASPARTATE + GLUCOSE b

0

80 70

-

st

Pt

~

~

60 .

T

50-

T

P P

I

2

4

8 DAYS OF AGE

Fig. I . Oxygen uptake of immature rabbit brain slices.

* **

Nuclear-Chicago Corporation. Metrohm, Switzerland.

16

167

OXIDATIVE DECAKBOXY LATION I N IMMA'TURE BRAIN

is the sole oxidizable substrate. A particularly great increase is noted between the 8th and 16th day of life. Values comparable to the adult are obtained at 16 days. The addition of L-glutamate or L-aspartate to media containing glucose did not affect the rate of oxygen uptake. When glutamate was the sole oxidizable substrate the rate of oxygen consumption did not differ from that obtained with glucose until the 8th day, and no striking changes are observed until the 16-day-old animal is employed. At this point the effect of high potassium concentration described by Ashford and Dixon (1935) is quite clear. Studies utilizing L-aspartate alone show L-aspartate to bea poor substrate for oxidation at all ages studied.

2

4

1

SUBSTRATE L

IS)

U-"C

L41"TAMlllt

. d

. 0

,

: a

I

L GtUlP.MbTE

LABELEU SUBSTRATE

GLUCOSE

GLUCOSE

L-GLUTAMATE 1

GLUCOSE

L-GLUTbMITE

L GLUTAMATE *GLUCOSE

GLUCOSE

i IrSPbRTATE

L-ISPARTblE

L 4SPARTATE L ASPARTATE

f

GLUCOSE

GLUCOSF

GLUCOSE

L ASPbRTITE

I

.

,

2

4

Fig. 2. Relative specific activity of COz evolved from various U-14C labeled substrates.

Oxidative decarboxylation When [U-14C]glucose was utilized approximately 40 % of the COZ stemmed from the glucose in the 2-, 4- and 8-day animal brain (Fig. 2A). Studies of the 16-day-old brain revealed 60 % of the COz to stem from the labeled glucose. Addition of radioinert L-glutainate to the labeled glucose media markedly depressed the R.S.A. of the CO:! collected at all ages studied, despite the fact that oxygen uptake was rising (Fig. 2A). When [U-14C]labeled L-glutamate was utilized along with radio-inert glucose 45 % References p . 1681169

168

K. F. S W A I M A N et

a/.

to 50% of the COZ was labeled (Fig. 2B). Thus when glucose and glutamatc werc employed together there appeared to be a large amount of glutamate being convcrted to COz. In the 16-day animal glucose was oxidized at a slightly greater rate (Fig. 2A). When ~-[U-l~C]glutamate was utilized alone about 45 % of the COZoriginated from the glutamate (Fig. 2B). When [U-l4C]aspartate was employed as the sole oxidizable substrate in the media about 40% of the CO2 originated from the aspartate (Fig. 2C), despite the low rate of oxygen uptake (Fig. 1). The addition of radioactive L-aspartate to the media depressed the oxidation of [U-14C]glucose in the 16-day-old animal as indicated by the R.S.A. of the C02 evolved (Fig. 2A). When the media contained UJ4C labeled L-aspartate and non-radioactive glucose there was little change in the per cent of C02 originating from aspartate compared to experiments when aspartate was utilized alone (Fig. 2C). However, the addition of glucose to the media resulted in a great increase in oxygen uptake of the brain slices in all ages studied (Fig. 1). DISCUSS1 OK

Oxygen uptake in the presence of glucose as the only added oxidizable substrate increases as the animal matures and reaches adult levels by the 16th day. The addition of glutamate or aspartate to glucose in the media does not change the rate of oxygen consumption. Glutamate depresses glucose oxidation in all ages studied (Fig. 2A). Despite the increasing oxygen uptake with age, when glutamate and glucose are present 45 % of the COZ stems from glutamate (Fig. 2B). This phenomenon occurs at a time when glutamic acid decarboxylase is reported to have 30% to 40% of activity of mature brain (Himwich et a/., 1961). This fact suggests that glutamate is not principally oxidized through a pathway involving the formation of y-aminobutyric acid in the developing brain. SUMMARY

( I ) Various combinations of UJ4C labeled and non-radioactive glucose, L-glutamate, and L-aspartate are utilized as oxidizable substrates of immature rabbit brain slices. (2) Glutamate depresses glucose oxidation beginning with the youngest brain studied. Aspartate depresses glucose oxidation in the 16-day-old animal. (3) Glutamate is more readily oxidized than glucose when both are present in the media. This occurred at all ages studied. ( 4 ) The relative specific activity of COz collected when UJ4C labeled aspartate or UJ4C labeled glucose are utilized as sole oxidizable substrates is comparable in the most immature animals. However, aspartate alone poorly supports respiration of the slices. REFERENCES ASHFORD, C. A., AND DIXON,K. C., (1935); The effect of potassium on the glucolysis of brain tissue with reference to the Pasteur effect. Biochem. J., 29, 157-168.

OXIDATIVE DECARBOXY L A T l O N I N IMMATURE BRAIN

I69

CHAIN,E. B., COHEN,M. M.. AND POCCHIARI, F., (1962); Interrelationship of glucose, glutamate and aspartate metabolism in rat cerebral cortical slices. Proc. roy. Soc. B, 156, 163-167. COHEN,M. M., COHEN,H. P., AND CHAIN,E. B., (1962); Effect of glutamic acid on phosphorylative activity in cerebral tissue in virro. Acra neurol. scnnd., 38, Suppl. 1, 12. COHEN,M. M., SIMON,G. R.. BERRY, J. F., AND CHAIN.E. B., (1962); Conversion of glutaniic acid into aspartic acid in cerebral cortex slices. Biochem. J., 84, 43-44. HIMWICH, W., PETERSEN, J., AND GRAVES, J., (1961 ): Recent Advances in Bic~~ogicalPsyc/zratry. Vol. 111. New York, Grune and Stratton (p. 218-226). M C ~ L W A IH., N ,(195 I ) ; Metabolic response in vitro to electrical stimulation of sections of mammalian brain. Biochem. J., 49, 382-393. RAPOPORT, M., (1959); Textbook of Pediatrics. W. E. Nelson, Editor. 7th Edition. Philadelphia. Saunders (p. 121 5). SWAIMAN, K. F., MILSTEIN, J. M., AND COHEN,M. M., (1963); Interrelationships of glucose and glutamic acid metabolism in developing b r i n . J. Neurochem.. 10, 635.

170

Biochemical Studies of the Central Nervous System of the Dog during Maturation A. R. D R A V I D

AND

W. A. H I M W I C H

Tliudicl~uniPsycliiatric Research Laboratory, Calesbirrg State Research Hospital, Galesburg, Ill. (U.S.A.)

In the course of the development of the nervous system profound biochemical changes occur along with differentiation of the structural components. In our laboratory research has been centered on the developing dog brain for studies of the concomitant maturation of neurological functions and chemical composition. The present work on free amino acids and phospholipids in the developing dog brain was undertaken hoping that eventually a correlation could be established between the chemical composition and function of the central nervous system. METHODS

Young dogs at various ages from birth were sacrificed by decapitation, the brains removed quickly in a cold room and frozen on dry ice. Adult dogs were anesthetized with intravenous pentobarbital, the brains removed as quickly as possible and frozen on dry ice. Extraction of amino acids was carried out with 70 % ethanol, the extracts evaporated to dryness and dissolved in a small quantity of water. Two dimensional paper chromatography was used for the estimation of the amino acids. Solvent systems: 1st phase, isopropanol : acetic acid : water (10 : 2.4 : 2); 2nd phase, phenol : water (4 : 1). The lipids were extracted by the method of Folch-Pi (1955). Silicic acid column chromatography as described by Kishimoto and Radii1 (1959) was used with modifications to fractionate the lipids. RESULTS

Glutamic acid, glutamine, y-aminobutyric acid and aspartic acid were determined in whole brains of puppies at various ages after birth and in adult dog brains. Two fetal brains of known prenatal age were also used for the above analyses. The data summarized in Table I indicate a gradual increase in glutamic, aspartic and y-aminobutyric acids with age, reaching the adult levels by the age of 70 days. The latter two substances, however, show little change after 20 days of age. Glutamine, on the other hand was found to be present in considerably higher amounts in the two fetal brains

171

BIOCHEMICAL STUDIES OF T H E DOG BRAIN

TABLE 1 F R E E AMINO ACIDS I N DOG BRAIN D U R I N G MATURATION

pmoleslg wet weight Figures in parentheses are numbers of samples ._____ -~

*

Age in days

Clufaniic acid

Cluraniine

5 Days (2)* 1 Days (9) 10 Days ( 5 ) 20 Days (4) 30 Days ( 5 ) 40 Days (3) 70 Days (2) Adult (2)

3.18 3.89 4.50 5.78 6.95 7.85 10.57 11.02

6.15 4.49 4.54 4.17 4.10 3.28 3.64 3.90

y- A minobufyiYc

acid

Aspartic acid

0.99 0.92 1.13 2.13 2.00 2.20 1.87 2.37

1.41 I .01 1.48 2.38 2.17 2.18 2.42 2.51

Prenatal.

studied and shows a slight decrease from birth to adulthood, again reaching the adult level by 70 days of age. The studies on brain lipids were performed on whole brains from I-30-day-old dogs and adult dogs (Fig. 1). The phosphatides are present i n the brains on the first

= CHOLESTEROL

P. \a

'I

__10

'15

20

30

-, J-o

=LECITHIN P. = SPHINGOMYELIN P. =SERINE P.

- ADULT

AGE IN DAYS

Fig. 1. Cholesterol and phosphatides in dog brain during maturation.

day of postnatal life and little change is observed in the deposition of these lipids up to the age of 15 days. By 20 days of age the values of phosphatides, with the exception of sphingomyelin, are significantly decreased ; by 30 days of age ethanolamine and Refrrpnces p. 173

I72

A. R. D R A V I D A N D

W.

A. H I M W I C H

lecithin values return to the one day level. The adult whole brain values for phosphatides are slightly lower than 30-day-old brains. Cerebrosides were first detected in measurable quantities in dog brains 20 days after birth, however, traces of it were observed in 15-day-old brains. The deposition of cholesterol during the period under consideration shows little change, however, some decrease was observed in dog brains 20 days after birth. D I S C U S S I ON

Amino acids in the developing brains of various mammalian species have been previously reported (Himwich and Petersen, 1959; Waelsch, 1951 ; Vernadakis and Woodbury, 1962). A gradual increase with growth was observed in most of the species studied. Considering the magnitude of metabolic functions subserved by these amino acids in the brain, the increase seems to parallel the overall increase in the metabolism of the brain, however, it is possible that a part of these amino acids are present in an inactive pool. The pattern of the sum of glutamic acid and glutamine is similar to that reported by Himwich and Petersen (1 959) using different methods. They postulated a 65% of the adult compliment of these two substances as a necessary level for the appearance of an adult type EEG. However, in the companion paper by DiPerri et al. (this volume, p. 89) a qualitatively adult type EEG was observed at 4 weeks at a time when the glutamic acid and glutamine level is 74% of the adult. Since the appearance of such an EEG seems to be dependent on the growth of cell processes the attainment of a given level of these substances probably serves only as an indicator of cellular development. The changes in GABA concentration during maturation are worthy of mention. Pylkko and Woodbury (1961) have shown a correlation between GABA concentration and convulsant potencies of strychnine and of brucine with age. The role of GABA asan inhibitory substancein the nervous system has been extensively studied but is still poorly understood. However, further work is necessary to elucidate the function of GABA or a closely related metabolite of it. The lipid concentration of the brain increases markedly during the period of early development as a concomitant of myelination (Folch-Pi, 1955). Sperry (1955), however, pointed out that the amount of lipids deposited in the process of myelination was relatively small in proportion to the increase as a result of growth. The results presented here agree with those of Sperry (1955) on developing rat brain, however, the period from 15-30 days after birth seems to be of critical importance in the maturation of the dog brain in terms of lipid content. Cerebrosides and sphingomyelin are believed to be typical myelin lipids (Folch-Pi, 1955; Davidson and Wajda, 1959). Our results suggest that extensive myelination does not occur in the dog until 20 days of age. The appearance of the adult type of EEG would seem to indicate the approach of the adult chemical composition. It occurs near the end of the third week (15-30 days of age) (DiPerri et al. this symposium) which is of critical importance in considering the lipid content and at a time when nearly the adult levels of GABA and aspartic acid are present and the glutamic acid content is 63 % of that in the mature animal.

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

173

SUMMARY

Amino acids and various lipid fractions were determined in whole brains of puppies at various ages from birth and in adult dog brains. The studies on amino acids indicate a gradual increase in glutamic, aspartic and y-aminobutyric acids with age, reaching the adult levels by the age of 70 days. Glutamine, on the other hand, was found to be present in higher amounts in the two fetal brains studied and in the newborn animals, showing a decrease from birth to adulthood, again reaching the adult levels by 70 days of age. The results on brain lipids show the presence of phosphatides on the first day of postnatal life and little change is observed in relative proportions of these lipid fractions expressed as percent of lipid up to the age of 15 days. By 20 days of age, however, values of phosphatides with the exception of sphingomyelin, are significantly decreased. Cerebrosides were first detected in measurable quantities in 20-day-old dog brains. The deposition of cholesterol during the period under consideration shows little change. The results are discussed in terms of neurological maturation of the dog. REFERENCES DAVIDSON, A. N., A N D WAJDA,M., (1959): Metabolism of myelin lipids. Estimation and separation of brain lipids in the developing rabbit. J . Neurochem., 4, 353-359. FOLCH-PI,J., ( I 955); Biochemistry of the Developing Nervous Sysfem. New York. Academic Press. HIMWICH, W. A., AND PETERSEN, J. C., (1959); BiologiculPsychiutry, New York. Grune and Stratton. KISHIMOTO, Y., AND RADIN, N. S., ( I 959); Isolation and determination methods for brain cerebrosides, hydroxy fatty acids, and unsaturated and saturated fatty acids. J . Lipid Res., 1, 72-78. PYLKKO, 0. O., AND WOODBURY, D. M., (1961); The effect of maturation on chemically induced seizures in rats. J. Pharmucol. exp. Ther., 131, 185-190. SPERRY, W. M., (1955); Biochemistry o f t h e Developing Nervous System. New York. Academic Press. VERNADAKIS, A., AND WOODBURY, D. M., (1962); Electrolytes and amino acid changes in rat brain during maturation. Amer. J . Physiol., 203, 748-752. WAELSCH, H., (1951); Advunces in Protein Chemistry. Vol. VI. New York. Academic Piess.

174

Effect of Acute and Subacute Administration of Diphenylhydantoin on Electroshock Seizure Threshold in Developing Rats ANTONIA VERNADAKIS

AND

D I X O N M. W O O D B U R Y

Deportment of Pharmacology, University of Utah College of Medicine, Salt Lake City, Utah (U.S.A.)

Previous studies in this laboratory (Woodbury, 1954) have demonstrated that when diphenylhydantoin (Dilantin) is administered acutely in intact adult rats it does not affect the electroshock seizure threshold (EST). However, if Dilantin is given acutely in adrenalectomized rats or chronically in intact rats it significantly elevates the threshold (Woodbury, 1954). It has also been shown (Woodbury, 1954) that Dilantin stimulatcs the pituitary-adrenal system. The adrenocortical steroids released as a result of this stimulation may antagonize the threshold-elevating effects of Dilantin. Since the pituitary-adrenal axis in developing rats is not functioning completely during the first two weeks after birth (Jailer, 1950), the EST-elevating effect of Dilantin should be more marked than in mature animals. The following experiments were designed to investigate this hypothesis. METHODS

Experimental design. All experiments were performed on Sprague-Dawley rats. Animals younger than 21 days of age were kept with their mothers. The following experiments were performed : ( 1 ) Acute administration of Dilantin. Dilantin, 20 mg/kg body weight, was administered subcutaneously in rats 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, and 19 days of age. Each age group consisted of 20 rats and was compared to appropriate nonmedicated controls. The electroshock threshold for minimal seizure was measured 45 min after administration of the drug. (2) Subacute administration of Dilantin. Dilantin, 20 mg/kg body weight, was administered subcutaneously daily in rats from the 8th to 11th day after birth. The electroshock seizure threshold was measured daily, from the 12th to 26th day in both treated and control rats. Electroshock Procedure. Minimal electrical stimulus was applied by corneal electrodes for 0.2 sec with a 60-cycle, alternating-current electroshock apparatus (Woodbury and Davenport, 1952). The current for evoking a minimal convulsion in

175

DILANTIN ON ELECTROSHOCK SEIZURE THRESHOLD

50 % of animals together with the 95 % fiducial limits was calculated by the method o f Litchfield and Wilcoxon (1949). RESULTS

Fig. 1 illustrates the thresholds in control rats and rats treated with a single dose of Dilantin 45 min prior to the measurement of thresholds. Dilantin increased the EST

W

- 604

w 50

1

N

I

W J

08

9

10 II

12 13 14 15

19 20 21 22 23 24 25 26

31 32 33 34

40

W

DAYS AFTER BIRTH

Fig. 1. Electroshock seizure threshold (EST) in control ( 0 )and Dilantin-treated (45 min) (0)rats during development. Each point represents the EST 50 (see text for explanation). Only when the thresholds in the Dilantin-treated rats differ significantly from those in the control animals are the 95 % fiducial limits indicated (by the vertical bracketed lines). Ordinate is electroshock seizure threshold in mA and abscissa is days after birth.

in rats during the first 2 weeks. The markedly elevated EST in 13- and 14-day old rats at present cannot be explained. After the first 2 weeks the EST was not affected by Dilantin. Fig. 2 illustrates the thresholds in control and rats treated with Dilantin for 4 days prior to the measurement of thresholds. The EST was lower in the Dilantin-treated rats than in the controls from the 12th to the 22nd day of age and did not differ thereafter. DISCUSSION

The present results suggest an age-dependent biphasic effect of Dilaiitin on the electroshock seizure threshold of maturing rats. The rnechanisin of action of Dilantin on this parameter of brain excitability can at present only be speculated. If we assume that Dilantin stimulates both inhibitory and excitatory systems, and that during the first 2 weeks the inhibitory system is predominant, then its depressant effect would be evident during this period. Evidence that the inhibitory system is predominant during early development is provided by the fact that in normal newborn animals the threshold is very high and decreases with maturation. After the first 2 weeks the RC(C).EIIWF p. 177

176

A. V E R N A D A K I S A N D D.

M. W O O D B U R Y

threshold-elevating effect of Dilantin may be antagonized by developing subcorticalcortical systems that are stimulated by Dilantin. One of these subcortical excitatory systems is the hypothalamo-adenohypophysealadrenal (HAA) system. In adult rats Dilantin has been shown to stimulate this system 24

n I

a

20 19

g

3

18 17

VJ Y

0

l6 15

v)

2

14

2 13 I-

w

12 II

10 12 13 14

15 16

17 18 19 20 21 22 23 24 25 26

DAYS AFTER BIRTH

Fig. 2. Electroshock seizure threshold (EST) in control (e) and Dilantin-treated (8th-I lth day) (0) rats during maturation. (See legend for Fig. 1).

(Woodbury, 1954; Woodbury et al., 1957). Furthermore, when cortisone is given to Dilantin-treated animals the threshold-elevating effect of Dilantin is not apparent (Woodbury, 1954). In addition, in adrenalectomized rats Dilantin elevates the threshold (Woodbury, 1954). Thus, the adrenocortical steroids released as a result of Dilantin-induced HAA stimulation antagonize the direct threshold-elevating action of Dilantin. When Dilantin was given chronically (8th to 1 1th day of age) to the maturing rats, the threshold was lowered (Fig. 2). It can be speculated that administration of Dilantin for 3 days induces precocious functional development of the HAA system. Hence, the excitatory action of the adrenocortical steroids on the nervous system appears earlier as a result of the Dilantin-induced HAA stimulation. It was postulated (Vernadakis and Woodbury, 1963) that cortisol, given during the period between 8 and 12 days after birth, significantly lowers the electroshock seizure threshold by enhancing myelination of higher nervous centers. Myelination confers upon a nerve fiber the properties of lowered threshold. Thus, the threshold-lowering effect of Dilantin, when given from the 8th to 1 lth day after birth, is thought to be secondary to its excitatory action on the HAA system.

D I L A N T I N O N ELECTROSHOCK S E I Z U R E T H R E S H O L D

SUMMARY

AND

177

CONCLUSIONS

The following tentative conclusions can be drawn from the above results: ( I ) during early development when subcortical excitatory areas have not fully matured, the threshold-elevating effect of Dilantin is apparent ; (2) Dilantin given chronically during early development exerts a threshold-lowering effect which lasts after the period of treatment. It can be speculated that, in this case, Dilantin influences some subcortical excitatory system (for example, H A A ) which in turn antagonizes its effect on the cortex. REFERENCES JAILER,J. W., (1950); The maturation of the pituitary-adrenal axis in the newborn rat. Endrocrinology, 46,420425. LITCHFIELD, J . T., JR., AND WILCOXON,F., (1949); A simplified method of evaluating dose-effect experiments. J . Pharmacol. exp. Ther., 96, 99-1 13. VERNADAKIS, A., AND WOODBURY,D. M., (1963); Effect of cortisol on the electroshock seizure thresholds in developing rats. J . Pharmacol. exp. Ther., 139, 110-1 13. WOODBURY, D. M., (1954); Effect of hormones on brain excitability and electrolytes. Recent Progr. Hormone Res., 10, 65-107. WOODBURY, D. M., TIMIRAS,P. S., AND VERNADAKIS, A., (1957); Hormones, Brain Function and Behavior. New York. Academic Press. WOODBURY, L. A., AND DAVENPORT, V . D., (1952); Design and use of a new electroshock seizure apparatus, and analysis of factors altering seizure threshold and pattern. Arch. int. Pharmacodyn., 92, 97-109.

178

Postnatal Changes in Animo Acid Metabolism of Kitten Brain SOLL BERL Depar fmrnt of Neurological Surgery, College qf Pliysicians and Snrgeons, Columbia Unirer.si/.v, New York, N. Y . (U.S. A,)

The complexities of the functioning central nervous system in vivu, obviously, will be understood only when sufficient information is available which will correlate the morphology, physiology and biochemistry of the organ. One approach that adds materially toward this goal is ontogenetic studies; these provide data which make possible the integration of structural developmeiit with physiological and biochemical changes. The work of the Flexners (cfi Flexner, 1955) on the developing guinea-pig brain, among others, may be cited as an example of studies designed to provide such correlations. In further fulfilment of this aim a series of biochemical investigations on the developing cat brain has been initiated in which the major objective is to specify factors which are associated with morphological and physiological maturation of various brain areas (Berl and Purpura, 1963). The cat was chosen for these studies because considerable information is already available on the morphological (Noback and Purpura, 1961 ; Voeller et al., 1963) and physiological (Purpura et al., 1960; Purpura, 1961) development of its cerebral cortex. In this paper the postnatal changes in brain content of glutamic acid, glutamine, aspartic acid, y-aminobutyric acid (GABA) and glutathione (GSH) are presented. In addition, preliminary results on the fate of tracer amounts of ~-[1~C]glutamic acid administered into the cisterna magna of newborn and adult animals have been obtained and are herein discussed. METHODS

Postnatal changes in the metabolites investigated were followed in 36 kittens ranging in age from 6 11 to 35 days. Under light ether anesthesia the cerebral hemispheres were exposed and the cerebral cortex removed and frozen in solid carbon dioxide. In the isotope studies ~-[14C]glutamicacid, uniformly labeled, was administered into the cisterna magna of 1-day-old kittens (I pC, 1.24 pg in 0.1 ml of saline) as well as into the cisterna magna of adult animals (10 pC, 12.4 pg in 1 .O nil of saline replacing I ml of spinal fluid). The kittens were decapitated after 3 min, the cerebral hemispheres removed, rinsed in ice-cold saline, the excess fluid absorbed on filter paper and frozen in liquid nitrogen. In the adult animals the brain was exposed prior to intracisternal

P O S T N A T A L C H A N G E S I N AhllNO A C I D METABOLIShl

179

administration of the isotope. Cerebral cortex was taken after 10 min and was similarly rinsed, blotted and frozen. Glutamic acid, glutamine, aspartic acid, GABA and GSH in brain samples (0.2-1 .O g) were isolated and analyzed by resin column methods previously described (Berl et al., 1961 ; Berl et al., 1962). The radioactive samples were counted at infinite thinness in a low background gas flow Nuclear-Chicago instrument. For dry weight determinations tissue samples were oven-dried to constant weight at 1 10-1 20". RESULTS AND DISCUSSION

The postnatal changes in ths metabolites investigated are presented in Fig. 1. It is apparent that adult levels of these substances (Berl et al., 1959; Berl et al., 1961) are 0

GSH

GLUTAMIC ACID

?

:I, 0

5

ASPA R T l C ACID

,

,

10

15

,

20 AGE (days)

I

,

,

25

30

35

Fig. 1. Changes in glutamic acid, glutamine, aspartic acid, y-aminobutyric acid (GABA) and glutathione (GSH) in the developing cortex of the kitten.

reached at different rates. In newborn kittens glutamic acid was approximately 40 %, glutamine loo%, aspartic acid 60%, GABA 70% and GSH 80% of the levels found in mature animals on a wet weight basis. The concentrations of glutamic and aspartic acids increased during 3 to 4 postnatal weeks while GABA and GSH achieved adult levels by the end of the 2nd week. By the 3rd-4th week after birth all the compounds had attained values found in fully developed cat cortex. There did not appear to be any direct correlation between the rates of increase of the metabolites and the dry weight, the former usually increasing at a more rapid rate than the latter. Data on glutamic acid and glutamine levels in the brain of developing animals have Rqferences p . 182

180

S. B E R L

been reported previously. Waelsch (1 95 1) showed a marked increase in glutamic x i d and a smaller change in glutamine on a wet weight basis in the maturing rat. Himwich and Petersen (1959) have compared the total concentration of glutamic acid plus glutamine in several species of animals. All animals, except the guinea-pig showed increases with postnatal development ; however, the representative curve was much flatter for the kitten than for the other animals. Developing rat brain studies of Vernadakis and Woodbury (1 962) have shown progressive increases in glutamic and aspartic acids up to the 25th postnatal day. Glutarnine increases occurred primarily during the first 5 days and GABA attained mature levels prior to the end of the 3rd postnatal week. With the exception of glutamine, all the compounds in the present study are lower in newborn kittens than in adult animals. It is now also quite clear from the work in Purpura’s laboratory (Noback and Purpura, 1961 ; Purpura et al., 1960) that in the cat neuronal elements develop at different rates. The apical dendrites are relatively well developed in the neonatal kitten, whereas basilar dendrites are very poorly developed at birth. The development of the basiler dendritic system of cortical pyramidal neurons in the kitten occurs essentially after birth, whereas apical dendritic growth occurs before birth as well as postnatally. The period of maximal pyramidal neuron growth and differentiation occurs during the 5th-14th days and is completed by the end of the 3rd postnatal week. Stellate cells show less dramatic changes. By the 3rd week the fine structure of the cortical neurons and synapses in the kitten (Voeller et a]., 1963) can no longer be differentiated from that in the adult cat (Pappas and Purpura, 1961). Developmental alterations in a variety of evoked electrocortical activities (Purpura, 1961) occur along with the morphogenetic changes. It should also be recalled that the maturation of superficial and deep neuropil is accompanied by a decrease in the density of cell bodies (Smith, 1934; SchadC and Baxter, 1960; Voeller et al., 1963). These maturational changes occur during the period of maximal increase of glutamic and aspartic acids in particular, as well as of GABA and GSH and suggest that these metabolites are associated with dendritic and axonal development as well as with the cell bodies of the cortical neurons. The level of GABA in the brain of the newborn kitten is greater than that found at birth in the mouse (Roberts et al., 1951), rabbit (Baxter et al., 1960) and chick (Sisken et aZ., 1961). Mature levels are also more rapidly attained in the kitten than in these animals. These differences probably reflect differences in the developmental characteristics of neuronal elements in the newborn cat, rabbit, rodent (Noback and Purpura, 1961) and chick. It may also be pointed out that the GABA levels in newborn kittens are no lower than that frequently encountered in adult cats (Bed et al., 1959; Berl et al., 1961). This suggests that the amino acid may be associated mainly with cell bodies and perhaps apical dendrites in newborn as well as in adult animals. In this connection it is of interest that Baxter et al. (1960) have reported that in the rabbit the largest developmental increases in levels of GABA coincided with the largest increases in the volume of neuronal cell bodies. Although the changes in glutamic acid, aspartic acid, GABA and GSH parallel the morphological development of neocortical elements in the kitten, the constant level

P O S T N A T A L C H A N G E S IN A N I M O A C I D M E T A B O L I S M

I81

of glutamiiie throughout the first month is in striking contrast. One explanation is suggested by previous findings that in adult animals glutamiiie arises from a small but ‘active’ compartment of glutamic arid which is not in rapid equilibrium with the total tissue glutamate (L&ha et al., 1959; Berl et al., 1961 ; Berl et al., 1962). The adult value for glutamine in the newborn in the presence of a greatly reduced glutamic acid level suggests that in these animals glutamate is present mainly in the ‘active’ compartment. If this is so then it is not unlikely that glutamine synthesis occurs predominantly in cell bodies in which the ‘active’ glutamate pool is localized, whereas during development the relatively less active pool of glutamic acid becomes distributed in the dendrites and perhaps elso in the non-neuronal glial elements. Preliminary findings strongly support this hypothesis. Following intracisternal administration of [14C]glutarnic acid in the adult cat, within 10 min the glutaniine specific activity (counts/min/pmole) is 3 times that of the glutamic acid. These findings are in accord with simila,r results obtained in the adult rat and monkey (Berl et al., 1961).Tncontrast. following intracisternal administration of [14C]glutamic acid to one-day-old kittens, after 3 min, the specific activity of the glutamine was approximately 1/3 that of the glutamic acid; the glutamine specific activity relative to that of glutamic acid specific activity was only approximately one-tenth that of the relative specific activity obtained in the adult animal. The physiological significance of these latter results remains to be elucidated. Since glutamine formation, however, is the main pathway for removal of ammonia in brain (Du Ruisseau et al., 1957; Berl et al., 1962), the findings suggest that the maintenance of low brain ammonia levels is of major importance to the neonatal animal. The data also underline the necessity of integrating structure with metabolism for an understanding of metabolic events in v i m . I n this paper conjecture has centered mainly on the relationship of amino acid metabolism to neuronal development. Undoubtedly maturational changes in nonneural elements such as glia must also be taken into account. Lack of such information limits further considerations at the present time. SUMMARY

In the developing cerebral cortex of the kitten, mature levels of glutamic acid, glutamine, aspartic acid, y-aminobutyric acid and glutathione are achieved at different rates; by the 4th postnatal week the concentrations of these metabolites are equal to those found in adult animals. At birth glutamine values are comparable to those present in fully developed animals. A compartment for the glutamic-glutamine system is demonstrable in the adult animal but not in the neonatal animal. ACKNOWLEDGEMENTS

This investigation was supported in part by Public Health Service Research Career Program Award NB-K3-5117 from the National Institute of Neurological Diseases References p . 182

182

S. R E R L

and Blindness, and by grants B-226, B-556 and NB-04064-01 from the National Institute of Neurological Diseases and Blindness, National Institutes of Health. REFERENCES BAXTER, C. F., SCHADB, J. P., AND ROBERTS, E., (1960); Maturational changes in cerebral cortex. 11. Levels of glutamic acid decarboxylase, gamma-aminobutyric acid and some related amino acids. Inhibition in the Nervous System and y-Aniinobutyric Acid. E. Roberts, Editor. New York, Pergamon Press (p. 214-218). H., (1961); Amino acid and protein metabolism. VI. Cerebral BERL,S., LAJTHA,A., AND WAELSCH, compartments of glutamic acid metabolism. J. Neurochem., 7, 186-197. D. P., (1963); Postnatal changes in amino acid content of kitten cerebral BERL,S., AND PURPURA, cortex. J . Neurochem., 10, 237-240. D. P., GIRADO,M., AND WAELSCH, H., (1959); Amino acid metabolism in BERL,S., PURPURA, epileptogenic and non-epileptogenic lesions of the neocortex (cat). J. Neurochem., 4, 31 1-317. G., CLARKE, D. D., AND WAELSCH, H., (1962); Metabolic compartments in vivo: BERL,S., TAKAGAKI, ammonia and glutamic acid metabolism in brain and liver. J. biol. Chem., 237, 2562-2569. BERL,S., TAKAGAKI, G., AND PURPURA, D. P., (1961); Metabolic and pharmacological effects of injected amino acids and ammonia on cortical epileptogenic lesions. J . Neurochem., 7 , 198-209. D u RUISSEAU, J. P., GREENSTEIN, J. P., WINITZ,M., AND BIRNBAUM, S. M., (1957); Studies on the metabolism of amino acids and related compounds in vivo. VI. Free amino acid levels in the tissues of rats protected against ammonia toxicity. Arch. Biochem. Biophys., 68, 161-171. FLEXNER, L. B., (1955); Enzymatic and functional patterns of the developing mammalian brain. Biochemistry ofthe Developing Nervous System. H. Waelsch, Editor. New York, Academic Press (pp. 281-300). HIMWICH,W. A., AND PETERSEN, J. C., (1959); Correlation of chemical maturation of the brain in various species with neurologic behavior. Biological Psychiatry. J. Masserman, Editor. Vol. I. New York, Grune and Stratton (p. 2). LAJTHA,A., BERL,S., AND WAELSCH, H., (1959); Amino acid and protein metabolism of the brain. IV. The metabolism of glutamic acid. J. Neurochem., 3, 322-332. D. P., (1961); Postnatal ontogenesis of neurons in cat neocortex. NOBACK,C. R., AND PURPURA, J. comp. Neurol., 117, 291-308. PAPPAS,G. D., AND PURPURA, D. P., (1961); Fine structure of dendrites in the superficial neocortical neuropil. Exp. Neurol., 4, 507-530. PURPURA, D. P., (1961); Analysis of axodendritic synaptic organizations in immature cerebral cortex. Ann. N . Y. Acad. Sci., 94, 601-654. E. M., (1960); Physiological and anatomical PURPURA, D. P., CARMICHAEL, M. W., A N D HOUSEPIAN, studies of development of superficial axodendritic synaptic pathways in neocortex. Exp. Neurol., 2, 324-347. S., (1951); Gamma-aminobutyric acid content and ROBERTS, E., HARMAN, P. J., AND FRANKEL, glutamic decarboxylase activity in developing mouse brain. Proc. Soc. exp. Biol. ( N . Y.), 78, 799-803. SCHAD~ J. ,P., AND BAXTER, C. F., (1960); Maturational changes in cerebral cortex. I. Volume and surface determinations of nerve cell components. Inhibition in the Nervous System and y-dminobutyric Acid. E. Roberts, Editor. New York, Pergamon Press (p. 207-213). SISKEN,B., SANO,K., A N D ROBERTS, E., (1961); Gamma-aminobutyric acid content and glutamic decarboxylase and gamma-aminobutyric transaminase activities inthe:optic lobe of the developing chick. J. biol. Chem., 236, 503-507. SMITH,C. G., (1934); The volume of the neocortex of the albino rat and the changes it undergoes with age after birth. J. comp. Neurol., 60, 319. VERNADAKIS, A,, AND WOODBURY, D. M., (1962); Electrolyte and amino acid changes in rat brain during maturation. Amer. J . Physiol., 203, 748-752. K., PAPPAS, G. D., AND PURPURA, D. P., (1963); Electron microscope study of development VOELLER, of cat superficial neocortex. Exp. Neurol., 7, 107-130. H., (1951); Glutamic acid in cerebral function. Advanc. Prorein Chem., 6, 301-341. WAELSCH,

183

General Discussion*

O’NEILL:I would like to address a question to Dr. Jilek. This is in relation to his observations on the effects of low oxygen tension. I am curious whether or not he has considered the possibility that in the relatively young and developing nervous system one might find alternate metabolic pathways even though we know with some certaint y this cannot be demonstrated under ordinary circumstances in the adult cerebral cortex. This would help to explain for example some of the effects that he sees with cyanide and iodoacetate. In lactating maminary glands and other tissues one can demonstrate a by-pass of iodoacetate block when you stimulate pentose pathway activity. I would like to rephrase my question to make it clearer perhaps. I wondered if there was a possibility that during this very early developmental stage when there seems to b: a relative resistance to anoxia and stagnant hypoxia whether there is pentose pathway activity operative? Certainly in what we consider to be adult guinea pig or rat cerebral cortex there seems to be little evidence of such an active pathway but during myelinization process when lipogenesis is high you might expect a relatively high activity of the pentose pathway. J ~ L E: K Our experiments show that in the metabolic adaptation in oligemia, stagnant hypoxia, of the nervous tissue the greatest importance has the stimulation of the anaerobic glycolysis. There could be, of course, also the stimulation of another metabolic pathway, as Dr. O’Neill has mentioned. COHEN:I was quite fascinated by some of Dr. Jilek’s neuropathological specimens where it showed that in the developing animal, which did not survive, the principal damage was in the brain stem, whereas in the adult animal the principal damage was in the cortex. Many of us have considered for a long time, that one of the principal reasons that the younger animals may survive in the presence of anoxia was due to some glycolytic mechanism in operation for providing energy. Now it occurred to me in looking at these pictures that in these animals the cortex is not functioning to any great degree and thus had lower energy requirements, The animals that did die did so when the energy requirement of the brain stem was inadequate. When you reach the age of the older animals, then at this time, the cortex is functioning and has a higher energy requirement and I wonder if this difference in energy requirement may not be the key factor in the fatality of these animals rather than a different pathway of metabolism? JLLEK:I agree with Dr. Cohen. But there are at least two factors, which are in play. One of them is a different pathway of metabolism in the nervous tissue of the imma-

*

This discussion refers to the papers of Dr. J. Scherrer and Dr. A. Fourment, Dr. L. Jilek et a[., Dr. J. P. SchadC and Dr. E. G. Pascoe and Dr. S. Berl.

184

GENERAL DISCUSSION

ture animals. The other one is the functional, structural and metabolical maturation of the central nervous system as a whole and especially of its different parts. HARRIS:I n connection with the question which has been raised concerning the absolute dependence of brain upon glucose as a substrate with its utilization largely by way of the glycolytic pathway, I would like to submit results of a study which are pertinent here. This work which was started at Albany Medical College, continues collaboratively there with Dr. Moss and with myself at the Barrow Neurological Institute in Phoenix. The relative participation of the glycolytic and hexose monophosphate shunt was studied in situ in calf brain using a benign differential pressure perfusion technic developed by Moss. By perfusing labelled glucose, glucose-6-14C, glucose- 1J4C, as well as tritium labelled glucose in position 6, and measuring arterial-venous differences of the labelled 14C02 liberated, and the tritiated water (HTO) which was formed, it was found that the shunt pathway was evident to the extent of 70% and upwards to 85%. The results of the HTO which paralleled those of 14C02 from glucose-6-14C thereby offer confirination of this. Although the exact quantitation may be open to question, the data, without doubt, show that the hexose monophosphate shunt is a major pathway for cerebral glucose metabolisin in the young mammal such as the calf. Aside from age and species differences, we feel that previous work indicating a dominant glycolytic pathway suffers because of experimental difficulties. Firstly, by not obviating the participation of the systemic circulation, recycling of the administered labelled glucose with resultant randomization of the label by the liver would yield false low values for the hexose monophosphate shunt pathway. Further, in those instances wherein cerebral isolation was attempted or achieved, the trauma of the surgical manipulations involved leave open the questionable effects that this could have on sensitive brain tissue. Both of these objections have been eliminated with the benign surgical procedure of Moss, in which complete arterial isolation of normal intact functioning brain has been achieved. By perfusing through the common carotid arteries at a differential pressure 20 mm (Hg) higher than mean arterial pressures, cerebral perfusions can be conducted while excluding systemic blood from the cerebral circulation. Indeed, the technic permits investigating the precise role of the liver in cerebral glucose metabolism and in particular to check Geiger’s important observation that the liver (or cytidine and uridine) is required for glucose utilization in the brain. POPE:I have one question that I would like to ask Dr. Jilek. How adequate is the vertebral circulation and the collateral circulation in general after bilateral carotid occlusion? Is the developmental aspect of the cerebral vascularization in any way a factor in the experimental results you have described? J ~ L E KFrom : the work of Craigie it is known that the development of circulation, vascularization, goes parallel with the development of intensity of metabolism in the nervous tissue. Also histopathological changes caused by oligemia in the CNS show that most important is the intensity of metabolism, energetic, turnover and not the eventual developmental changes in the circulation in carotid and vertebral arteries. WEINSTEIN: I would like to ask Dr. SchadC the following question: Having once

G E N E R A L DISCUSSION

185

established the mathematical formulae for obtaining the volume index, it is necessary to make measurements of the area of the dendritic processes which lie within each of the concentric rings, or will the measurements within a fewer number of such rings suffice to obtain the answer? S C H A D In ~ : order to obtain exact data on the area of the dendritic processes one should measure and calculate the surface area within each of the concentric rings. In view of the decremental conduction in dendrites it is important to know the contribution of the surface area at any given distance to the whole receptive pole of the neuron. WEINSTEIN: In some of the graphs which Dr. SchadC showed, the intercept of the lines with the ordinate varied. What effect did this have on the final answers? S C H A DIn ~ : the graphs I showed, the logarithmic values of the number of dendritic intersections per unit area of the concentric spheres were plotted against the distance of this surface from the perikaryon. For all neuron populations lines could be drawn, fitting to the logarithmic form of y

= ae-kx

In this equation the factor a has no biological significance but indicates the point at which the regression line intersects the y-axis. : Dr. Scheibel presented evidence for rates of maturation of numerous WEINSTEIN types of neurons within the cortex. What relevance might this have to your studies? S C H A DI~perfectly : agree with Dr. Scheibel that there are different rates of growth and maturation of various types of neurons in the cerebral cortex. We have also found, for example that the pyramidal cells and stellate cells of a given layer in the middle frontal gyrus of the human brain show a different rate of differentiation and growth. Since we are analyzing the dendritic branches quantitatively we can only work with a large number of cells. This is the reason why we have limited our studies to an analysis of pyramidal and stellate cells and did not take into account other types of cells such as the fusiform cells. POPE:Are there any further general questions? DRAVID:I have a question for Dr. Berl. The amino acid values which I myself presented and those which you did in kitten brains were expressed in terms of wet weight but I remember a paper by Dr. Waelsch who reported glutamic acid and glutamine in developing rat brains and his values, expressed in terms of dry weight, do not show any change as the rat grows. I wonder if Dr. Berl would like to comment on it? BERL: Well, those are the values that Dr. Waelsch reported some time ago on developing rat brain. Our studies were done on kitten brain and there are some species differences apparent from the dry weight determinations obtained in this study. There did not seem to be this correlation in the kitten brain. The water content during the first week or so postnatally did not change very much, wereas during this period the amino acids were increasing rather considerably. COHEN: In line with this particular question of whether to use dry weight or wet weight, each procedure has its own individual problems. Dry weight is not only in-

186

G E N E R A L DI SC U SSION

fluenced by variations of intra-cellular liquids but in the developing brain by unequal increases in the intra-cellular solid constituents and particularly by laying down of myelin. In the work that we have done previously, we assumed that we were working with cellular phenomena which could best be expressed in terms of tissue cellularity. We considered the best way to do this was in the terms of DNA. As it turned out, the DNA and wet weight of rabbit brain paralleled each other exactly. Thus we considered wet weight as indicative of cellular content. As I recall, Uzman also demonstrated the same relationship in his studies on, I believe, mice. I would thus consider that Dr. Berl’s values represent an increase in the materials studied as he indicated. I would like to ask Dr. Berl if there is any information on glutamine synthetase in these young animals in which there has been a lower incorporation of radio activity into the glutamine? BEIIL:I do not know of any such data. I have plans to look into it. POPE:Are there any more questions? WEINER:I would like to ask Dr. Berl if he has measured the amount of radioactivity in the TCA insoluble fraction of the brain tissues, the idea, of course, being that the radioactive glutamic acid may be shunted, either via glutamine or directly, into protein and/or other insoluble components? This alternate pathway may be utilized to a greater extent during earlier stages of development. BERL:No, we did not. 1 suppose perhaps we should, but in such short time experiments as these we would not expect to find very much incorporated in protein. GUROFF: I would like to ask one of the discussants, Dr. Swaiinan, a question. I wonder if you have any data on the endogenous respiration comparing slices from adults and from newborn animals? SWAIMAN: We do have such data. The rate of oxygen uptake in developing rabbit brain slices utilizing endogenous substrate is similar to the rate noted when aspartate is utilized as the sole oxidizable substrate. Studies utilizing L-alanine and L-glycine yield similar rates of oxygen uptake at the various ages studied. Endogenous adult brain slice respiration proceeds at a rate comparable to that found in the 16-day-old slices when L-aspartate is the only oxidizable substrate present.

187

The Uptake of Aromatic Amino Acids by the Brain of Mature and Newborn Rats GORDON GUROFF

AND

SIDNEY U D E N F R I E N D

Laboratory of Clinical Biochetriistry, National Heart Institute, Bethestla, Mrl. ( U . S . A . )

Several studies have indicated that the penetration of ions and metabolites from the blood stream into the brain is faster in newborn animals than in adults (Bakay, 1953; Himwich et al., 1957; Lajtha, 1957, 1958, 1961; Waelsch, 1955). It has been suggested that the ‘blood-brain barrier’ is either reduced or missing in young animals. However, no direct demonstration of reduced blood-brain barrier function, even in foetal animals, has been possible using the classical indicator, trypan blue (Grazer and Clemente, 1957; Millen and Hess, 1958). Although the difficulties involved in making meaningful comparisons of immature and adult brain have been stressed (Dobbing, 1961), it is of importance to attempt to discover the nature of the changes giving rise t o these anomalous results. The first question resolves into the following: Does increased uptake of metabolically active materials by developing brain depend upon a decreased barrier function or on some increased active transport mechanism? Evidence to be presented indicates that, at least for amino acids, a change in the latter mechanism is the most likely cause. AROMATIC AMINO ACID UPTAKE I N ADULT RAT BRAIN

in

ViVO

A study of the in vivo uptake of the aromatic amino acids in adult rats reveals that the tyrosine, tryptophan, or phenylalanine content of brain rises in response to elevated blood levels of these amino acids (Fig. 1) (Chirigos el al., 1960; Guroff and Udenfriend, 1962). Previous work with other amino acids under similar conditions has indicated that, while equilibration between blood and brain amino acids is rapid, as revealed by tracer studies, (Lajtha, 1958; Lajtha et al., 1959; Roberts et al., 1959) elevated concentrations of amino acids in blood are reflected poorly or not at all in brain (Lajtha, 1958; Schwerin et al., 1950; Gaitonde and Richter, 1955; Dingman and Sporn, 1959; Kuttner et a/., 1961; Friedman and Greenberg, 1947; Kamin and Handler, 1951; Goldstein, 1961). The aromatic amino acids, then, are as well taken up by brain as any group studied and tyrosine appears to be the best of the aromatic group. It can readily be shown that tyrosine uptake by brain is remarkably stereospecific Rrferences p . 1961197

188

G.

G U R O F F A N D S. U D E N F R I E N D

Fig. 1. Increases in aromatic amino acid content of adult rat brain following intraperitoneal administration. L-tyrosine was given as a single injection (500 mg/kg), L-phenylalanine and L-tryptophan = brain; were given repeatedly throughout the experiment in doses of 25 mg/rat. 0-0 0-0 = plasma.

Fig. 2. Increases in tyrosine content of adult rat brain following intraperitoneal administration of the or L-isomer (500 mg/kg). A---A and 0-0 = following D-tyrosine injections; 0--0 and A-A = following L-tyrosine injections.

D-

(Fig. 2). However, the small amount of tyrosine entering the brain after administration of the D-isomer was shown to be in the D-form so the stereospecificity is not absolute. It was found, in addition, that the presence of the D-isomer does not interfere with the uptake of the L-form. This stereospecificity has also been demonstrated with respect to tryptophan uptake and so is most likely a general phenomenon. Certain amino acids can inhibit the entrance of tyrosine into the brain tissue (Table 1) probably by a competitive mechanism. The other aromatic amino acids and the long-chain aliphatics are excellent inhibitors but basic or acidic amino acids

T H E U P T A K E OF A R O M A T I C AMINO ACIDS

189

TABLE I E F F E C T O F O T H E R A M I N O A C I D S O N T H E D I S T R l n U T I O N OF L - T Y R O S I N E B E T W E E N A D U L T RAT BRAIN A N D PLASMA

Amino acid

in vivo* 3i.ain-to-plasmavatin (30 min)

L-Tyrosine L-Tryptophan D-Tryptophan 7 p-Fhoro-~~-phenylalanine I L-Leucine i L-Histidine L-Alanine L-Arginine t L-Lysine + L-Glutamate

0.72 0.18 0.42 0.24 0.24 0.38 0.88 0.84 0.83 0.8 1

L-Tyrosine -1- L-Tryptophan -I DL-Norleucine p-Hydroxyphenylacetic acid Isovaleric acid

0.60

L-Tyrosine ‘Cycloleucine’ (I-aminocyclopentane carboxylic acid)

0.65

+ +

+

+ +

+

0.20 0.29 0.59 0.44

0.37

* Tyrosine (500 mg/kg) and competitors (1000 mg/kg) were injected intraperitoneally at the same time except cycloleucine which was injected 30 min before tyrosine. have no effect. Acid congeners of inhibitory amino acids do not inhibit tyrosine uptake. The demonstration that cycloleucine is a competitor may have special significance because this amino acid remains in the blood stream for long periods following administration (Christensen and Clifford, 1962). It may be possible through the use of this analog to induce a chronic inhibition of amino acid transport into the brain. The direct demonstration of inhibition of amino acid uptake by phenylalanine is difficult because of the concomitant increase i n blood tyrosine. It can be shown indirectly, though, that tyrosine uptake is inhibited by phenylalanine (Fig. 3), since the distribution ratio of tyrosine arising from phenylalanine administration is much less than that found after tyrosine itself is given. By implication, then, all the aromatic amino acids are taken up by a common pathway which also extends to the long-chain aliphatics such as leucine and valine. Several structural analogues of tyrosine are taken up by brain (Table 11) but to a lesser extent than tyrosine itself. p-Hydroxyphenylacetic acid, however, is excluded completely. It appears that an amino group is necessary for uptake into the brain substance. Clearly the brain takes up aromatic amino acids by an active mechanism. The uptake is concentrative, structurally and sterically specific, and is subject to competitive inhibition. Comparative studies with some other tissues, primarily muscle, indicate References p . 1961197

190

G. G U R O F F A N D S. U D E N F R I E N D

I

I

I

I

1

I

I

1

E

3.

I00

Plasma -

-

80

. 1

-A

Plasmo

-

Brain

0

30

6o

Min

90

Fig. 3. Increases in the tyrosine content of brain and plasma of the adult rat following intraperitoneal administration of L-tyrosine or of L-phenylalanine. 0--0 = following L-phenylalanineinjections; n--A = following L-tyrosine injections. T A B L E I1 DISTRIBUTION OF VARIOUS T Y R O S I N E CONGENERS BETWEEN ADULT R A T B R A I N A N D P L A S M A it? vivo*

Compound

Brain-to-plasma ratio (30 niin)

L-Tyrosine Tyrarnine a-Methyl-DL-tyrosine D-Tyrosine p-Hydroxyphenylacetic acid

*

0.66 0.23 0.11

0.09 0.01

Materials were injected intraperitoneally (500 mg/kg).

that no such mechanism is present. Brain, then, is an organ, like kidney and intestine, whose specialized function requires highly specific regulatory mechanisms at the transport level of metabolism. AROMATIC AMINO A C I D U P T A K E BY A D U L T RAT BRAIN

in vitro

The uptake of aromatic amino acids by brain slices (Guroff et al., 1961) has been studied in an attempt to localize and describe the mechanism. It can be seen that the uptake of tyrosine by brain slices is rapid, concentrative, and metabolically linked (Fig. 4). The competitive relationships between amino acids are exactly the same as those found in vivo (Table 111), the aromatic and long-chain aliphatic amino acids being the best inhibitors. The structural specificity for concentrative uptake by the slice is identical with the structural requirements for uptake into brain in vivo with the one striking exception that the in vitro system exhibits no stereospecificity (Table IV).

191

THE UPTAKE OF AROMATIC AMINO ACIDS 2.00

1.80

1.60 1.40

1.20

1.00

.80 .60

0

20

40

60

80

100

120 140 Min

Fig. 4. Uptake of L-tyrosine by slices from adult rat brain. :C

=0 2 ;

0

Nz.

=

T A B L E 111 E F F E C T O F OTHER A M I N O A C I D S O N T H E D I S T R I B U T I O N O F L-TYROSINE B E T W E E N INTRACELLULAR WATER O F A D U L T R A T B R A I N SLICES A N D

Distribution ratio (60 min)

Amino acid

L-Tyrosine L-Glutamic acid -1- L-Arginine L-Histidine L-Tryptophan L-Phenylalanine L-Valine p-Fluoro-DL-phenylalanine

+ + + + + +

MEDIUM*

,ug/ml ce[l wafer ,ug/ml medium

)

(

2.60 2.51 2.55 1.77 1.28 1.15 1.15 0.90

*

Brain slices were incubated at 37" in Krebs-Ringer bicarbonate buffer Mconcentrationsofthecornpetitors. containing 1 . 10-3M~-tyrosineand1 . T A B L E IV D I S T R I B U T I O N OF V A R I O U S T Y R O S I N E C O N G E N E R S BETWEEN A D U L T RAT B R A I N SLICES A N D MEDIUM*

Compound L-Tyrosine D-Tyrosine a-Methyl-L-tyrosine Tyrarnine p-Hydroxyphenylacetic acid

Distribution ratio (60 min)

(,u:EZ::m)

1.86 1.67 1.61 1.35 0.61

* Brain slices were incubated at 37" in Krebs-Ringer bicarbonate buffer M tyrosine congener. containing 1 . References p . 1961I97

192

G . G U R O F F A N D S. U D E N F R I E N D

Work of this nature has been pursued in many laboratories (Stern et al., 1949; Terner et al., 1950; Takagaki et a/., 1959; Schanberg and Giarman, 1960; Neame, 1961,1962; Abadom and Scholefield, 1962). The results with other amino acidsparallel these findings with tyrosine. It can be concluded that the in vitro system is a useful model for the study of amino acid uptake by the whole brain. However, by comparing the in vivo and in vitro data a suggestion can be made that two mechanisms are involved in the uptake of amino acids by brain. First, a barrier function which prohibits the entry of acidic molecules, reduces the speed of entry of the amino acids themselves, and possesses great stereospecificity. Second, a cellular concentrating mechanism which is metabolically linked and subject to competitive inhibition. Both mechanisms are operative in vivo but the first is not found in slice experiments and may be part of the ‘blood-brain barrier’. A R O M A T I C A M I N O A C I D U P T A K E BY N E W B O R N R A T B R A I N

in vivo A N D in vitro

When tyrosine is administered to newborn rats, brain levels of this amino acid rise (Fig. 5). The rate of increase is comparable to that observed in adult animals but the final distribution ratio (brain/plasma) obtained in newborn animals is greater. Also, the endogenous brain-plasma ratio of tyrosine is higher in newborn animals than in adults. These results indicate that the total transport system for tyrosine in the newborn rat is more active than in the adult. The structural specificity of the uptake into brain is the same in the newborn as in the adult (Table V), p-hydroxyphenylacetic acid being completely excluded in both cases. The same competitive relationships exist (Table VI) regardless of age. Finally, the marked stereospecificity observed in the adult was found to be just as dramatic in

=c E 120

0

1 0

15

I 30

I 45

I

I

, 60

Min

Fig. 5. Uptake of L-tyrosine by brain of newborn rat following intraperitoneal injection (500 mg/kg). 0 = brain; 0 = plasma.

THE UPTAKE OF AROMATIC AMINO ACIDS

193

the newborn (Table V). It is clear, then, that the properties postulated to be part of the barrier function, i.e., the stereospecificity and the exclusion of acidic congeners, are as marked in the newborn as in the adult. The one characteristic which perhaps indicates a balance between the barrier and the concentrating mechanism, i.e., the distribution ratio, is altered. TABLE V D I S T R I B U T I O N OF V A R I O U S C O M P O U N D S

BE-rWEEN B R A I N in viva*

A N D PLASMA

OF NEWBORN A N D ADULT RATS

Disrriburion ratio (30 min)

L-Tyrosine D-Tyrosine L-Tryptophan D-Tryptophan p-Hydroxyphenylacetic acid

*

Newborn

Adult

1.13 0.31 0.74 0.30 0.05

0.63 0.12 0.26 0.08

0.01

Compounds were injected intraperitoneally (500 mg/kg).

T A B L E VI E F F E C T S O F O T H E R A M I N O A C I D S O N T H E U P T A K E OF L - T Y R O S I N E B Y BRAIN OF N E W B O R N R A T S

in vivo*

Increase in L-ryrosine content (30 niin)

L-Tyrosine p-Fluoro-DL-phenylalanine L-Tryptophan

+ +

Plasma (pg/nil)

Brain (pggig)

35

32 40 0

81 69

* L-tyrosine (500 mggikg) and competitor (1000 mg/kg) were injected intraperi toneally. An examination of the uptake of tyrosine by slices from the brains of newborn rats (Fig. 6) shows that the concentrating mechanism in the newborn is much more active than in the adult. The mechanism of the amino acid uptake is unknown so the basis of the difference between newborn and adults is also obscure. It is interesting, however, that amino acid transport in the brain, as in other systems (Noall et al., 1957), is most active in the rapidly growing tissues of young animals.

CONCLUSIONS

The data presented above appear to answer the original question. The barrier function of the brain as related to amino acids is unchanged in newborn animals. The more References p . 1961197

194

G . G U R O F F A N D S. U D E N F R I E N D 350 I

Of

0

1

I

I

I

10

I

20

I

30

1

I

I

I 40

I 50

I 60

I

Min

70

Fig. 6. Uptake of L-tyrosine by slices of brain from adult and newborn rats. 0 = newborn; 0 = adult.

rapid rate of amino acid uptake by the brains of newborn animals seems to be due to an increased concentrating mechanism which is unrelated to the barrier function. Another question concerning these studies is the following: What are the consequences of a high blood level of one amino acid on the over-all metabolism of the brain? It is apparent from the competition data that high chronic levels of one amino acid could limit the uptake of related amino acids by the brain. A careful examination of such data further shows (Table VII) that high blood levels of inhibitory amino acids will also lower the endogenous tyrosine content of the brain. In addition, there is ample evidence that high levels of one amino acid will lower the amounts of metabolites of related amino acids found in the brain (Renson et al., 1962; Louttit, 1962; Wang et al., 1962). For example (Renson et al., 1962) (Table VIII), an injection of phenylalanine lowers the amount of brain serotonin formed from an injected dose of 5-hydroxytryptophan. It can be concluded that an amino acid present in elevated amounts in the blood can limit the uptake and, thus, the further metabolism of related amino acids in the brain. T A B L E VII EFFECT OF INJECTIONS O F AMINO ACIDS O N T H E ENDOGENOUS LEVELS OF

TYROSINE IN ADULT RAT BRAIN

in vivo*

Tissue tyt-osine (30 min) Aniino acid injected

None L-Tryptophan p-Fluoro-DL-phenylalanine L-Leucine

*

Brain (pglg)

Plasma (pglml)

19 11

9

14 16 12

11

9

Amino acids were injected intraperitoneally (1000 mg/kg)).

T H E U P T A K E OF A R O M A T I C AMINO ACIDS

195

We would suggest, then, that in conditions in which abnormally large amounts of amino acids occur chronically in the blood stream, e.g., phenylketonuria, maple syrup urine disease, competition exists at the level of transport of amino acids into the brain. T A B L E VllI E F F E C T O F L - P H E N Y L A L A N I N E O N C O N V E R S I O N 01. 5 - H Y D R O X Y T R Y P T O P H A N TO S E R O T O N I N I N R A T B R A I N

iii vivo*

Brain serotonin (/.gig)

Uninjected L-Phenylalanine

Conirols

After 5-HTP

0.43 0.31

1.41 0.47

* L - P h e n y l a l a n i n e (1 g/kg) was injected 6 , 4 and 2 h before s a c r i f i c e . 5-HTP (300 mg/kg) was i n j e c t e d 1 h before s a c r i f i c e . Under such conditions the levels of metabolites derived from these amino acids are lowered due to decreased uptake of the precursor. It is possible that this phenomenon is responsible for the mental aberrations which occur in children afflicted with these diseases. The demonstration that the transport reactions are of major importance in the newborn animal makes this postulate even more likely. SUMMARY

Previous studies froin this laboratory coiicerning the uptake of aromatic amino acids by brain have indicated that certain characteristics of the uptake may be functions of the ‘blood-brain barrier’. Specifically these may be the marked stereospecificity and the exclusion of acidic congeners from the brain substance. Reports indicate that the ‘blood-brain barrier’ to various other ions and metabolites is absent or reduced in young animals even though the exclusion of the classical indicator of barrier function, trypan blue, is complete even in fetal animals. Studies on tyrosine uptake in newborn animals were therefore carried out to see if the stereospecificity was reduced or if acidic congeners penetrated the brain substance of newborn animals. It was observed that stereospecificity and barrier function for aromatic amino acids is as pronounced in newborn animals as in adults. However, the uptake of L-tyrosine by rat brain in vivo and in vitro was found to be faster and more concentrative in the newborn animals. It can be concluded that the transport mechanisms for various ions and metabolites are more active in newborn brain while the ‘blood-brain barrier’ remains unaffected. During these studies it was also observed that all the aromatic amino acids share a common transport pathway and compete for entry into the brain. Perhaps as a result of the competitive relationship, a high blood level of one amino acid will lower the endogenous brain level of a related one. Further, a high blood level of one amino acid can be shown to lower the endogenous metabolic products from a related amino acid. High blood levels of amino acid can alter drastically the metabolic sequences References p . 1961197

196

G. G U R O F F A N D S. U D E N F R I E N D

of the brain by competing with various amino acid precursors for entry into the brain substance. The implications of these experiments in the problem of the mental retardations associated with defects of amino acid metabolism, as in phenylketonuria, are discussed. REFERENCES ABADOM, P., AND SCHOLEFIELD, P. G., (1962); Amino acid transport in brain cortex slices, 1. IT. III. Canad. J . Biochem., 40, 1575-1618. BAKAY, L.,(1953); Studies on the blood-brain barrier with radioactive phosphorus. 111. Embryonic development of the barrier. A.M.A. Arch. Neurol. Psychiai., 70, 30-39. CHIRIGOS, M. A., GREENGARD, P., A N D UDENFRIEND, S.,(1960); Uptake of tyrosine by rat brain in vivo. J . biol. Chem., 235, 2075-2079. CHRISTENSEN, H . N . , AND CLIFFORD, J. A., (1962); Excretion of I-aminocyclopentane carboxylic acid in man and the rat. Biochim. biophys. Acta ( A m s t . ) , 62, 160-162. DINGMAN, W., AND SPORN,M. B., (1959); The penetration of proline and proline derivatives into brain. J . Neurochem., 4, 148-153. DOBBING. J.. (1961); The blood-brain barrier. Physiol. Rev., 41, 130-1 88. FRIEDMAN, F., AND GREENBERG, D.M.,(1947); Endocrine regulation of amino acid levels in blood and tissues. J . biol. Chem., 168, 405-413. GAITONDE, M. K., AND RICHTER,D., (1955); The uptake of S3j into rat tissues after injection of S35 methionine. Biochem. J., 59, 690-696. GOLDSTEIN, F. B., (1961); Biochemical studies on phenylketonuria. I. Experimental hyperphenylalanemia in the rat. J . biol. Chem., 236, 2656-2661. GRAZER, F. M., AND CLEMENTE, C. D.,(1957); Developing blood-brain barrier to trypan blue. Proc. SOC.exp. Biol. ( N. Y , ) , 94, 758-760. GUROFF, G., KING,W., AND UDENFRIEND, S., (1961); The uptake of tyrosine by rat brain in vitro. J . biol. Chem., 236, 1773-1777. GUROFF, G., AND UDENFRIEND, S.,(1962); Studies on aromatic amino acid uptake by rat brain in vivo. J . biol. Chern., 237, 803-806. HIMWICH, W. A., PETERSEN, J. C., AND ALLEN,M. L., (1957); Hematoencephalic exchange as a function of age. Neurology (Minneap.), 7 , 705-710. KAMIN,H., AND HANDLER, P., (1951); The metabolism of parenterally administered amino acids. 11. Urea synthesis. J. biol. Chem., 188, 193-205. KUTTNER, R., SIMS,J. A,, AND GORDON, M. W.. (1961); The uptake of a metabolically inert amino acid by brain and other organs. J. Neurochem., 6, 31 1-3 17. LAJTHA, A,, (1957); The development of the blood-brain barrier. J . Neurochem., 1, 216-227. LAJTHA, A., (1958); Amino acid and protein metabolism of the brain. 11. The uptake of L-lysine by brain and other organs of the mouse at different ages. J. Neurochem., 2, 209-215. LAJTHA, A., (1961); The brain-harrier system. 11. Uptake and transport of amino acids by the brain. J . Neurochem., 8,216-225. LAJTHA, A., BERL,S., AND WAELSCH, H., (1959); Amino acid and protein metabolism of the brain. IV.The metabolism of glutamic acid. J. Neurochem., 3, 322-332. LOUTTIT,R. B., (1962); Effect of phenylalanine and isocarboxazid feeding on brain serotonin and learning behavior in the rat. J . comp. physiol. Psychol., 55, 425428. MILLEN,J. W., AND HESS,A., (1958);:The blood-brain barrier: An experimental study with vital dyes. Brain, 81, 248-257. NEAME, K. D., (1961); Uptake of amino acids by mouse brain slices. J. Neurochem., 6, 358-366. NEAME, K. D., (1962); Uptake of L-histidine, L-proline, L-ornithine, L-lysine, and L-methionine by brain tissue in viiro: A comparison with uptake by sarcoma RD3 and other tissues. J. Neurochem., 9, 321-324. NOALL, M. W., RIGGS,T. R., WALKER, L. M., AND CHRISTENSEN, H. N., (1957); Endocrine control of amino acid transfer. Science, 126, 1002-1005. RENSON, J., WEISSBACH, H., AND UDENFRIEND, S., (1962); Hydroxylation of tryptophan by phenylalanine hydroxylase. J . biol. Chem., 237, 2261-2264. ROBERTS,R. B., FLEXNER, 3. B., AND FLEXNER, L. B., (1959); Biochemical and physiological differentiation during morphogenesis. XXIII. J . Neurochem., 4,78-90.

THE U P T A K E OF AROMATIC AMINO ACIDS

197

SCHANBERG, S., AND GIARMAN, N. J . , (1960); Uptake of 5-hydroxytryptophan by rat brain. Biochim. biophys. Acta (Amst.), 41, 556-558. SCHWERIN, P., BESSMAN, S. P., AND WAELSCH, H.. (1950); The uptake of glutan~icacid and glutamine by blain and other tissues of the rat and mouse. J . hiol. Chem., 184, 3 7 4 4 . STERN,J. R., EGGLESTON, L. V., HEMS,R., A N D KREBS,H . A., (1949); Accumulation of glutamic acid in isolated brain tissue. Biochem. J., 41, 41041 8. TAKAGAKI, G., HIRANO, S., AND NAGATA, Y., (1959); Some observations on the effect of D-glutamate on the glucose metabolism and the accumulation of potassium ions in brain cortex slices. J . Neurochem., 4, 124-134. TERNER, C., EGGLESTON, L. V., AND KREBS,H. A., (1950); The role of glutamic acid in the transport of potassium in brain and retina. Biochem. J., 47, 139-149. WANG,H. L., HARWALKER, V. H., A N D WAISMAN, H. A., (1962); Effect of dietary phenylalanine and tryptophan on brain serotonin. Arch. Biochem., 97, 181-184. WAELSCH, H., (1955); The turnover of components of the developing brain. The blood-brain barrier. Biochemistry of rhe Developin: Nervous System. H. Waelsch, Editor. New York, Academic Press (p. 187).

198

Tryptophan Metabolism in the Brain of the Developing Rat* G E R T R U D E M. T Y C E , E U N I C E V. F L O C K

AND

C H A R L E S A. O W E N , JR

Mayo Clinic and Mayo Foundation, Rochester, Minn. (U.S.A.)

I t is known that oiily very small amounts of 5-hydroxytryptamiiie are present in the brain of the rat during early development (Kato, 1960; Karki et al., 1960; Nachmias, 1960) and that monoainine oxidase activity in homogenates of the brain of the newborn is one third that found in the young adult rat (Nachmias, 1960). Preliminary experiments showed that at this age, when only small amounts of 5-hydroxytryptamine were present, the concentration of tryptophan in the brain was considerably higher than i n the adult. We have made a study of changes in the concentration of 5-hydroxytryptamine and its metabolite, 5-hydroxyindoleacetic acid, in relation to the concentration of the precursor amino acid, tryptophan, during the early development of the rat.

METHODS

Male Sprague-Dawley rats were used. Tissues and blood of fetal rats i n the last 3 days of pregnancy, and of rats up to 21 days of age were compared with those of adult rats weighing 230 to 280 g. Newborn rats in this series were rats born within the previous 15 h ; further days i n age were measured from this time. The animals were killed by decapitation. Tissues were frozen in dry ice and stored at -4" until the determinations were carried out; 6 to 10 determinations were made at each age. The number of brains used for each determination depended on their weight; as many as 8 brains of very young animals were pooled, whereas only 1 or 2 brains of the 21-day-old rats were used. Fluorometric methods were used to measure 5-hydroxytryptamine, 5-hydroxyindoleacetic acid, tryptophan, and tryptamine (Bogdanski et al., 1956; Hess and Udenfriend, 1959; ROOS,1962). Because the fluoroinetric method used does not distinguish between tryptophan and tryptamine, the latter was extracted with benzene from alkaline tissue homogenates and determined separately (Hess and Udenfriend, 1959). In a number of our experiments the concentration of tryptophan was compared with that of another aromatic amino acid, namely, tyrosine. Tyrosine was measured in trichloracetic acid filtrates b y the method of Waalkes and Udenfriend (1957).

* This investigation was supported in part by research grant B-4004 from the National Institutes of Hedth. Public Health Service.

I99

TRYPTOPHAN METABOLISM

The uptake of tryptophan by the brain after intraperitoneal injection of tryptophan was studied in newborn, 3-day-old, 10-day-old and adult rats. L-tryptophan in amounts of 33 and 66 pg/g of body weight was dissolved in saline and neutralized to pH 7.0 with 0.1 N sodium hydroxide. The tryptophan was injected intraperitoneally in a final volume of 0.2 ml into infant rats and 2.0 ml into adult rats. After 30 min the animals were killed ; brain, skeletal and cardiac muscle, and blood were removed for tryptophan assay. RESULTS

In Table I are shown the concentrations of 5-hydroxyindoles in rats during infancy. I n confirmation of previous reports, only low concentrations of 5-hydroxytryptaniine TABLE I 5-HYDROXYlNDOLtS IN T H E DEVELOPINC R A T *

Age

(days)

Nc,vborn 1 3 5 10 21 Adult

5-Hyduoxytryptarnine in brain, ,wig

0.24 0.23 0.23 0.21 0.29 0.40 0.52

4 0.01

*d= 0.02 * 0.03 0.02 0.05 f 0.03 -C 0.02

(6) (6) (6) (6) (6) (6) (10)

5-Hydroxyindoles in hloorl, pgliiil 0.16

+ 0.01

0.12,

0.14

(5) (2)

0.43, 0.41 (2) 0.75 5 0.03 (4) I S O 0.1I (5) 1.69 0.15 ((1)

**

5- Hy~luoxyindoleacetic acid in brain, pgig

0.38 t 0.03 0.35 i 0.02 0.38 i 0.02 0.35 & 0.02 0.45 -C 0.01 0.49 t 0.01 0.41 -k0.01

(6) (6) (6) (6) (6) (6) (12)

* The average concentrations of 5-hydroxyindoles i n blood and brain of infant and adult rats. The number after the is the standard error of t h e mean. and the number of determinations is in paren theses. were found in the brains of infant rats; the concentration of total 5-hydroxyindoles in blood was also low in the newborn. Total 5-hydroxyindoles increased in the blood during the 1st week of life; increase in 5-hydroxytryptamine occurred in the brain somewhat later, between 10 and 21 days. Pepeu and Giarnian in 1962 noted that in the goat the concentration of 5-hydroxytryptamine was three times higher in the blood of the newborn than in the mother, while in the rabbit the reverse was true. In contrast, the concentration of 5-hydroxyindoleacetic acid in the brains of our newborn rats war not markedly less than that found in the adult (Table I). Although monoamine oxidase activity has been found to be reduced in the brain of the newborn rat, apparently some turnover of 5-hydroxytryptamine does occur. I n Table I1 is shown the concentratioii of tryptophan and tyrosine in brain, skeletal and cardiac muscle, and plasma of rats during the first 21 days of life. The rats u p to 21 days were not fasted before they were killed; comparison is made with the concentration of these amino acids in noiifasted adult rats and in adult rats that had been fasted for 24 h. In fetal and newborn rats the concentration of tryptophan in the brain was about three times that found in the adult. During the first 3 days of life References p . 2021203

G. M. T Y C E , E. V. F L O C K A N D C. A. O W E N , JR.

200

T A B L E 11 TRYPTOPHAN A N D TYROSINE I N THE DEVELOPING RAT*

T,.yptoplian or tyrosine, pglg or nil in: Age (days)

Brain

Fetal Newborn 1 3 5 10 21 Adult, fasted Adult, nonfasted

19.9 & 0.5 21.6 f 1.4 12.0 1.6 7.0 & 0.6 10.5 f 1.0 9.5 i 0.5 8.5 f 0.4

Fetal Newborn 1 3

56.9 & 1.6 36.6 f 2.1 38.7 & 2.6 44.1 f 1.7 43.5 & 2.7 56.8 f 2.2 26.3 f 2.2

5

10 21 Adult, fasted Adult, nonfasted

Plasma

(7) (6) (6) (6) (6) (6) (6)

21.4, 21.8 19.0 & 1.3 18.0 & 2.1 15.5 & 0.7 20.5 & 1.5 14.9 & 0.7 15.0 410.8

Skeletal muscle Tryptophan (2) (6) 31.4 7.8 (9) 12.6, 20.5 (10) 13.9, 9.8 (9) (6) 15.4 f 1.6 (5) 7.0 f 0.4

+

6.6 f 0.3 (10)

12.0 & 0.6 (10)

6.7 f 0.4 (10)

21.7, 19.5 (2)

(7) (6) (6) (6) (6) (6) (6)

42.8, 45.9 24.6 k 0.6 29.9 & 1.0 39.8 & 1.5 42.9 & 0.7 46.3 & 3.8 18.8 & 2.0

Cardiac muscle

(6) (2) (2)

24.0 I 4 . 7 (4) 14.3, 18.3 (2) 12.2, 13.0 (2)

(5) (4)

18.9 20.7 (2) 8.3 4I 1.9 (6)

8.2 5 0 . 4 (3)

10.9 & 0.4 (8)

10.5

0.5 (4)

Tyrosine (2) (6) 51.2 i~3.8 (6) 43.2, 50.5 (7) 66.1, 60.4 (6) (6) 74.6 f 4.0 (6) 37.5 2.1

10.6, 12.1 (2)

(6) (2) (2)

40.9 i 3.4 (4) 45.2, 44.3 (2) 72.2, 51.8 (2)

(5) (4)

66.8, 83.7 (2) 22.9 i 5.5 (6)

19.3 f 0.9 (10)

15.5 & 1.0 (10)

25.9 L- 0.7 (8)

20.5 k 0.5 (8)

22.8 f 1.4 (10)

27.5, 52.6 (2)

41.2 & 4.8 (4)

45.1, 27.6 (2)

-

* Mean concentrations of tryptophan and tyrosine in brain, plasma, skeletal and cardiac muscle of infant rats compared to fasted and nonfasted adults. Rats up to 21 days were not fasted. The number after the 5 is the standard error or the mean, and the number of determinations is in parentheses. there was a rapid decrease in concentration of tryptophan, followed by a modest increase during the next week, and the adult level was almost reached in the 21-day-old weanlings. No tryptamine could be demonstrated in 4 pools (6 brains each) of newborn rats. In the fetal and the newborn rat the concentration of tryptophan in the plasma approximated that found in the brain; from 1 day onward the concentration of tryptophan in the plasma was greater than that in the brain, and the final 1 : 2 ratio of brain-to-plasma tryptophan appeared to be established by the 3rd day. In the first 3 days of life it would appear that the blood-brain barrier for tryptophan is not established. However, it is noteworthy that much greater variation was found in the concentrations of tryptophan in the skeletal and cardiac muscles of the newborn than in the brain: in skeletal muscle, concentrations ranged from 16-68 pg/g, in cardiac muscle, from 15-38 pg, but in the brain, from only 17-25 pug. Tyrosine was present in fetal brains in concentrations three times greater than in the adult; the brains of the newborn rats contained somewhat less tyrosine. From

20 1

TRYPTOPHAN METABOLISM

birth until 10 days of age there was a steady increase in concentration of tyrosine. By the 21st day the concentration of tyrosine in the brain had decreased to levels only slightly higher than in the adult. The concentration of tyrosine in the plasma was always lower, but it generally paralleled that in the brain; at each age the concentrations of tyrosine in skeletal and cardiac muscle were higher than in plasma and brain. T A B L E 111 U P T A K E O F T R Y P T O P H A N BY T l l E B R A I N 1 N l N F A N T A N D A D U L T R A T S *

Age (clays)

Newborn* *

3 10

Adult

Tryptophan injected, Pgk

Tryptophan, p g / g or nil in

Plasma

*

19.0 1.3 40.5 40.5 71.2 15.5 0.7 43.0 t 4.0 59.9 f 10.0 14.9 0.7 39.1 _t 3.9 55.0 i 1.7 12.0 5 0.6 40.7 f 4.6 69.8 i 4.2

+

+

Brain 21.6 1.4 36.8 42.4 51.7 7.0 0.6 27.2 3.2 43.3 2.8 9.5 5 0.5 28.7 t 1.7 43.0 1.0 6.6 0.3 16.1 & 1.7 28.2 2.9

* *

*

Skeletal

muscle

Cardiac muscle

15.4 & l.6"** 18.9, 20.7t 35.1 2.7 39.9 & 2.3 49.4 i 2.9 49.9 i 3.1 10.9 0.4t-i8.2 0.4j-t 23.1 22.0 C 2.6 3.0 45.9 i 2.1 49.6 :t 2.5

+

* *

* Average concentrations of tryptophan in plasma, brain, skeletal and cardiac muscle 30 mill after a single intraperitoneal injection of L-tryptophan i n concentrations of 33 and 66 pg/g of body weight. The number aftei the -C is the standard error of the mean, and the number of determinations is in parentheses. ** Two animals used for each determination. * * * Mean of 5 determinations. Two determinations. tt Mean of 8 determinations. In Table 111 is shown the concentration of tryptophan in brain, skeletal and cardiac muscle, and plasma of rats 30 min after intraperitoneal injection of L-tryptophan i n amounts of 33 or 66 pg/g of body weight. In both infant and adult rats an increase occurred in the concentration of tryptophan in the brain after the injection of these relatively small amounts of tryptophan. This increase was apparently greatest in the newborn rats and least in the adult rats. In 10-day-old and adult rats substantial increases in the concentrations of tryptophan were noted in skeletal and cardiac muscle. I n both age groups the final concentrations in skeletal and cardiac muscle were higher than in the brain. COMMENT

High concentrations of tryptophan and tyrosine were found i n the plasma and tissues of infant rats. A number of other amino acids or derivatives have been reported to Rrferences p . 202j203

202

C. M . T Y C E , E. V. F L O C K A N D C . A. O W E N , JR.

be present in immature brains in higher concentrations than in the adult. These include taurine, alanine, and lysinc in mice (Roberts et al., 1950; Lajtha, 1958). On the other hand glutamic acid, aspartic acid, or y-amino butyric acid have been found in reduccd concentrations in the brains of newborn mice, frogs, chicks, kittens or dogs (Roberts and Simonsen, 1962; Berl, 1963; Dravid and Himwich, 1963), and the concentration of glutamine found in newborn kittens was similar to that in the adult (Berl, 1963). The changes that occurred in the concentrations of these two aromatic amino acids followed a different pattern during the first 21 days of life in our rats; the decrease in concentration of tryptophan was rapid in the first 3 days whereas thc decrease in the concentration of tyrosine occurred after the 10th day. After thc intraperitoneal injection of L-tryptophan the amino acid entered the brain more rapidly in the infant than i n the adult rat. It was, however, surprising that increases were apparent in the brain of adult rats after the injection of such small amounts of tryptophan. In previous experiments much larger quantities of tryptophan have been injected in order to demonstrate increases in brain (Guroff and Udenfriend, 1962, 1963). Entry of tryptophan into the infant brain may be even more rapid than was apparent from our experiments. It is likely that tryptophan is incorporated into proteins and proteolipids that have been shown to be synthesized rapidly in the first weeks of life in rats and mice (Clouet and Gaitonde. 1956; Folch-Pi, 1955). During the time when the concentration of tryptophan was high in the brain of the infant rat, only very low concentration of 5-hydroxytryptamine could be found. This is in confirmation of previous reports (Karki et al., 1960; Kato, 1960; Nachmias, 1960). The concentration of the metabolite, 5-hydroxyindoleacetic acid, in brains of infant rats was not markedly less than in adults. This suggests that some turnover of 5-hydroxytryptamine occurred in brains of infant rats. SUMMARY

The concentrations of 5-hydroxytryptamine and its metabolite, 5-liydroxyindoleacetic acid, were related to the concentration of the precursor amino acid, tryptophan, during the early development of the rat and in the adult rat. High concentrations of tryptophan and tyrosine were found in the plasma and other tissues of infant rats compared to those of adult rats. Low concentration of 5-hydroxytryptamine was found in the brain of the infant rat when the concentration of tryptophan was high. The finding of similar concentrations of 5-hydroxyindoleacetic acid in the brains of infant and adult rats indicates some turnover of 5-hydroxytryptamine in the brains of the infant rats. REFERENCES BERL,S., (1964); Postnatal changes in amino acid content of kitten brain. Progress in Brain Research, The developing Brain, Vol. 9. W. A. Himwich and H. E. Himwich, Editors. Amsterdam, Elsevier (p. 178). BOGDANSKI, D. F., PLETSCHER, A., BRODIE,B. B., AND UDENFRIEND. S., (1956); Identification and assay of serotonin in brain. J. Phaumacol. exp. Ther., 117,82-88. M. K.; (1956); The changes with age in the protein composition of CLOUET,D. H., AND GAITONDE, the rat brain. J. Neurochem., 1. 126-133.

TRYPTOPHAN METABOLISM

203

DRAVID,A . , AND HIMWICH,W. A., (1964); Biochemical studies of the central nervous system of the dog during maturation. Progress in Brain Research, The developing Brain.Vol.9. W. A. Himwich and H. E. Himwich, Editors. Amsterdam, Elsevier (p. 170). FOLCH-PI, J., (1955); Composition of the brain in relation to maturation. Biochemistry of the Developing Nervous System. H. Waelsch, Editor. Proceedings of the First International Neurochemical Symposium. New York, Academic Press (p. 121). S., (1962); Studies on aromatic amino acid uptake by rat brain in GUROFF,G., AND UDENFRIEND, vivo: Uptake of phenylalanine and of tryptophan : Inhibition and stereoselectivity in the uptake of tyrosine by brain and muscle. J . biol. Chem., 237, 803-806. G., AND UDENFRIEND, S., (1964); The uptake o f aromatic amino acids by the brain of mature GUROFF, and newborn rats. Progress in Brain Research, The developing Brain, Vol. 9. W. A. Himwich and H. E. Himwich, Editors. Amsterdam, Elsevier (p. 187). H ~ s sS. , M., A N D UDENFKIEND, S., (1959); A fluorometric procedure for the measurement of tryptamine in tissues. J . Pharmacol. exp. Ther., 127, 175-177. R., AND BRODIE,B. B., (1960); Norepinephrine and serotonin brain levels KARKI,N. T., KUNTZMAN, at various stages of ontogenetic development. Fed. Proe., 19, 282. KATO,R., (1960); Serotonin content of rat brain in relation to sex and age. J . Neurochem., 5,202. LAJTHA,A , , (1958); Amino acid and protein metabolism of the brain. 11. The uptake of L-lysine by brain and other organs of the mouse at different ages. J . Neurochem., 2, 209-215. V. T., (1960); Amine oxidase and 5-hydroxytryptamine in developing rat brain. J . NeuroNACHMIAS, chern., 6, 99-104. N. J.: (1962); Serotonin in the developing mammal. J . gen. Physiol., 45, PEPEU,G., AND GIARMAN, 575-583. E., FRANKEL, S., AND HAKMAN, P. J., (1950); Amino acids of nervous tissue. Proc. Soc. ROBERTS, exp. Biol. ( N . Y . ) , 74, 383-387. E., AND SIMONSEN, D. G., (1962); Free amino acids in animal tissue. Aiiiino Acid Pools: ROBERTS, Distribution, Formation and Function of Free Aniino Acids. J. T. Holden, Editor. New York, Elsevier (p. 284). Roos, B., (1962); On the occurrence and distribution of 5-hydroxyindoleacetic acid in brain. Life Sciences, I, 25-27. T. P., AND UDENFRIEND, S., (1957); A fluorometric mcthod for the estimation of tyrosine WAALKES, in plasma and tissues. J . Lab. clin. Med., 50, 733-736.

204

Tissue Acid-Base Changes During Maturation C. D. W I T H R O W

AND

D I X O N M. W O O D B U R Y

Depurtment of Pharnmcology, Uiiiversity of Utah College of Medicine, Salt Lake City 12. Utah (U .S.A .)

It is becoming increasingly apparent that a complete knowledge of acid-base metabolism is possible only if intracellular, as well as extracellular, acid-base parameters are understood (Robin, 196 I). Although many determinations of tissue intracellular hydrogen ion concentrations have been done in adult animals, no studies have been reported in which tissue cell pH was studied in immature animals. Therefore, tissue acid-base changes during maturation were studied by comparing acid-base patterns of 8-day-old, 25-day-old, and adult rats. METHODS

Micro methods were used to obtain individual values for blood pH and for C02 content of plasma, brain, and skeletal muscle. The tissue total COe contents of muscle and brain in all three age groups were fractionated into intracellular and extracellular components, by use of chloride space as a measure of extracellular volume. Additional tissue acid-base information was obtained by determining the distribution of the 13C-labeled dimethyloxazolidinedione (DMO) in similar groups of animals (Waddell and Butler, 1959). RESULTS A N D DISCUSSION

The results of cell pH measurements derived from the distribution of C02 are given in Table I. Although muscle cell pH decreased significantly between 8 and 25 days of age, consideration of all the pH changes reported here for both muscle and brain leads to the conclusion that cell pH does not markedly change during maturation. However, it is obvious that a marked decrease in intracellular bicarbonate concentration occurs during early growth. Additional data concerning cell pH changes during maturation are presented in Table 11. The DMO measurements reported here confirm the C02 observation that muscle cell pH does not vary widely during maturation despite the striking change in cellular total C02 content. Although not reported, similar data have been obtained for brain. However, cell pH measured by the DMO method is more acid in all age groups than is cell pH determined from COz distribution.

T I S S U E ACID-BASE

205

CHANGES

The above results have been interpreted in the following manner. Maintenance of a constant cell pH during growth indicates that the ratio between intracellular bicarbonate and carbonic acid concentrations does not change during maturation. However, the absolute amounts of each change markedly during growth. It has been suggested that the decrease in brain total COZduring growth is caused by an increase in carbonic anhydrase activity in this tissue during that period (Millichap et al., 1958). The present results are not in agreement with this suggestion since skeletal muscle, which contains no carbonic anhydrase (Van Goor, 1940), also decreased in COZ content during maturation. TABLE I INTRACELLULAR

pH

D A T A F O R CEREBRAL CORTEX A N D SKELETAL MUSCLE OF IMMATURE AND A D U L T RATS

Means & S.E.M.

1.63*** *0.09 (26)

8-Day

25-Day

Adult

33.1**

5 2.9 (22)

0.98 50.07 (20)

14.8 & 0.5

1.04 10.05 (20)

15.4 & 0.6 (18)

(20)

7.39* +0.04 (22)

1.63*** k0.09 (26)

f 1.3 (25)

7.29 k0.03 (20)

0.98 ~t0.07 (20)

f 0.8 (20)

7.37 &0.04 (20)

7.28 50.03

1.04 50.05 (20)

19.4 & 1.2 (18)

7.37 50.04 (18)

(18)

33.1***

17.9

7.41 lk0.02 (25)

in mmole/l cell HzO. The number in parentheses is the number of animals used in each experiment providing data for the indicated calculations. Asterisks denote significant difference from 25-day-old animals. none p = >0.05; *p = 0.01-0.05; **p = 0.0014.01; ***p = rences p. 212

212

H. A. W A I S M A N

et al.

known, but may be likely, that the enzymes which are ordinarily located on the cristae of the mitochondria are not formed or not functioning when the cristae are absent. At the present time it is not possible to correlate brain enzyme activity with morphological changes. Additional studies need to be made on the enzymes of the various portions of the brain as well as specialized stains on brain material to determine whether enzymes or enzyme components are altered by experimental phenylketonuria. SUMMARY

Preliminary results on the brains of rats and one monkey show that alterations in the brains from these phenylketonuric animals were primarily restricted to glial cells. The cytoplasm was apparently not involved and immature appearance of myelin was interpreted as typical of retarded or interrupted myelin formation. The loss of cristae in the mitochondria of these tissues implies some loss of enzymes which are located on the cristae. Further work on the enzyme content of various portions of the brain together with specialized staining procedures is now underway. REFERENCES AUERBACH, V. H., WAISMAN, H. A., AND WYCKOFF, L. B., (1958); Phenylketonuria in the rat associated with decreased temporal discrimination learning. Nature, 182, 871-872. BOGGS, D. E., AND WAISMAN, H. A., (I 962); Effects on the offspring of female rats fed phenylalanine. Life Sciences, 8, 373-376. F e t L I N c , A., (1934): Uber Ausscheidung von Phenylbrenztraubensaure in den Harn als Stoffwechsel Anomalie in Verbindung mit Imbezillitat. Hoppe-Seylers Z. physiol. Chern., 227, 169-1 76. GARROD, A. E., (1909); Znborn Errors of Metabolism. London. Henry Frowde. Poser, C . M., AND BOGART,VON, L., (1959); Neuropathologic observations in phenylketonuria. Brain, 82, 1-9. WAISMAN, H. A., WANG,H. L., PALMER, G., AND HARLOW, H. F., (1962); Experimental phenylketonuria in infant monkeys. Proceedings London Conference on the Scientific Study of Mental Retardation. Dagenham. May and Baker (p. 126-141). WANG,H. L., AND WAISMAN, H. A., (1961); Experimental phenylketonuria in rats. Proc. SOC.exp. Biol. ( N . Y . ) , 108, 332-335.

213

Effects of Reserpine and Isocarboxazid in the Frog G. R. PSCHEIDT Thudichum Psychiatric Research Laboratory, Calesburg State Research Hospital, Calesburg, Ill. (U.S.A.)

We have previously studied the effects of monoamine oxidase inhibitors and reserpine

in several species. In this paper we wish to report comparable observations on the frog. The frogs (Rana pipiens) were housed in concrete tanks and allowed free movement in running tap water. Either reserpine (5 mg/kg) or isocarboxazid (Marplan, 125 mg/kg) was injected into the dorsal lymph sac once a day for 3 days and the animals were killed by decapitation on the 3rd day. Three animals were included in each group and the brains pooled for analysis of serotonin and catecholamines by the method of Shore and Olin (1958) as modified by Mead and Finger (1961). In two experiments (6 frogs) the brains were divided into the following regions in order to gain some idea of the relative distribution of the amines: hemispheres, di- and mesencephalon, and rhombencephalon. Adrenalin was found to be the major catecholamine present in the brain. Neither reserpine nor isocarboxazid in the relatively large doses employed had any significant effect on the brain levels of adrenalin (Table I). In contrast serotonin levels were increased some 50 % above control values by isocarboxazid and reduced some 35 % TABLE I A M I N E CONTENT OF F R O G B R A I N

Whole brain (4.5 animals) ~

~

Adrenalin (pguglg) Isocarboxazid (125 mg/kg for 3 days) Control Reserpine (5 mg/kg for 3 days)

Serotonin (,ug/g)

2.2 0.4 2.2 rt 0.3

4.2 i- 0.4 2.8 0.4

2.0 f 0.4

1.8 rt 0.3

Normal brain parts (6 animals) Hemispheres Adrenalin (,ug/g) Serotonin (pg/g) References p , 2I6

1.o 0.9

Di-and mesencephalon

Rhombencephalon

2.8 3.5

1.5 2.0

214

G . R. P S C H E I D T

Fig. 1. Pupillary constriction following reserpine injection. The frog on the right with the smaller pupil received 5 mg/kg of reserpine each day for 3 days. The frog on the left received 125 mg/kg of isocarboxazid each day for 3 days. Photograph taken on the 3rd day.

Fig. 2. Color changes induced by reserpine and isocarboxazid. C = control frog; R = reserpine, = isocarboxazid, 125 mg/kgdaily for 3 days. Photograph taken on 3rd day.

5 mg/kg daily for 3 days; N

by reserpine. Both serotonin and adrenalin were found to be selectively concentrated in the di- and mesenceplialon. The rhoinbencephalon contained about half the

EFFECTS O F A M I N E S I N THE F R O G

215

mesencephalic concentration of each amine, while the hemispheres contained the least amounts of either amine. Reserpine consistently induced a marked pupillary constriction by the end of the 3rd day of injection. This is illustrated in Fig. 1 where a frog treated with reserpine is compared with a frog receiving isocarboxazid. The eyes of control frogs were indistinguishable from those treated with isocerboxazid. However, in one experiment (3 animals) where both isocarboxazid and reserpine were administered simultaneously the average pupillary diameter was intermediate between that of the reserpine-treated animals and controls. This indicates that isocarboxazid was capable of antagonizing this effect of reserpine. Reserpine and isocarboxazid had opposite effects on the coloration of the frogs. Reserpine-treated frogs became uniformly dark while those receiving isocarboxazid invariably became light. This is shown in Fig. 2 where one animal from each group is represented. The lightening effect of isocarboxazid is clearly depicted but it was difficult to suitably photograph the darkening effect of reserpine. Microscopic examination of the frog skin revealed that the melanocyte granules in the frogs receiving reserpine were completely dispersed while the granules in the isocarboxazid-treated frogs were aggregated. These effects were obtained in a wet environment at environmental temperatures of 15-17" and it became of interest to determine whether or not similar results would be obtained with frogs maintained at higher temperatures and in drier surroundings. Accordingly frogs were transferred to slightly moistened sand at room temperature and then given 5 mg/kg of reserpine daily for 3 days. These animals were somewhat darker than controls but became very light colored compared t o controls allowed to remain in water. Our values for frog brain amines agree with those recently published by Bogdanski et a]. (1963) (serotonin 3.7 pg/g, adrenalin 2.1 ,ug/g) allowing for differences in methodology. Khazan and Sulman (1961) studied the melanocyte dispersing activity of reserpine in frogs using higher doses of drug than were used in this study and suggested that reserpine caused an intensified secretion of melanocyte stimulating hormone. Our results agree with their findings completely. The lightening effect of the monoamine oxidase inhibitor isocarboxazid in the frog may be due to a direct antagonism of reserpine action on the frog pituitary or may involve increased availability of melatonin, the skin lightening agent found in amphibia. The chemical data show that in the frog the serotonin levels in the brain may be selectively increased by isocarboxazid or selectively decreased by reserpine without any significant alteration in adrenalin levels. Higher species do not exhibit this plienomenon which suggests that the frog possesses special mechanisms for regulating catecholamine metabolism.

SUMMARY

The normal content and distribution of serotonin and catecholamines in frog brain was determined. Adrenalin was the major catecholamine present. Both amines were present i n higher concentrations in the di- and mesencephalic regions. Reserpine and References p . 216

216

G . R. P S C H E I D T

isocarboxazid had opposite effects on the serotonin content, the former reducing and the latter elevating the amount present. Other effects of reserpine were pupillary constriction and skin darkening. These phenomena could be counteracted with simultaneous administration of isocarboxazid. Neither reserpine nor isocarboxazid in the doses employed had any effect on the adrenalin content of the frog brain. REFERENCES BOGDANSKI, D. F., BONOMI,L., AND BRODIE,B. B., (1963); Occurrence of serotonin and catecholamines in brain and peripheral organs of various vertebrate classes. Life Sci.,1, 8C-84. KHAZAN,N., AND SULMAN, F. G., (1961); Melanophore-dispersing activity of reserpine in Rana frogs. Proc. SOC.exp. Biol. (N. Y.), 107, 282-284. MEAD,J. A. R., A N D FINGER, K. F., (1961); A single extraction method for the determination of both norepinephrine and serotonin in brain. Biochem. Pharmacol., 6, 52-53. SHORE, P. A., AND OLIN,J. S., (1958); Identification and chemical assay of norepinephrine in brain and other tissues. J. Pharniacol. exp. Ther., 122, 295-300.

217

Effect of Diet on Lipid Composition of Brain M. K. HORWITT L. B. Mendel Research Laboratory, Elgin State Hospital, Elgin, 111. (U.S.A.)

Up to this point in the symposium, attention has been focused on the composition of amino acid metabolites in the brain so it may be worthwhde to record a brief comment related to the lipid composition of the brain. The brain is unique in that it has far more lipid than any other tissue, except adipose tissue. It was quite natural, therefore, for Dr. Thudicum to have concentrated so much of his energy toward the development of techniques for the analyses of phospholipids, cerebrosides and sulfolipids which, together with cholesterol, account for most of the brain lipids. In recent years, especially since the advent of gas chromatographic techniques, more attention has been paid to the fatty acid composition of some of the lipid components of the brain, especially after it became apparent that increasing the unsaturated fatty acid composition of the diet could be at least partially related to the production of encephalomalacia (Century et al., 1959; Horwitt and Bailey, 1959) under conditions where insufficient antioxidants were provided. Our first studies (Horwitt et al., 1959) on the effect of diet on the fatty acid composition of the chick brain was somewhat handicapped by the inadequacy of methods available in the early days of gas chromatography but two facts were already apparent: one, that the linoleic acid content of the brain was unusually low and two, that the type of fat fed could alter the fatty acid composition of nervous tissue. The latter was not surprising to the nutritional scientist who would expect the fatty acids of every phospholipid in the body to be affected by diet but it was considered unexpected by many in other disciplines. Subsequent analyses with better techniques (Horwitt et al., 1960) showed that the percentage of linoleic acid to total fatty acid in the brain mitochondrial fraction from one day old chicks could be raised from 0.4% to 2 % in 21 days by adding ethyl linoleate to a diet in which the fats were essentially all saturated. Adding 7 % cod liver oil to such a diet raised the docosahexanoic acid content from 8 to 16 % of the total fatty acids in the chick brain mitochondrial fraction, respectively. It should be noted that the so-called mitochondrial fraction, obtained by the centrifugal techniques used, contained considerable myelin. The fatty acids in rat brain mitochondrial fraction have been studied in greater detail by Witting et al., (1961). The lipids of brain mitochondria from weanling rats contained 1.7 % linoleic acid, 11 % arachidonic acid and 9 % docosahexanoic acid. By Refurences p. 219

TABLE I L.W. (Died of cardiovascular occlusion; diet for last 9 months contained 72 g safflower oil per day) PERCENTAGE COMPOSITION OF TOTAL FATTY A C I D S I N V A R I O U S TISSUES OF

Fatty Acid*

12 : o 14 : O 16 : aldehyde* * 16 : O 16 : 1 16:2 18 :aldehyde** 18 : O 18:l 18 : 2 (linoleic) 18 : 3 20 : 0 20 : 1 20 : 2 20 : 2 20 : 3(A5,8,11) 20 : 3(A8,11,14) 8, 11,14) 20 : 4 (As, 20:4(A8,11,14,17) 20 :5(A5,8,11,14,17) 22:2(A10,13) 22 :2(A13,16) 22 : 4 (A7,10,13,16) 22 : 4 ( A 10,13,16,19) 22:5(A4,7,10,13,16) 22 : 5 ( A7,10,13,16,19) 22 : 6 (A4,7,10,13,16,19) Others

Erythroeytes

Adipose f Buttock)

Adipose (Abdominal)

0. I 0.4 2.4 15.0 I .6 0.5 3.7 15.4 12.3 14.3

3.4 5.9 0.1 14.4 9.8 0.4 0.4 1.2 34.1 24.5 -

7.6 9.4 1.9 14.5 6.1 0.8 0.4 2.2 30.7 21.6 -

-

Stomach

-

5.2 7.8 1.6 17.8 6.3 0.8 0.5 3.4 25.3 24.4 0.6 0.8 0.5 0.3

0.5

-

-

-

-

0.1 0.6 0.9 0.3

0.1

-

0.2 1.1 0.1 0.2 0.2

-

-

-

0.5 0.2 0.8 0.2 1.9 14.9 I .5 0.9 0.2 0.1

0.4 0.1 0.3 tr 0.2 0.4 0. I

I .2

-

-

5.0

0.2

0.4

0.3 1.2 1.2 2.1 3.3

0.1

0.5 0.1

0.2 0.2 0.2

-

0.1 0.1 3.7

Stomach Mueosa

1 .1

2.2 18.6 1.7 0.3 1.1 13.9 18.4 20.1 0.6 0.2 0.6 0.9 -

-

Duodenum

Kidney

Heart

Liver

2.5 3.8 0.4 16.6 3.3 0.4 0.5 7.3 23.8 32.5 0.4

2.1 2.5 4.6 12.0 4.7 2.1 0.9 11.7 18.3 23.8

0.1 0.8 0.7 17.8 2. I 1.4

0.4 0.4

3.2 4.9 I .9 15.6 4.6 I .O 0.2 8.8 25.1 23.2 0.6 0.3 tr 0.4

-

-

-

0.1 0.6 7.4 0.1 0.1 0.2 0.1 0.3

0.2

-

tr tr 0.3 0.4 13.8 0.1

1.6 10.5 0.3 0.6 0.6 2.2

0.4 4.0 tr tr 0.1

-

1 .o

-

-

-

0.2 0.3 0.4 1.6

1.2 0.6

0.5 0.3 0.4 1.1

0.1 0.8 0.4 0.0

0.2 0.5 0.4 0.9

-

1 .o

0.7

-

0.7

0.1 0.1 -

0.5

-

18.6 12.9 23.2 0.5 -

0.8 -

2.4 13.0 0.2 -

0.2 1.1 1.o

-

0.4 2.2 0.6

Brain

lFrontalj -

0.4 2.1 17.0 1.6 0.4 2.6 23.1 24.7 1.1 0.2 0. I 1.1 0.2 0.2 tr 0.7 7.9 -

0.2 0.4 0.2 5.2

positions of double bonds. Saturated aliphatic aldehydes. tr (trace) =